No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Adhesion, invasion and evasion: The many functions of the surface proteins of Staphylococcus aureus
Abstract
Staphylococcus aureus is an important opportunistic pathogen and persistently colonizes about 20% of the human population. Its surface is 'decorated' with proteins that are covalently anchored to the cell wall peptidoglycan. Structural and functional analysis has identified four distinct classes of surface proteins, of which microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) are the largest class. These surface proteins have numerous functions, including adhesion to and invasion of host cells and tissues, evasion of immune responses and biofilm formation. Thus, cell wall-anchored proteins are essential virulence factors for the survival of S. aureus in the commensal state and during invasive infections, and targeting them with vaccines could combat S. aureus infections.
... During primary attachment of staphylococcal cells to biotic surfaces, microbial surface components recognizing matrix molecules (MSCRAMMs) that bind to host factors such as fibronectin (FnBpA, FnBpB, Embp), fibrinogen (ClfA, ClfB), and collagen (Cna, SdrF) play an essential role (Foster, 2020;Foster et al., 2014). Proteins such as autolysins and the biofilm-associated protein (Bap) on the other hand mediate attachment to abiotic surfaces (Cucarella et al., 2001;Heilmann et al., 1997). ...
... Since surface proteins are known to be essential in adherence and biofilm formation of other coagulase-negative staphylococci (Foster, 2020;Foster et al., 2014), we have screened the genomic sequences of two S. xylosus strains (TMW 2.1023 and TMW 2.1523) for open-reading frames (ORFs) encoding surface proteins that could act as Bap alternatives mediating biofilm formation in this organism. Special emphasis was laid on the presence of an N-terminal YSIRK-G/S signal peptide, a C-terminal LPxTG cell wallanchoring motif, and a hydrophobic transmembrane segment, as most surface proteins involved in biofilm formation described for other staphylococci share these motifs (Bowden et al., 2002;Mazmanian et al., 2001). ...
... Over the past years, many cell wall-anchored (CWA) proteins of S. aureus and S. epidermidis have been functionally characterized and current knowledge on staphylococcal biofilm formation is mainly based on the results obtained for these two prominent species (Foster, 2020;Foster et al., 2014;Speziale et al., 2014). Little research has been done on biofilm formation of other CNS such as S. xylosus, except for some valuable studies on S. xylosus strain C2a, focusing on the eDNA part of biofilm matrices though (Leroy et al., 2021;Planchon et al., 2006). ...
Biofilm formation of staphylococci has been an emerging field of research for many years. However, the underlying molecular mechanisms are still not fully understood, and vary widely between species and strains. The aim of this study was to identify new effectors impacting biofilm formation of two Staphylococcus (S.) xylosus strains. We identified a novel surface protein conferring cell aggregation, adherence to abiotic surfaces and biofilm formation. The S. xylosus surface protein A (SxsA) is a large protein occurring in variable sizes. It lacks sequence similarity to other staphylococcal surface proteins but shows similar structural domain organization and functional features. Upon deletion of sxsA, adherence of S. xylosus strain TMW 2.1523 to abiotic surfaces was completely abolished, and significantly reduced in TMW 2.1023. Macro‐ and microscopic aggregation assays further showed that TMW 2.1523 sxsA mutants exhibit reduced cell aggregation compared to the wildtype. Comparative genomic analysis revealed that sxsA is part of the core genome of S. xylosus, S. paraxylosus and S. nepalensis and additionally encoded in a small group of S. cohnii and S. saprophyticus strains. This study provides insights into protein‐mediated biofilm formation of S. xylosus and identifies a new cell wall‐associated protein influencing cell aggregation and biofilm formation. This paper describes a novel surface protein of Staphylococcus xylosus which was named SxsA and found to mediate cellular aggregation and biofilm formation. The importance of the protein in these processes was evaluated by generation of knockout mutants. Thereby mutant strains showed a clearly distinct phenotype to wildtype strains.
... Furthermore, they can, among a plethora of other illnesses, give rise to implant-related infections on catheters, or joint prostheses or be the cause of sexually transmitted diseases such as gonorrhoea [5,6,7]. Since Staphylococcus aureus, as the main culprit for device-related infections, as well as Neisseria gonorrhoeae, as the causative agent of gonorrhoea, can be resistant to antibiotics, it is paramount to understand the initial steps of attachment to a substrate and subsequent colony growth [6,8]. In the native setting, the aspect of colony growth and adhesion is imminently linked and certainly a better understanding of adhesion benefits a greater understanding of colony growth. ...
... A stochastic process is a collection of random variables S t with a possible continuous set of indices t representing time. 8 This variable is defined by the family of distribution functions p(s n , t n , · · · , s 0 , t 0 ) 9 specifying the probability 10 of (S t 0 , . . . , S tn ) taking values (s 0 , . . . ...
... All these macromolecules contribute to the adhesion to abiotic and biotic material by electrostatic, hydrophilic or hydrophobic interactions. CWAs constitute a broad family and S. aureus expresses up to 24 different proteins depending on growth conditions [8]. First, it was thought that the binding to host surfaces is mediated by a class of CWAs termed "microbial surface components recognizing adhesive matrix molecules" (MSCRAMMs), which are binding to ECM proteins like collagens, fibronectins and fibrinogen. ...
... There are some important cell-surface proteins on S. aureus that mediate the virulence and adhesion of the organism to host cells. These proteins include an elastin-binding protein and clumping factors A and B (clfA and clfB genes) [46,47], matrix adhesion proteins, and extracellular matrix fibrinogen and collagen proteins [48]. Besides, protein A (spa gene) is a multifunctional surface protein located on a fragment crystallizable portion of immunoglobulin G (IgG). ...
... Besides, protein A (spa gene) is a multifunctional surface protein located on a fragment crystallizable portion of immunoglobulin G (IgG). This protein produces immune evasion and hinders the antibody-mediated immune clearance of the pathogen, so it can act as a superantigen of B cells [48]. Accordingly, protein A antagonizes the phagocytosis of opsonized bacteria through binding with IgG [49]. ...
Staphylococcus aureus is a Gram-positive coccus normally present on the skin and internal organs of animals, birds, and humans. Under certain conditions, S. aureus could produce septicemia and affection of the skin, joints, and heart, as well as sepsis and death. The pathogenicity of S. aureus is associated with the presence of some virulent surface proteins and the production of some virulent toxins and enzymes. This pathogen is considered one of the most important and worldwide foodborne causes as it is incriminated in most cases of food poisoning. The hazardous use of antibiotics in the veterinary field leads to the development of multidrug-resistant S. aureus strains that can be transmitted to humans. The incidence of methicillin-resistant S. aureus (MRSA) strains has increased globally. These resistant strains have been detected in live animals, poultry, and humans. In addition, retail animal products, especially those of avian origin, are considered the main source of MRSA strains that can be easily transmitted to humans. MRSA infection is regarded as nosocomial or occupational. Humans get infected with MRSA strains through improper handling or preparation of contaminated animals or poultry carcasses or improper cooking with contaminated meat. Live birds also can transmit MRSA to close-contact workers in poultry farms. Transmission of MRSA infection in hospitals is from an infected individual to a healthy one. Prevention and control of MRSA are based on the application of hygienic measures in farms as well as proper processing, handling, and cooking of retail poultry products. The cooperation between veterinary and human practitioners is a must to avoid the possibility of zoonotic transmission. Accordingly, this review focused on the sources and transmission of MRSA infection, virulence and resistance factors, incidence and prevalence in poultry and different products, antibiotic resistance, and prevention and control strategies.
... In terms of the composition of biofilm, the majority of EPS components within S. aureus biofilm is the polysaccharide intercellular adhesin (PIA) [58], which plays an important role in several aspects, including colonization, biofilm generation and biofilm-associated infections, immune evasion, and resistance to antimicrobials and phagocytosis [59]. What is more, S. aureus EPS also connects to the following proteins with different effects: accumulation-associated proteins (Aap) interact with PIA to facilitate the maturation of biofilm [58]; the surface-binding proteins, Spa and SasG, are related to surface attachment and infections [60]; the fibronectin-binding proteins, FnbA and FnbB, can afford the pathogen the ability to attach to cellular integrins and further trigger the internalization into the host cells [61]; and the cell wall-anchored (CWA) proteins facilitate the adhesion among EPS, the membrane of the host cell, and the CWA proteins on adjacent cells, thus promoting the accumulation of biofilm [62]. Furthermore, it is accepted that amyloid fibers can provide a scaffold for biofilm to form a basis of matrix and maintain its structural stability [63,64]. ...
... During the process of infection, the biofilms provide a strong safeguard from handicaps, including the host immune response and antibiotic therapy. As biofilm acts as a strong physical barrier to conceal pathogenassociated molecular patterns (PAMPs), those pathogens are capable of resisting the host immune response for a long time [44,62,113,114]. Moreover, biofilm can also protect the invading bacteria from the immune system of the host by disrupting the activation of phagocytes and the complement system [115,116]. ...
Recently, the drawbacks arising from the overuse of antibiotics have drawn growing public attention. Among them, drug-resistance (DR) and even multidrug-resistance (MDR) pose significant challenges in clinical practice. As a representative of a DR or MDR pathogen, Staphylococcus aureus can cause diversity of infections related to different organs, and can survive or adapt to the diverse hostile environments by switching into other phenotypes, including biofilm and small colony variants (SCVs), with altered physiologic or metabolic characteristics. In this review, we briefly describe the development of the DR/MDR as well as the classical mechanisms (accumulation of the resistant genes). Moreover, we use multidimensional scaling analysis to evaluate the MDR relevant hotspots in the recent published reports. Furthermore, we mainly focus on the possible non-classical resistance mechanisms triggered by the two important alternative phenotypes of the S. aureus, bio-film and SCVs, which are fundamentally caused by the different global regulation of the S. aureus population, such as the main quorum-sensing (QS) and agr system and its coordinated regulated factors, such as the SarA family proteins and the alternative sigma factor σB (SigB). Both the biofilm and the SCVs are able to escape from the host immune response, and resist the therapeutic effects of antibiotics through the physical or the biological barriers, and become less sensitive to some antibiotics by the dormant state with the limited metabolisms.
... Staphylococcus aureus (S. aureus) is a major opportunistic pathogen of humans and animals and one of the most pathogenic microorganisms in bovine mastitis [5][6][7]. S. aureus can secrete large amounts of virulence factors, such as fibronectin-binding protein A (FnBPA), coagulase (Coa), β-hemolysin (Hlb), glyceraldehyde-3-phosphate dehydrogenase (GapC) and leukotoxin [8][9][10]. The interaction between virulence factors is the main reason for the pathogenicity of S. aureus [1,11]. ...
... Studies have shown that S.aureus can cleave bovine mammary epithelial cells (MAC-T cells) and immune cells by secreting leukotoxins, resulting in the damage and inflammation of mammary gland tissues. The rapid recruitment of neutrophils to the infected site is the key to limiting S. aureus infection in bovine mastitis [10]. Previous studies have shown that LukMF is the most abundant leukocidin secreted by bovine mastitis isolates and plays an important role in the pathogenesis of bovine mastitis [12,13]. ...
Staphylococcus aureus is a vital bovine mastitis pathogen causing huge economic losses to the dairy industry worldwide. In our previous studies, leukotoxin ED (LukED) was detected in most S. aureus strains isolated from bovine mastitis. Here, four single-chain fragment variables (scFvs) (ZL8 and ZL42 targeting LukE, ZL22 and ZL23 targeting LukD) were obtained using purified LukE and LukD proteins as the antigens after five rounds of bio-panning. The complementarity-determining region 3 (CDR3) of the VH domain of these scFvs exhibited significant diversities. In vitro, the scFvs significantly decreased LukED-induced cell killing by inhibiting the binding of LukED to chemokine receptors (CCR5 and CXCR2) and reduced the death rates of bovine neutrophils and MAC-T cells caused by LukED and S. aureus (p < 0.05). In an S. aureus-induced mouse mastitis model, histopathology and MPO results revealed that scFvs ameliorated the histopathological damages and reduced the infiltration of inflammatory cells (p < 0.05). The ELISA and qPCR assays showed that scFvs reduced the transcription and expression levels of Tumor Necrosis Factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8 and IL-18 (p < 0.05). The overall results demonstrated the protective anti-inflammatory effect of scFvs in vitro and in vivo, enlightening the potential role of scFvs in the prevention and treatment of S. aureus-induced mastitis.
... S. aureus surface is enriched with various adhesin proteins, which are covalently attached to peptidoglycan layer, and involved in adhesion and colonization step. According to the presence of motifs identified through structure function analysis, cell wall anchored proteins (CWA) are divided into four groups [16]. The first and most predominant group is the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) family, which includes collagen-binding protein, fibronectin-binding proteins A and B, clumping factor A and B proteins and Sdr proteins (from SD Repeat) [16,17]. ...
... According to the presence of motifs identified through structure function analysis, cell wall anchored proteins (CWA) are divided into four groups [16]. The first and most predominant group is the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) family, which includes collagen-binding protein, fibronectin-binding proteins A and B, clumping factor A and B proteins and Sdr proteins (from SD Repeat) [16,17]. SdrC, SdrD, and SdrE are the three proteins encoded by the sdr locus, but not all S. aureus strains have all three genes [18]. ...
... ClfA promotes clumping, while ClfB aids binding to cytokeratin 10 and 8, and locirin. (Foster et al., 2014;Paharik and Horswill, 2016) Fibronectin binding protein (FnBP)A, FnBPB ...
... Desquamated epithelial cell (nasal)/ fibrinogen adherence (E). Self-associates within biofilms (Foster et al., 2014) Serine-aspartate repeat proteins (Sdr)C, SdrD, SdrE (Yang et al., 2014) Serine rich adhesin for platelets (Sra)P Cell dispersal occurs with physical shearing at biofilm surfaces, forcibly removing cells from the community, or it can be cell-mediated via quorum signaling. Quorum signals are connected to oxygen or nutrient paucity, or waste and secondary metabolite accumulation which reduce environmental suitability (Lapointe et al., 2019;Solano et al., 2014). ...
The control of bacterial contaminants on chicken meat is a key area of interest in the broiler industry. Microbes that pose a significant food safety risk on chicken include Campylobacter spp., Salmonella enterica, Listeria monocytogenes and Escherichia coli. In addition, microbes including Pseudomonas spp., Brochothrix thermosphacta and Lactic Acid Bacteria must be controlled to ensure product quality and maintain shelf-life. Poultry meat processing challenges including cold and chemical exposure are employed to control the microbiota of the end-product, as well as to maintain environment hygiene. Exposure to these stresses can also induce adaptive shifts in the transcriptome and proteome of foodborne bacteria. This review will explore the complex interactions at play in the poultry processing environment and explain how bacteria exposed to such stresses behave in this environmental niche through the production of heat and cold-shock proteins, the expression of efflux pumps, sporulation, and the formation of mono- and mixed-species biofilms within the production environment.
... There is a vast diversity of biofilm infections 8 but an exhaustive account of these infections is beyond the scope of this Review. Generally, staphylococci are the most common cause of infections associated with indwelling medical devices 16,17 . If caused by a bacterial species with no or few virulence factors (such as, for example, S. epidermidis), the infection is characterized by a prolonged course with discrete, or negligible, signs of systemic inflammation 12,13 , and these infections are often dominated by local symptoms related to continuous inflammatory damage around foreign bodies 12,13 . ...
... S. aureus SCVs are isolated from chronic infections, such as osteomyelitis, endocarditis and recurrent abscesses, and are characterized by a high capacity to enter and survive within host cells, thus evading the immune system 16,75 . S. aureus SCVs are characterized by the downregulation of QS, decreased production of virulence factors, alterations in metabolic pathways and tolerance to antimicrobials, and are able to survive for long periods and sustain infections 75 . ...
Chronic infections caused by microbial biofilms represent an important clinical challenge. The recalcitrance of microbial biofilms to antimicrobials and to the immune system is a major cause of persistence and clinical recurrence of these infections. In this Review, we present the extent of the clinical problem, and the mechanisms underlying the tolerance of biofilms to antibiotics and to host responses. We also explore the role of biofilms in the development of antimicrobial resistance mechanisms. Chronic infections caused by microbial biofilms represent an important clinical challenge owing to the recalcitrance of microbial biofilms to antimicrobials and the immune system, causing persistence and clinical recurrence of these infections. In this Review, Ciofu and colleagues discuss our current understanding of the mechanisms of tolerance of such biofilms to the immune system as well as of tolerance and resistance to antimicrobials.
... The microbial surface component recognizing adhesive matrix molecule (MSCRAMM) family of proteins is the largest class of S. aureus virulence factors involved in adhesion and invasion [31]. The MSCRAMM family is comprised of S. aureus surface proteins that mediate binding to host extracellular matrix (ECM) components, including fibrinogen, collagen, and fibronectin. ...
... Fibronectin-binding proteins (FnBPs) are members of the MSCRAMM family that bind, with high affinity, to host fibronectin. The major mechanism of S. aureus invasion into host cells utilizes FnBPs [31][32][33]. S. aureus mutants that do not express FnBPs lose their invasive ability [32]. Furthermore, the ectopic expression of FnBPs in non-invasive bacterial species, such Staphylococcus carnosus and Lactococcus lactis, confers invasiveness [32,33]. ...
An emergent approach to bacterial infection is the use of host rather than bacterial-directed strategies. This approach has the potential to improve efficacy in especially challenging infection settings, including chronic, recurrent infection due to intracellular pathogens. For nearly two decades, the pleiotropic effects of statin drugs have been examined for therapeutic usefulness beyond the treatment of hypercholesterolemia. Interest originated after retrospective studies reported decreases in the risk of death due to bacteremia or sepsis for those on a statin regimen. Although subsequent clinical trials have yielded mixed results and earlier findings have been questioned for biased study design, in vitro and in vivo studies have provided clear evidence of protective mechanisms that include immunomodulatory effects and the inhibition of host cell invasion. Ultimately, the benefits of statins in an infection setting appear to require attention to the underlying host response and to the timing of the dosage. From this examination of statin efficacy, additional novel host-directed strategies may produce adjunctive therapeutic approaches for the treatment of infection where traditional antimicrobial therapy continues to yield poor outcomes. This review focuses on the opportunistic pathogen, Staphylococcus aureus, as a proof of principle in examining the promise and limitations of statins in recalcitrant infection.
... Staphylococcus aureus is a highly effective human pathogen that causes a wide-range of infections including those of the skin and soft tissue, bone, heart and joints [1], resulting in significant morbidity and mortality worldwide. Antibiotic resistance, especially to clindamycin, is increasing for both methicillin-sensitive and methicillin-resistant S. aureus (MRSA) [2]. ...
... These virulence factors target the formation or function of C3-convertase, thereby affecting opsonization and downstream effects [5,9,10]. S. aureus protein A, a well-known immune-evasive protein, binds the Fc region of immunoglobulin thereby providing a two-pronged inhibitory function to effectively impede C1q-mediated activation of the classical pathway of complement and block FcγR-mediated phagocytosis [1]. ...
Staphylococcus aureus employs a multitude of immune-evasive tactics to circumvent host
defenses including the complement system, a component of innate immunity central to controlling
bacterial infections. With antibiotic resistance becoming increasingly common, there is a
dire need for novel therapies. Previously, we have shown that S. aureus binds the complement
regulator factor H (FH) via surface protein SdrE to inhibit complement. To address the need for
novel therapeutics and take advantage of the FH:SdrE interaction, we examined the effect of a
fusion protein comprised of the SdrE-interacting domain of FH coupled with IgG Fc on complement-
mediated opsonophagocytosis and bacterial killing of community associated methicillinresistant
S. aureus. S. aureus bound significantly more FH-Fc compared to Fc-control proteins
and FH-Fc competed with serum FH for S. aureus binding. FH-Fc treatment increased C3-
fragment opsonization of S. aureus for both C3b and iC3b, and boosted generation of the anaphylatoxin
C5a. In 5 and 10% serum, FH-Fc treatment significantly increased S. aureus killing
by polymorphonuclear cells. This anti-staphylococcal effect was evident in 75% (3/4) of clinical
isolates tested. This study demonstrates that FH-Fc fusion proteins have the potential to mitigate
the protective effects of bound serum FH rendering S. aureus more vulnerable to the host
immune system. Thus, we report the promise of virulence-factor-targeted fusion-proteins as an
avenue for prospective anti-staphylococcal therapeutic development.
... Other authors also confirmed that adhesion of S. aureus to collagen promoted infection and the initiation of biofilm formation 30 . The ability of adhesion to fibronectin is important in the disease development process, because fibronectin is a ubiquitous host protein present in soluble form in the blood and in fibrillar form in cellular matrices 31 . ...
... MRSA isolates. A total of 120 MRSA isolates from human clinical materials such as swabs from wound (30), anus (15), nose (8), blood samples (11), respiratory tract (48), and other samples: swabs from tracheostomy tube, endotracheal tube and catheter and urine (8) were used in this study. The MRSA isolates were obtained from hospitals in Siedlce (83) and Warsaw (37) (Poland) in 2015-2017 (Table 5). ...
Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for hard-to-treat infections. The presence of 19 virulence genes in 120 MRSA isolates obtained from hospitalized patients and genetic relationships of these isolates were investigated. The eno (100%) and ebps (93.3%) genes encoding laminin- and elastin binding proteins, respectively, were ubiquitous. Other adhesion genes: fib (77.5%), fnbB (41.6%), bbp (40.8%), cna (30.8%) encoding proteins binding fibrinogen, fibronectin, bone sialoprotein and collagen, respectively, and map / eap (62.5%), encoding Eap, were also frequent. The etB and etD genes, encoding exfoliative toxins, were present in 15.6% and 12.5% isolates, respectively. The splA , splE and sspA , encoding serine protease were detected in 100%, 70.8% and 94.2% isolates, respectively. The tst gene, encoding toxic shock syndrome toxin-1 was found in 75% isolates. The cna , map/eap and tst genes were the most common in wound isolates and much less common in blood isolates. We identified 45 different spa types, t003 (21.7%) and t008 (18.8%) being the most common. The t003 was the most frequent among isolates from the respiratory tract (35.5%), while t008 in blood isolates (40%). Identification of virulence factors of MRSA is important for evaluation of pathogen transmission rate and disease development.
... Colonization is a key step in bacterial pathogenesis and bovine S. aureus has evolved in many ways to facilitate adhesion to different host cell types [98,99]. One family of S. aureus adhesins comprises the microbial surface component recognizing adhesive matrix molecules (MSCRAMMs), which are cell-wall anchored proteins that share structural features like an N-terminal folded domain responsible for ligand binding, and a wall-spanning region followed by a sorting signal located at the C-terminal that anchor the protein to the cell wall [100]. ...
... Bovine isolates of S. aureus produce other MSCRAMMs that may promote colonization [109,116,117]. For example, clumping factors A and B (ClfA and ClfB) are fibrinogen-binding proteins that act as adhesins and have several defined roles in colonization and pathogenesis [100,118,119]. Although adherence to human endothelial cells requires fibrinogen to mediate the interaction between ClfA and the host integrin α5β3 [120], adherence to bovine epithelial cells occurs in a fibrinogen-independent manner via the annexin A2 receptor [121]. ...
Staphylococcus aureus is a leading cause of bovine mastitis worldwide. Despite some improved understanding of disease pathogenesis, progress towards new methods for the control of intramammary infections (IMI) has been limited, particularly in the field of vaccination. Although herd management programs have helped to reduce the number of clinical cases, S. aureus mastitis remains a major disease burden. This review summarizes the past 16 years of research on bovine S. aureus population genetics, and molecular pathogenesis that have been conducted worldwide. We describe the diversity of S. aureus associated with bovine mastitis and the geographical distribution of S. aureus clones in different continents. We also describe studies investigating the evolution of bovine S. aureus and the importance of host-adaptation in its emergence as a mastitis pathogen. The available information on the prevalence of virulence determinants and their functional relevance during the pathogenesis of bovine mastitis are also discussed. Although traits such as biofilm formation and innate immune evasion are critical for the persistence of bacteria, the current understanding of the key host-pathogen interactions that determine the outcome of S. aureus IMI is very limited. We suggest that greater investment in research into the genetic and molecular basis of bovine S. aureus pathogenesis is essential for the identification of novel therapeutic and vaccine targets.
... The name sortase is derived from its role in sorting the proteins passing through the secretory pathway [154]. After identifying the sortase enzyme, they were widely studied and considered medicinal targets due to their role in binding cell wall pathogenic factors in pathogens [155]. These enzymes are also involved in the polymerization of pili units in bacteria, which is one of the key factors that bind bacteria to surfaces [156]. ...
... Teichoic acid, lipoteichoic acid, exopolysaccharide, enzymes, S-layer proteins, and other cell surface proteins are found in adhesions and pililike structures, and are specifically associated with host binding [162]. Pathogenic bacterial surface proteins like protein A in Staphylococcus aureus and internalin A in Listeria monocytogenes have an important role in their infection and pathogenicity [155,163]. Instead, the surface structures in probiotic bacteria have a key role in benefiting the host, which is placed on the surface of the bacteria during the surface display process. The surface display is a combination of two processes: protein targeting and protein binding to the extracellular membrane. ...
Due to the importance of using cost-effective methods for therapeutic purposes, the function of probiotics as safe microorganisms and the study of their relevant functional mechanisms have recently been in the spotlight. Finding the mechanisms of attachment and stability and their beneficial effects on the immune system can be useful in identifying and increasing the therapeutic effects of probiotics. In this review, the functional mechanisms of probiotics were comprehensively investigated. Relevant articles were searched in scientific sources, documents, and databases, including PubMed, NCBI, Bactibace, OptiBac, and Bagel4. The most important functional mechanisms of probiotics and their effects on strengthening the epithelial barrier, competitive inhibition of pathogenic microorganisms, production of antimicrobials, binding and interaction with the host, and regulatory effects on the immune system were discussed.
In this regard, the attachment of probiotics to the epithelium is very important because the prerequisite for their proper functioning is to establish a proper connection to the epithelium. Therefore, more attention should be paid to the binding effect of probiotics, including sortase A, a significant factor involved in the expression of sortase-dependent proteins (SDP), on their surface as mediators of intestinal epithelial cell binding. In general, by investigating the functional mechanisms of probiotics, it was concluded that the mechanism by which probiotics regulate the immune system and adhesion capacity can directly and indirectly have preventive and therapeutic effects on a wide range of diseases. However, further study of these mechanisms requires extensive research on various aspects.
... Bacteria have extensive tools-from single monomeric proteins to multimeric macromolecules-used for adhesion and invasion [239]. The infection starts with the adhesion of a bacterial pathogen to the host cell's surface. ...
Novel biosensors already provide a fast way to detect the adhesion of whole bacteria (or parts of them), biofilm formation, and the effect of antibiotics. Moreover, the detection sensitivities of recent sensor technologies are large enough to investigate molecular-scale biological processes. Usually, these measurements can be performed in real time without using labeling. Despite these excellent capabilities summarized in the present work, the application of novel, label-free sensor technologies in basic biological research is still rare; the literature is dominated by heuristic work, mostly monitoring the presence and amount of a given analyte. The aims of this review are (i) to give an overview of the present status of label-free biosensors in bacteria monitoring, and (ii) to summarize potential novel directions with biological relevancies to initiate future development. Optical, mechanical, and electrical sensing technologies are all discussed with their detailed capabilities in bacteria monitoring. In order to review potential future applications of the outlined techniques in bacteria research, we summarize the most important kinetic processes relevant to the adhesion and survival of bacterial cells. These processes are potential targets of kinetic investigations employing modern label-free technologies in order to reveal new fundamental aspects. Resistance to antibacterials and to other antimicrobial agents, the most important biological mechanisms in bacterial adhesion and strategies to control adhesion, as well as bacteria-mammalian host cell interactions are all discussed with key relevancies to the future development and applications of biosensors.
... During the colonization process by S. aureus, binding to the host cell surface in a reversible or irreversible manner is mediated by the so-called microbial surface component, recognizing adhesive matrix molecules (MSCRAMM) [22]. ...
The widespread use of mobile phones (MP) among healthcare personnel might be considered as an important source of contamination. One of the most pathogenic bacteria to humans is Staphylococcus aureus, which can be transmitted through the constant use of MP. Nevertheless, which specific type of strains are transmitted and which are their sources have not been sufficiently studied. The aim of this study is to determine the source of contamination of MP and characterize the corresponding genotypic and phenotypic properties of the strains found. Nose, pharynx, and MP samples were taken from a group of health science students. We were able to determinate the clonality of the isolated strains by pulsed-field gel electrophoresis (PFGE) and spa gene typing (spa-type). Adhesin and toxin genes were detected, and the capacity of biofilm formation was determined. Several of the MP exhibited strains of S. aureus present in the nose and/or pharynx of their owners. methicillin-susceptible Staphylococcus aureus (MSSA), hospital-acquired methicillin-resistant S. aureus (HA-MRSA), and community-acquired methicillin-resistant S. aureus (CA-MRSA) strains were found, which indicated a variety of genotypes. This study concludes that MP can be contaminated with the strains of S. aureus present in the nose and/or pharynx of the owners; these strains can be of different types and there is no dominant genotype.
... According to World Health Organization (WHO) reports, MRSA was one of the top published lists of 12 pathogens that pose public health threat (Reardon 2014) requiring global concern for discovering of new antibacterial agents and development of new control strategies (Duval et al. 2019;Tong et al. 2016;Turner et al. 2019). Targeting MRSA virulence such as biofilm, adhesion and tissue colonization are considered one of innovative strategies to combat its pathogenesis and prevent complications (Marra 2004;Donlan and Costerton 2002;Foster et al. 2014).Various in-vitro and in-vivo studies had documented the use of natural crude extracts of garlic and royal jelly (RJ) as anti-virulence agents for controlling MRSA infections (El-Gayar et al. 2016;Lin et al. 2020;Zhu and Zeng 2020;Nidadavolu et al. 2012). Further studies were conducted and showed that both allicin and ajoene are the two major active ingredients of the GE having antibiofilm, (Tsao et al. 2003;Girish et al. 2019), antimicrobial, (Li et al. 2015;Yadav et al. 2015;Fratianni et al. 2016) and antifungal properties (Li et al. 2015(Li et al. , 2016Fratianni et al. 2016;Wr et al. 2014). ...
The limited therapeutic options associated with methicillin-resistant Staphylococcus aureus (MRSA) necessitate search for innovative strategies particularly, use of natural extracts such as lyophilized royal jelly (LRJ) and garlic extract (GE). Therefore, out study aimed to formulate emulgels containing different concentrations of both LRJ and GE and to evaluate their activities using a murine model infected with MRSA clinical isolate. Four plain emulgel formulas were prepared by mixing stearic acid/yellow soft paraffin-based O/W emulsion formulae based on Carbopol 940, Na alginate, Na carboxymethylcellulose or Hydroxypropyl methyl cellulose E4. Sodium alginate-based emulgel was selected for preparation of four medicated emulgel formulations combining LRJ and GE at four different concentrations. The selected medicated emulgels were used for the in vivo studies. The emulgel formulated with Na alginate and HPMC (MF3) exhibited optimum smooth homogeneous consistency, neutral pH, acceptable viscosity, spreadability, extrudability values and best storage stability properties. In vivo results revealed that, the wounds infected with MRSA isolate in rates were wet (oozing) and showed pus formation when compared to injured uninfected wounds. MF3 formula containing 4% LRJ and 50% GE showed the maximum wound healing properties, both in the apparent physical wound healing measurements and in the histopathological examination. In conclusion, the medicated emulgel formulation (MF3) prepared with Na alginate was found optimum for topical application. MF3 formula containing 4% LRJ and 50% GE has shown the highest in vivo wound healing capacities. Further clinical studies should be conducted to prove both its safety and efficacy and the potential use in human.
... In S. aureus biofilm, poly-N-acetyl-β-(1-6)-glucosamine (PNAG; is also called polysaccharide intercellular adhesin PIA) helps biofilms form and enables bacteria to gain protection from the host immune system 161,162 . Adhesion to the surface and initiation of biofilm formation 163 aureus also produces polysaccharide-dependent biofilms, and the exopolysaccharide components, which have identical structures as PIA from S. epidermidis, were given the name of PNAG by Maira-Litran et al. 161 . The structure of PIA/PNAG polymer is shown in Fig. 12. ...
The purpose of our research was to design a new anti-biofilm strategy to effectively destruct mature biofilms formed by S. aureus and to explore the possible mechanisms which lead to the strain specificity of biofilm forming capacity at the molecular level. By confocal laser scanning microscopy, we found that subtilisin A and calcium ion significantly destroyed the biofilm matrix. Combining oxytetracycline with each of them enhanced the detachment of biofilm bacteria, while maintaining their destructive effect on the biofilm matrix. One hundred and five S. aureus ST398 isolates were then analyzed for their biofilm forming capacity and their biofilm-related genes. The results demonstrated that, two isolates with the same host source, the same type of biofilm-related genes, and the same spa-type could have significantly different biofilm-producing abilities. Further experiments will analyze the expression of biofilm-related genes and explore the possible mechanism of the difference in biofilm productivity between these two types of isolates. Our research proposed a new strategy to target biofilm matrix by combining antibiotics with non-antibiotics and may provide new targets for future research and development of antibiofilm strategies.
... In the case of surfaces that have been coated with a matrix in this way, bacterial cell wall anchored proteins that specifically recognize various host matrix components are important 14 . These cell wall anchored proteins include the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) which recognize and bind to numerous host substrates including fibrinogen, collagen and fibronectin [20][21][22] . MSCRAMMs OPEN are negatively regulated by the agr system, so it is argued that QSI will downregulate virulence factors but upregulate biofilm (reviewed in 6 ). ...
Staphylococcus aureus is an opportunistic, pathogenic bacteria that causes significant morbidity and mortality. As antibiotic resistance by S. aureus continues to be a serious concern, developing novel drug therapies to combat these infections is vital. Quorum sensing inhibitors (QSI) dampen S. aureus virulence and facilitate clearance by the host immune system by blocking quorum sensing signaling that promotes upregulation of virulence genes controlled by the accessory gene regulator ( agr ) operon. While QSIs have shown therapeutic promise in mouse models of S. aureus skin infection, their further development has been hampered by the suggestion that agr inhibition promotes biofilm formation. In these studies, we investigated the relationship between agr function and biofilm growth across various S. aureus strains and experimental conditions, including in a mouse model of implant-associated infection. We found that agr deletion was associated with the presence of increased biofilm only under narrow in vitro conditions and, crucially, was not associated with enhanced biofilm development or enhanced morbidity in vivo.
... The most common surface adhesins that are covalently bonded to the peptidoglycan cell wall are known as the MSCRAMM (microbial surface components recognizing adhesive matrix molecules) protein family. Well-characterized MSCRAMMs are ClfA and B, Cna, IsdA, B and H, FnBPA and B, and SdrC, D, and E [69][70][71]. The studies have shown that mutant strains of MSCRAMMs are less likely to cause infections in animal models [72]. ...
Staphylococcus aureus (S. aureus) is an opportunistic pathogen that causes various inflammatory local infections, from those of the skin to postinfectious glomerulonephritis. These infections could result in serious threats, putting the life of the patient in danger. Antibiotic-resistant S. aureus could lead to dramatic increases in human mortality. Antibiotic resistance would explicate the failure of current antibiotic therapies. So, it is obvious that an effective vaccine against S. aureus infections would significantly reduce costs related to care in hospitals. Bacterial vaccines have important impacts on morbidity and mortality caused by several common pathogens, however, a prophylactic vaccine against staphylococci has not yet been produced. During the last decades, the efforts to develop an S. aureus vaccine have faced two major failures in clinical trials. New strategies for vaccine development against S. aureus has supported the use of multiple antigens, the inclusion of adjuvants, and the focus on various virulence mechanisms. We aimed to present a compressive review of different antigens of S. aureus and also to introduce vaccine candidates undergoing clinical trials, from which can help us to choose a suitable and effective candidate for vaccine development against S. aureus. View Full-Text
... SrtA is the key enzyme that anchors pathogenesis-related proteins to the cell wall [8,9], a process that is mediated by recognition of the LPXTG amino acid sequence in the peptide chain and clipping [10,11], followed by anchoring of the peptide chain to the cell wall by transpeptidation [12,13]. This confers on S. aureus the ability for cell adhesion, biofilm formation, antiphagocytosis and invasion of host cells [14,15]. It has been demonstrated that srta gene deletion attenuated inflammatory response and tissue bacterial load in the early stages of host infection and attenuated renal abscesses caused by non-lethal MRSA infections [16]. ...
The strategy of targeting virulence factor has received great attention as it barely develops bacterial resistance. Sortase A (SrtA) and caseinolytic peptidase P (ClpP), as important virulence factors, are considered to be ideal pharmacological targets for methicillin-resistant Staphylococcus aureus (MRSA) infection. Through screening hundreds of compounds, we found scutellarin, a natural flavonoid, markedly inhibited SrtA and ClpP activities of MRSA strain USA300 with an IC50 of 53.64 μg/mL and 107.00 μg/mL, respectively. Subsequently, we observed that scutellarin could inhibit the SrtA-related virulence of MRSA. To demonstrate whether scutellarin directly binding to SrtA, fluorescence quenching assay and molecular docking were performed and the results indicated that scutellarin directly bonded to SrtA molecule with a KA value of 7.58 × 104 L/mol. In addition to direct SrtA inhibition, scutellarin could also inhibit hemolytic activity of S. aureus by inhibiting the expression of Hla in a SrtA-independent manner. Further assays confirmed that scutellarin inhibited hemolysis by inhibiting ClpP. The combination of scutellarin and vancomycin showed enhancing inhibition of USA300 in vitro and in vivo, evidenced by decreased MIC from 3 μg/mL to 0.5 μg/mL and increased survival and improvement of lung pathology in pneumonia mice. Taken together, these results suggest that scutellarin exhibited di-inhibitory effects on SrtA and ClpP of USA300. The di-inhibition of virulence factors by scutellarin combined with vancomycin to prevent MRSA invasion of A549 cells and pneumonia in mice, indicating that scutellarin is expected to be a potential adjuvant against MRSA in the future.
... However, it is worth noticing that each lineage expressed a different combination of adhesin genes, likely reflecting functional redundancy. Previous studies showed how these adhesin genes were frequently associated with invasive infections including bone infections (Palmqvist et al., 2005;Rieg et al., 2013;Foster et al., 2014;Post et al., 2014). Cna has previously been identified in few S. aureus clones (Peacock et al., 2002) and has been shown to play an important role in bone tropism in a murine model of haematogenous OM and in the development of septic arthritis (Patti et al., 1994;Elasri et al., 2002). ...
Staphylococcus aureus is the most common pathogen causing osteomyelitis (OM). The aim of this study was to explore the clonal complex (CC) distribution and the pattern of virulence determinants of S. aureus isolates from OM in Italy. Whole-genome sequencing was performed on 83 S. aureus isolates from OM cases in six hospitals. Antibiotic susceptibility tests showed that 30.1% of the isolates were methicillin-resistant S. aureus (MRSA). The most frequent CCs detected were CC22, CC5, CC8, CC30, and CC15, which represent the most common lineages circulating in Italian hospitals. MRSA were limited in the number of lineages (CC22, CC5, CC8, and CC1). Phylogenetic analysis followed the sequence type-CC groupings and revealed a non-uniform distribution of the isolates from the different hospitals. No significant difference in the mean number of virulence genes carried by MRSA or MSSA isolates was observed. Some virulence genes, namely cna, fib, fnbA, coa, lukD, lukE, sak, and tst, were correlated with the CC. However, different categories of virulence factors, such as adhesins, exoenzymes, and toxins, were frequently detected and unevenly distributed among all lineages. Indeed, each lineage carried a variable combination of virulence genes, likely reflecting functional redundancy, and arguing for the importance of those traits for the pathogenicity in OM. In conclusion, no specific genetic trait in the most frequent lineages could explain their high prevalence among OM isolates. Our findings highlight that CCs detected in OM isolates follow the epidemiology of S. aureus infections in the country. It is conceivable that any of the most common S. aureus CC can cause a variety of infections, including OM.
... In the herd, the infection is primarily spread from cow to cow, as the main reservoir of S. aureus are chronically infected mammary glands . However, S. aureus is able to colonize a wide range of extramammary sites, in part due to its impressive array of virulence factors, including adhesins, toxins, and immune evasion proteins (Foster et al., 2014;Thammavongsa et al., 2015). Some of them are controlled by the agr quorumsensing system (Gordon et al., 2013) (see further). ...
Staphylococcus aureus remains one of the most common causative agents of bovine mastitis because of its pathogenicity, contagiousness, capability to persist in the mammary gland, colonization of skin, and poor cure rates when causing intramammary infections with the currently available therapies. Therefore, alternative treatments and preventive measures are desirable to control S. aureus mastitis, especially when caused by methicillin-resistant strains (MRSA). In vitro and in vivo studies have suggested beneficial traits of non-aureus staphylococci (NAS) originating from bovine milk and teat apices (TA) against major pathogens such as S. aureus. More recent studies have speculated that interactions between S. aureus and other colonizing staphylococci via the quorum-sensing (QS) system might affect the ability of S. aureus to produce virulence factors such as biofilm, and cause infection, suggesting that NAS could be used as potential sources of metabolites to prevent or target S. aureus infections. The general aim of this thesis was to study some elements of the regulation of S. aureus growth and virulence, including biofilm formation, by bovine NAS, gauging for new venues for preventive or therapeutic measures against bovine S. aureus mastitis.
... 3.5 Mechanism of high infectivity of Cryptosporidium at low temperatures As shown in Fig. 7(a), the ESPs are postulated to mediate parasite attachment to host cells (Wanyiri and Ward, 2006;Foster et al., 2014). Several proteins have been shown to mediate invasion, cause direct tissue damage to the host, or directly act on the target site of the host to enhance pathogenicity (Okhuysen and Chappell, 2002;Rajapandi, 2020). ...
This study investigated the occurrence, species, infectivity and removal efficiency of Cryptosporidium spp. across typical wastewater treatment train. Samples from different process units were collected seasonally and synchronously from four wastewater treatment plants (WWTPs) in Northeastern China. Live Cryptosporidium oocysts were identified in most samples from both influent (97.50%) and effluent (90.00%) wastewaters of the four WWTPs, at an average density of 26.34 and 4.15 oocysts/L, respectively. The overall removal efficiency was 84.25%, and oocysts were mainly removed (62.01%) by the modified secondary sedimentation process. Ten Cryptosporidium species were identified in the effluent samples. C. andersoni, C. bovis, and C. ryanae were the three most prevalent species. Oocyst viability assays indicated no reduction of excystation rate during the primary and secondary wastewater treatments (varied in the range of 63.08%–68.50%), but the excystation rate declined to 52.21% in the effluent after disinfection. Notably, the Cryptosporidium oocysts showed higher infection intensity in the cold season (winter and spring) than that in summer and autumn. The influences of environmental temperature on virulence factors of Cryptosporidium were further examined. It was observed that more extracellular secretory proteins were bound on the oocyst surface and several virulence genes were expressed relatively strongly at low temperatures, both of which could facilitate oocyst adhesion, invasion, and host immune evasion. This research is of considerable interest since it serves as an important step towards more accurate panoramic recognition of Cryptosporidium risk reduction in WWTPs, and especially highlights the potential health risk associated with Cryptosporidium in cold regions/seasons.
The surface protein SasX, has a key role in methicillin-resistant Staphylococcus aureus (MRSA) colonization and pathogenesis, and has been associated with the epidemic success of some MRSA clones. To date, only one SasX homologous protein, named SesI, has been described in Staphylococcus epidermidis. In this work, we analyze the occurrence of the sasX gene and its genetic environment in Staphylococcus haemolyticus S. haemolyticus clinical strains (n = 62) were screened for the presence of the sasX gene and its carrier, the prophage Φ SPβ-like. A deep characterization was done in one strain (MD43), through which we determined the complete nucleotide sequence for the S. haemolitycus sasX-like gene. Whole genome sequencing of strain MD43 was performed, and the gene, termed here because of its unique attributes, shsA, was mapped to the Φ SPβ-like prophage sequence. The shsA gene was detected in 33 out of 62 strains showing an average identity of 92 and 96% with the sasX and sesI genes and at the amino acid level, 88% identity with SasX and 92% identity with SesI. The ~124Kb Φ SPβ-like prophage sequence showed a largely intact prophage compared to its counterpart in S. epidermidis strain RP62A, including the sesI insertion site. In conclusion, we identified a new sasX ortholog in S. haemolyticus (shsA). Its horizontal spread from this reservoir could represent an emergent threat in healthcare facilities since so far, no S. aureus sasX⁺ strains have been reported in Brazil.
Sortase A (SrtA) is a cysteine transpeptidase of most gram-positive bacteria that is responsible for the anchoring of many surface protein virulence factors to the cell wall. SrtA ablation has demonstrated to alleviate the infection without affecting the viability of bacteria. Herein, a series of benzofuran cyanide derivatives were synthesized and evaluated. Several compounds exhibited excellent inhibitory activity against SrtA with IC50 values from 3.3 μM to 21.8 μM compared with the known SrtA inhibitor pHMB (IC50 = 130 μM). Ⅲ-1, Ⅲ-15, Ⅲ-34 and V-2 showed potent inhibitory effects on biofilm formation with IC50 values from 2.1 μM to 54.2 μM. Invasion assays showed the four compounds caused a decrease of 4%–24.0% in the uptake of the S. aureus strain by 293T cells. Further assay showed that compound Ⅲ-15 decreased the amount of cell wall-associated protein A by 26.5%. Structure-activity relationship and docking studies demonstrated that the acrylonitrile moiety of the compounds played an important role in enhancing the activity. When the double bond of acrylonitrile changed to single bond, the activity was decreased significantly. This indicates that acrylonitrile, which is a Michael receptor, can inhibit the activity of SrtA by covalent binding effectively to the thiol group of Cys184.
Staphylococcus aureus is a major pathogen causing intramammary infection and mastitis in dairy cows. S. aureus genotypes (GT) can differ significantly in their ability to diffuse and persist in the herd; while the association of virulence gene carriage with epidemiological behavior remains unclear, a role for secreted proteins has been postulated. We characterized the secretome of six S. aureus strains belonging to two genotypes with opposite within-herd prevalence, GTB (high) and GTS (low), corresponding to sequence types (ST) 8 and 398, by high-resolution tandem mass spectrometry and differential analysis with Proteome Discoverer. Data are available via ProteomeXchange with identifier PXD029571. Out of 720 identified proteins, 98 were unique or more abundant in GTB/ST8 and 68 in GTS/ST398. GTB/ST8 released more immunoglobulin-binding proteins, complement and antimicrobial peptide inhibitors, enterotoxins, and metabolic enzymes, while GTS/ST398 released more leukocidins, hemolysins, lipases, and peptidases. Furthermore, GTB/ST8 released the von Willebrand factor protein, staphylokinase, and clumping factor B, while GTS released the staphylococcal coagulase and clumping factor A. Hence, GTB/ST8 secretomes indicated a higher propensity for immune evasion and chronicity and GTS/ST398 secretomes for cellular damage and inflammation, consistent with their epidemiological characteristics. Accordingly, GTS/ST398 secretions were significantly more cytotoxic against bovine PBMCs in vitro. Our findings confirm the crucial role of extracellular virulence factors in S. aureus pathogenesis and highlight the need to investigate their differential release adding to gene carriage for a better understanding of the relationship of S. aureus genotypes with epidemiological behavior and, possibly, disease severity.
Staphylococcus aureus is a Gram-positive bacterium that can persist in nature under hostile environmental conditions and interacts with humans as both commensal and pathogen. It is responsible for a multitude of hospital- and community-acquired infections. The bacterium is often found on the skin of the axilla, chest/abdomen, groin, and perineum as well as on the mucosa of pharynx and intestine [1, 2], but the main site of colonization in humans is the vestibulum nasi (anterior nares) [3, 4]. With regard to S. aureus colonization, humans are grouped into three categories: non-carriers, intermittent carriers, and persistent carriers [2]. Around 20% of human population are persistently colonized with S. aureus [5]. Nasal colonization depends on both host and microbial factors that will be described below. The differential rates of colonization among individuals are linked to their immune status, gene polymorphisms, and competition of S. aureus with other microorganisms [2]. Patients with airway diseases, such as asthma or chronic rhinosinusitis with nasal polyps (CRSwNP), show significantly higher rates of S. aureus colonization than healthy adults [6]. It has long been recognized that nasal carriage of S. aureus is a major risk factor for infections, which are mostly caused by the colonizing strain [7].
Background and Objective: Staphylococcus aureus is one of the most important human pathogens and common causes of nosocomial and acquired infections in the world. The aim of this study was to investigate antibiotic resistance in methicillin-resistant S. aureus (MRSA) strains and the molecular properties of S. aureus strains.
Materials and Methods: For this purpose, a descriptive study was performed in 2017 on 56 strains of S. aureus isolated from clinical specimens. MecA subclasses were also determined by Multiplex RCR reaction. The pvl and spa genes in these strains were determined by PCR reaction.
Results: The highest resistance of the strains was observed to be to the antibiotics oxacillin, cefoxytime and erythromycin, and vancomycin (12.5%). 100% of methicillin-resistant S. aureus strains contained mecA gene, and more than 50% of isolated strains were associated with nosocomial infections. In addition, 3SCCmec type and 1 SCCmec type were identified as the dominant subtypes in this study and (17.8%). Also, 10 isolated strains could not be typed.
Conclusion: The results showed that the spa gene distribution was present in 82% of the samples. Out of 46 samples, 40 samples belonged to wound samples. Also, pvl gene was observed in 12% (7 samples) of samples. There is an association between mecA gene and resistance to the antibiotics oxacillin, cefoxitin, and erythromycin in methicillin-resistant S. aureus strains.
Staphylococci are among the commonly isolated bacteria from intramammary infections in bovines, where Staphylococcus aureus is the most studied species. This species carries a variety of virulence genes, contributing to bacterial survival and spread. Less is known about non- aureus staphylococci (NAS) and their range of virulence genes and mechanisms, but they are the most frequently isolated bacteria from bovine milk. Staphylococci can also carry a range of antimicrobial resistance genes, complicating treatment of the infections they cause. We used Illumina sequencing to whole genome sequence 93 staphylococcal isolates selected from a collection of staphylococcal isolates; 45 S. aureus isolates and 48 NAS isolates from 16 different species, determining their content of antimicrobial resistance genes and virulence genes. Antimicrobial resistance genes were frequently observed in the NAS species as a group compared to S. aureus . However, the lincosamide resistance gene lnuA and penicillin resistance gene blaZ were frequently identified in NAS, as well as a small number of S. aureus . The erm genes conferring macrolide resistance were also identified in several NAS isolates and in a small number of S. aureus isolates. In most S. aureus isolates, no antimicrobial resistance genes were detected, but in five S. aureus isolates three to six resistance genes were identified and all five of these carried the mecA gene. Virulence genes were more frequently identified in S. aureus , which contained on average five times more virulence genes compared to NAS. Among the NAS species there were also differences in content of virulence genes, such as S. chromogenes with a higher average number of virulence genes. By determining the content of a large selection of virulence genes and antimicrobial resistance genes in S. aureus and 16 different NAS species our results contribute with knowledge regarding the genetic basis for virulence and antimicrobial resistance in bovine staphylococci, especially the less studied NAS. The results can create a broader basis for further research into the virulence mechanisms of this important group of bacteria in bovine intramammary infections.
Biofilm formation is an important and ubiquitous mode of growth among bacteria. Central to the evolutionary advantage of biofilm formation is cell-cell and cell-surface adhesion achieved by a variety of factors, some of which are diffusible compounds that may operate as classical public goods - factors that are costly to produce but may benefit other cells. An outstanding question is how diffusible matrix production, in general, can be stable over evolutionary timescales. In this work, using Vibrio cholerae as a model, we show that shared diffusible biofilm matrix proteins are indeed susceptible to cheater exploitation, and that the evolutionary stability of producing these matrix components fundamentally depends on biofilm spatial structure, intrinsic sharing mechanisms of these components, and flow conditions in the environment. We further show that exploitation of diffusible adhesion proteins is localized within a well-defined spatial range around cell clusters that produce them. Based on this exploitation range and the spatial distribution of cell clusters, we construct a model of costly diffusible matrix production and relate these length scales to the relatedness coefficient in social evolution theory. Our results show that production of diffusible biofilm matrix components is evolutionarily stable under conditions consistent with natural biofilm habitats and host environments. We expect the mechanisms revealed in this study to be relevant to other secreted factors that operate as cooperative public goods in bacterial communities, and the concept of exploitation range and the associated analysis tools to be generally applicable.
The rise of multidrug resistant bacteria is the biggest threat to human health globally as described by the World Health Organization (WHO). Mechano-bactericidal surfaces provides a sustainable approach to address this concern by eradicating pathogens, especially bacteria, “right-at-the-point” of first contacting the surface. However, the lack of a “design to manufacture” approach due to our limited understanding of the mechano-bactericidal mechanism has impeded engineering optimization to develop scalable exploitation routes in various healthcare applications. It can be argued that the reason, most particularly, is the limitations and uncertainties associated with the current instrumentation and simulation capabilities which has led to several streams of test protocols. This review highlights the current understanding on the mechano-bactericidal mechanisms in light of the contributing factors and various techniques which are used to postulate these mechanisms. The review offers a critique on the variations observed on how nanostructured surfaces found in literature have been evaluated such that the test protocols and the outcomes are incomparable. The review also shows a strong need of developing more accurate models of a bacterium as the currently reported experimental data is insufficient to develop bacteria’s material models (constitutive equations). The review also alludes to the scarcity of direct experimental evidence of the mechano-bactericidal mechanism suggesting a strong need for further in-situ monitoring as a future research direction.
The coagulation cascade represents a sophisticated and highly choreographed series of molecular events taking place in the blood with important clinical implications. One key player in coagulation is fibrinogen, a highly abundant soluble blood protein that is processed by thrombin proteases at wound sites, triggering self-assembly of an insoluble protein hydrogel known as a fibrin clot. By forming the key protein component of blood clots, fibrin acts as a structural biomaterial with biophysical properties well suited to its role inhibiting fluid flow and maintaining hemostasis. Based on its clinical importance, fibrin is being investigated as a potentially valuable molecular target in the development of coagulation therapies. In this topical review, we summarize our current understanding of the coagulation cascade from a molecular, structural and biophysical perspective. We highlight single-molecule studies on proteins involved in blood coagulation and report on the current state of the art in directed evolution and molecular engineering of fibrin-targeted proteins and polymers for modulating coagulation. This biophysical overview will help acclimatize newcomers to the field and catalyze interdisciplinary work in biomolecular engineering toward the development of new therapies targeting fibrin and the coagulation system.
Methicillin-resistant Staphylococcus aureus (MRSA) may persist for long periods due to biofilm formation. The objective of this study was to describe biofilm formation in association with the presence of S. aureus surface protein G (sasG) and its allelic variants in MRSA bacteraemia isolates from endemic (CC5, CC8, CC22) and sporadic clones in Spain (2008-2015). Crystal violet staining was used to assess biofilm formation; DNA microarray, RT-qPCR, and long-read whole genome sequencing were applied to determine the presence, expression and structure of sasG, respectively. The endemic CC5 and CC8 clones produced more biofilm than the sporadic clones; these endemic clones carried sasG allelic variant 1. Otherwise, sporadic clones, with less biofilm formation, showed either an absence of sasG (65%) or the presence of allelic variant 2 (35%). Variants 1 and 2 differed in the expression of sasG (1.56±1.20 and 0.37±0.32, respectively). The analysis of a large cohort of closed S. aureus genomes available on the NCBI database confirmed the distribution of the two allelic variants with low amino acid identity (68.1%) among endemic and sporadic clones. SasG variant 1 present in the major CC5 and CC8 clones was correlated with increased biofilm formation and may represent an important virulence determinant.
Actinomyces oris plays an important role in oral biofilm development. Like many Gram‐positive bacteria, A. oris produces a sizable number of surface proteins that are anchored to bacterial peptidoglycan by a conserved transpeptidase named the housekeeping sortase SrtA; however, the biological role of many A. oris surface proteins in biofilm formation is largely unknown. Here, we report that the glycoprotein GspA – a genetic suppressor of srtA deletion lethality – not only promotes biofilm formation but also maintains cell membrane integrity under cation stress. In comparison to wild‐type cells, under elevated concentrations of mono‐ and divalent cations formation of mono‐ and multi‐species biofilms by mutant cells devoid of gspA was significantly diminished, although planktonic growth of both cell types in the presence of cations was indistinguishable. Because gspA overexpression is lethal to cells lacking gspA and srtA, we performed a genetic screen to identify GspA determinants involving cell viability. DNA sequencing and biochemical characterizations of viable clones revealed that mutations of two critical cysteine residues and a serine residue severely affected GspA glycosylation and biofilm formation. Furthermore, mutant cells lacking gspA were markedly sensitive to sodium dodecyl sulfate, a detergent that solubilizes the cytoplasmic membranes, suggesting the cell envelope of the gspA mutant was altered. Consistent with this observation, the gspA mutant exhibited increased membrane permeability, independent of GspA glycosylation, as compared to the wild‐type strain. Altogether, the results support the notion that the cell wall‐anchored glycoprotein GspA provides a defense mechanism against cation stress in biofilm development promoted by A. oris. This article is protected by copyright. All rights reserved
Methicillin-resistant Staphylococcus aureus (MRSA) is a frontline human pathogen in which efflux pump activity confers high levels of antibiotic-resistance and poses a therapeutic challenge in the clinics. The present study illustrates the potential of urea-based ligand as an efflux pump inhibitor (EPI) in order to restore the efficacy of ciprofloxacin (CPX) against MRSA. Among eight structurally varying urea-based ligands, the ligand C8 could significantly inhibit efflux pump activity in the clinical MRSA strain S. aureus 4s and was superior to the known EPI reserpine. In combinatorial treatment, C8 enhanced cellular accumulation of CPX, rendered a 16× decrease in the MIC of CPX, and restored the susceptibility of S. aureus 4s to CPX. Notably, C8 downregulated the expression of norA gene coding for the efflux pump in MRSA and treatment with 10 μM C8 and 2.0 μM CPX prevented emergence of the CPX resistance trait and suppressed MRSA cell growth till 120 generations. For potential anti-MRSA therapy, C8-loaded poly(d,l-lactide-co-glycolide) nanocarrier (C8-PNC) was generated, which facilitated facile release of C8 in physiologically relevant fluid. C8-PNC (loaded with 50 μM C8) rendered efflux pump inhibition and eliminated MRSA in combination with only 2.0 μM CPX. Treatment with the non-toxic C8-PNC (loaded with 50 μM C8) and CPX (2.0 μM) also hindered MRSA adhesion on collagen manifold higher as compared to cells treated with 32 μM CPX and significantly downregulated norA gene expression in non-adhered MRSA cells. The urea-based ligand presented herein is a promising biocompatible therapeutic material for effective mitigation of MRSA infections.
The vaccine development strategies have evolved from using an entire organism as an immunogen to a single antigen and further towards an epitope. Since an epitope is a relatively tiny and immunologically relevant part of an antigen, it has the potential to stimulate more robust and specific immune responses while causing minimal adverse effects. As a result, the recent focus of vaccine development has been to develop multi-epitope vaccines that can target multiple virulence mechanisms. Accordingly, we designed multi-epitope vaccine candidates B (multi-B-cell epitope immunogen) and CTB-B (an adjuvant – cholera toxin subunit B (CTB) – attached to immunogen B) against S. aureus by employing immunoinformatics approaches. The designed vaccines are composed of B-cell epitope segments (20-mer) of the eight well-characterized S. aureus virulence factors, namely ClfB, FnbpA, Hla, IsdA, IsdB, LukE, SdrD, and SdrE connected in series. The designed vaccines were expressed, purified, and administered to C57BL/6 mice with Freund adjuvant to evaluate the immunogenicity and protective efficacy. The results revealed that the immunized mice showed high IgG titer for the immunogen, and the antibody titer increased significantly following the second immunization. However, the generated antibodies did not protect mice from infection. The interaction of anti-B antibodies with source virulence factors showed that the generated antibodies have no binding affinity with any of the corresponding virulence factors. Our results demonstrate the limitation of in silico designed B-cell multi-epitope vaccine and suggest that a protein domain carrying both linear and conformational B-cell epitopes might be a better choice for developing an effective multi-epitope vaccine against S. aureus.
Carried in the nasal passages by up to 30% of humans, Staphylococcus aureus is recognized to be a successful opportunistic pathogen. It is a frequent cause of infections of the upper respiratory tract, including sinusitis, and of the skin, typically abscesses, as well as of food poisoning and medical device contamination. The antimicrobial resistance of such, often chronic, health conditions is underpinned by the unique structure of bacterial biofilm, which is the focus of increasing research to try to overcome this serious public health challenge. Due to the protective barrier of an exopolysaccharide matrix, bacteria that are embedded within biofilm are highly resistant both to an infected individual’s immune response and to any treating antibiotics. An in-depth appraisal of the stepwise progression of biofilm formation by S. aureus, used as a model infection for all cases of bacterial antibiotic resistance, has enhanced understanding of this complicated microscopic structure and served to highlight possible intervention targets for both patient cure and community infection control. While antibiotic therapy offers a practical means of treatment and prevention, the most favorable results are achieved in combination with other methods. This review provides an overview of S. aureus biofilm development, outlines the current range of anti-biofilm agents that are used against each stage and summarizes their relative merits.
Endogenous Staphylococcus aureus sortase A (SrtA) covalently incorporates cell wall anchored proteins equipped with a SrtA recognition motif (LPXTG) via a lipid II-dependent pathway into the staphylococcal peptidoglycan layer. Previously, we found that the endogenous S. aureus SrtA is able to recognize and process a variety of exogenously added synthetic SrtA substrates, including K(FITC)LPMTG-amide and K(FITC)-K-vancomycin-LPMTG-amide. These synthetic substrates are covalently incorporated into the bacterial peptidoglycan (PG) of S. aureus with varying efficiencies. In this study, we examined if native and synthetic substrates are processed by SrtA via the same pathway. Therefore, the effect of the lipid II inhibiting antibiotic bacitracin on the incorporation of native and synthetic SrtA substrates was assessed. Treatment of S. aureus with bacitracin resulted in a decreased incorporation of protein A in the bacterial cell wall, whereas incorporation of exogenous synthetic substrates was increased. These results suggest that natural and exogenous synthetic substrates are processed by S. aureus via different pathways.
Staphylococcus aureus is a serious human pathogen that causes a wide variety of infectious diseases with high morbidity and mortality. Luteolin was recently shown to inhibit biofilm formation and reduce the production of virulence factors and the transcription of agrA in S. aureus. Given the broad impacts of the agr quorum-sensing system on the biofilm formation and virulence factors of S. aureus, this study aimed to investigate the effects of luteolin on the agr system and pathogenicity of S. aureus. Here, we show that at subminimal inhibitory concentrations (sub-MICs) that have no effect on bacterial growth, luteolin can markedly inhibit the adhesion and biofilm formation of both wild-type (WT) and agr mutant strains of S. aureus strain Newman. The hemolytic activity and toxin protein levels were markedly decreased in the culture supernatants of luteolin-treated WT strain but not the luteolin-treated agr mutant strain. qRT–PCR analysis showed that upon luteolin treatment, the expression of genes involved in virulence and biofilm formation was downregulated in the WT S. aureus strain, and the inefficacy of luteolin with respect to the virulence factors of only the agr mutant confirmed the agr-mediated antivirulence potential of luteolin. Furthermore, treatment with sub-MIC luteolin attenuated human alveolar epithelial A549 cell injury caused by the WT Newman strain and protected mice from pneumonia caused by the WT strain, but these effects were not observed with the agr mutant strain. These findings indicate that luteolin is a promising compound that interferes with the agr system and can be developed into novel therapeutic drugs against S. aureus infections.
Staphylococcus aureus (S. aureus) is a foodborne pathogen that endangers human health worldwide. Antimicrobial photodynamic inactivation (aPDI), mediated by titanium dioxide nanoparticles (TiO2NP), was recently used to control the growth of S. aureus, however, UV illumination had to be offered to initiate the photodynamic reaction. To overcome this drawback, a dual-photon system was established here based on TiO2NP and hypocrellin B (HB), a natural pigment with photocatalyst activity. Irradiated at the visible light (9 J/cm²) at the wavelength of 460 nm, the joint use of both photosensitizers (PS) caused a substantial decline of staphylococcal survival, reaching a maximum decrease of 4∼5 logs. When 10 nM HB and 100 µM TiO2NP were applied, a synergistic photokilling effect was achieved, only in the mixed phase of anatase/rutile, Degussa P25. Regarding the antimicrobial mechanisms, it was found that the membrane integrity of S. aureus was heavily disrupted, surface morphology was altered, intracellular substances like potassium and DNA were leaked, and biofilm formation was significantly circumvented due to the excitation of sensitized PS. In terms of antioxidant responsiveness of S. aureus, qPCR results showed that a series of genes encoding the membrane-associated cell death effectors were deregulated. Among them, LrgA was recognized as a key responsive element, due that the mutant strain harboring a constitutively-expressed LrgA strengthened the bactericidal effect of aPDI. Finally, the dual-photon strategy lowered the microbial contamination in the tested apple with its quality maintained, under the condition of visible light. Taken together, a new dual-photon system based on TiO2NP and HB was constructed and validated in photokilling S. aureus, providing a well-adapted technique to maintain food safety.
Pathogenic bacteria infection is a serious threat to human public health due to the high morbidity and mortality rates. Nano delivery system for delivering antibiotics provides an alternative option to improve the efficiency compared to conventional therapeutic agents. In addition to the drug loading capacity of nanocarriers, which is typically around 10%, further lowers the drug dose that pathological bacteria are exposed to. Moreover, nanocarriers that are not eliminated from the body may cause side effects. These limitations have motivated the development of self‐delivery systems that are formed by the self‐assembly of different therapeutic agents. In this study, a vehicle‐free antimicrobial polymer polyhexamethylene biguanide (PHMB, with bactericidal and anti‐biofilm functions) hybrid gold nanoparticle (Au NPs, with photothermal therapy (PTT)) platform (PHMB@Au NPs) is developed. This platform exhibits an excellent synergistic effect to enhance the photothermal bactericidal effect for Staphylococcus aureus under near‐infrared irradiation. Furthermore, the results showed that PHMB@Au NPs inhibit the formation of biofilms, quickly remove bacteria to promote wound healing through PTT in infection model in vivo, and even mediate the transition of macrophages from M1 to M2 type, and accelerate tissue angiogenesis. PHMB@Au NPs will have promising value as highly effective antimicrobial agents for patient management. A vehicle‐free antimicrobial polymer polyhexamethylene biguanide (PHMB, with bactericidal and anti‐biofilm functions) hybrid gold nanoparticle (Au NPs, with photothermal therapy) platform (PHMB@Au NPs) exhibits synergistic interaction and enhanced photothermal bactericidal effect and anti‐biofilm for Staphylococcus aureus under near‐infrared irradiation with superior biosecurity.
The a ccessory g ene r egulator ( agr ) quorum-sensing system is an important global regulatory system of Staphylococcus aureus and contributes to its pathogenicity. The S. aureus agr system is divided into four agr groups based on the amino acid polymorphisms of AgrB, AgrD, and AgrC. The agr activation is group-specific, resulting in variations in agr activity and pathogenicity among the four agr groups. Strains with divergent agr system always have different phenotypes. In the present report, we, respectively, exchanged the agr system of a certain S. aureus with other three agr alleles and assessed the corresponding phenotypes of these congenic strains. Replacement of the agr system led to significant variations in hemolytic activity, protein expression, and virulence gene expression comparing with that of the parental strain. Interestingly, we found that the biological characteristics of these agr congenic strains in the same strain background were highly similar to each other, and the allele-dependent differences of the agr systems were weakened. These findings indicate that the allele-dependent agr predilections of S. aureus are determined by some factors in addition to the polymorphisms of AgrB, AgrD, and AgrC. Future studies may reveal the novel mechanism to improve our understanding of the agr network.
Staphylococcus aureus is a foremost bacterial pathogen responsible for a vast array of human diseases. Staphylococcal superantigens (SAgs) constitute a family of exotoxins from S. aureus that bind directly to major histocompatibility complex (MHC) class II and T cell receptors to drive extensive T cell activation and cytokine release. Although these toxins have been implicated in serious disease, including toxic shock syndrome, the specific pathological mechanisms remain unclear. Herein, we aimed to elucidate how SAgs contribute to pathogenesis during bloodstream infections and utilized transgenic mice encoding human MHC class II to render mice susceptible to SAg activity. We demonstrate that SAgs contribute to S. aureus bacteremia by massively increasing bacterial burden in the liver, and this was mediated by CD4 ⁺ T cells that produced interferon gamma (IFN-γ) to high levels in a SAg-dependent manner. Bacterial burdens were reduced by blocking IFN-γ, phenocopying SAg-deletion mutant strains, and inhibiting a proinflammatory response. Infection kinetics and flow cytometry analyses suggested that this was a macrophage-driven mechanism, which was confirmed through macrophage-depletion experiments. Experiments in human cells demonstrated that excessive IFN-γ allowed S. aureus to replicate efficiently within macrophages. This indicates that SAgs promote bacterial survival by manipulating the immune response to inhibit effective clearing of S. aureus . Altogether, this work implicates SAg toxins as critical therapeutic targets for preventing persistent or severe S. aureus disease.
Aim: To investigate the function of broad-spectrum racemases in Aeromonas hydrophila (BsrA). Results: The A. hydrophila gene encoding BsrA ( bsr) mutants (AHΔbsr) exhibited a significant decrease in growth, motility, extracellular protease production and biofilm formation compared with the wild-type. Furthermore, bsr gene knockout instigated cell wall damage compared with the wild-type strains. The survival rate and replication capability in the blood and organs of the AHΔbsr-infected mice were significantly decreased. The degree of tissue injury in the AHΔbsr-infected group was lower than that of the wild-type-infected group. Moreover, there was a significant decrease in the expression of 12 AHΔbsr virulence genes. Conclusion: The bsr gene is essential for the viability and virulence of A. hydrophila.
Human milk N-glycome was previously identified to have strong antipathogenic activities. This study is aimed to characterize the detailed antibacterial properties and the potential function mechanism of human milk N-glycome against Staphylococcus aureus. A serials of traditional antibacterial assays showed that human milk N-glycome possessed both bacteriostatic and bactericidal activities, which was further confirmed by the cell structure disruption including the change of transmembrane potential and leakage of intracellular contents. The results of the bacterial surface zeta potential and hydrophobicity, bacterial binding assay, gel shift assay, and fluorescence spectra and the different synergistic effects of human milk N-glycome combined with different antibiotics indicated that the bacterial surface proteins could be the targets of human milk N-glycome. Moreover, human milk N-glycome also showed antibiofilm activity. In conclusion, human milk N-glycome exhibited good potential for acting as an antibacterial substance against S. aureus and the antibacterial mechanism was a cell surface targeting action.
Les plaies chroniques sont un réel problème de santé publique. L’une des principales complications est l’évolution fréquente vers l’infection avec un risque sous-jacent d'amputation. La difficulté de prise en charge repose sur la présence d’un biofilm polymicrobien au niveau du lit de la plaie. L'objectif de cette thèse était de promouvoir l'amélioration de l'état de l'art relatif à la compréhension de ce phénomène physiopathologique important mais également de développer de nouvelles approches concernant le traitement thérapeutique des plaies chroniques pour réduire les risques d'infections morbides. Ce travail de doctorat a permis : i) de parfaire les outils diagnostic de par la création d’un nouveau modèle in vitro mimant l’environnement dans lequel les bactéries évoluent au niveau des plaies chroniques ; ii) d’étudier le comportement (virulence, fitness, génomique, morphologie, formation de biofilm précoce, expression de gènes clés pour la formation de biofilm) de souches de références et de souches cliniques de Staphylococcus aureus (SA) et Pseudomonas aeruginosa (PA). Ces souches, isolées au sein d’une même plaie, étaient exposées de façon courte ou prolongée, à un milieu classique ou mimant l’environnement chronique d’une plaie. Les résultats ont montré qu’une exposition dans des conditions environnementales stressantes réduisaient la virulence et le fitness des souches au profit d’un comportement plus tourné vers la formation de biofilm ; iii) de démontrer l’impact de la présence de plusieurs bactéries au sein d’un environnement. Ce travail a particulièrement mis en évidence la baisse de pathogénicité et l’évolution de l’expression de certains gènes impliqués dans le biofilm de souche de SA en présence de PA. Enfin iv) la mise au point d’une technique de formation de biofilm dynamique et d’imagerie confocale permettant la visualisation d’un biofilm polymicrobien en trois dimensions et l’évaluation de molécules antimicrobiennes sur chaque espèce bactérienne composant le biofilm polymicrobien après un débridement mécanique automatisé. La combinaison de notre nouveau milieu « plaie chronique » et du système de formation en flux et visualisation en 3D du biofilm polymicrobien représente des outils puissants pour améliorer la compréhension des interactions régissant les biofilms mais aussi pour évaluer l’efficacité de molécules candidates dans la prise en charge des plaies chroniques.Mots clés : Plaies chroniques, infections, biofilms, coopération bactérienne
Oral diseases caused by infectious pathogens raises significant concerns in public health. In the light of side effects of current antibiotics therapy and growing drug resistance of pathogenic bacteria, natural products have become attractive alternatives for antibiotics agents in dental practice. This current study investigated the effects of polysaccharides extracted from Zizyphus jujuba Mill. on three major oral biofilm-forming pathogenic bacteria including caries-inducing Streptococcus mutans, lesions-causing MRSA, and periodontitis-related Porphyromonas gingivalis, as well as general oral microbiota. Our results demonstrated that jujube polysaccharide prepared in this study was mainly composed by galacturonic acid with an average molecular weight 242 kDa, which were further characterized for structural features by FT-IR spectra and NMR spectroscopy analysis. This jujube polysaccharide was shown to exhibit remarkable inhibitory effects against all the tested oral bacterial pathogens through various mechanisms including growth inhibition, biofilm prevention and disruption, intervention of bacterial infection (adhesion and invasion), attenuation of cytotoxicity, modulation of excessive inflammatory response of LPS-stimulated and MRSA-infected macrophages as well as positive regulation of oral microbiota. The present study paves the way to explore jujube polysaccharides for the prevention and treatment of oral infectious diseases.
Graphic Abstract.
Objective
To compare the clinical efficacy of vancomycin calcium sulfate implantation and fenestration decompression in the treatment of sclerosing osteomyelitis.
Method
A retrospective analysis for 46 cases of sclerosing osteomyelitis
were admitted to our department between June 2010 to June 2020. Twenty-one patients were treated with fenestration decompression, twenty-five patients were treated with vancomycin calcium sulfate implantation. The postoperative hospital stay, days of drainage tube placement, visual analogue scale scores, C-reactive protein and erythrocyte sedimentation rate were compared between the two groups.
Results
The visual analogue scale scores of both groups were significantly lower than before treatment ( p < 0.05), but the difference between them was not statistically significant. Patients treated by vancomycin calcium sulfate implantation had shorter postoperative hospital stay and days of drainage tube placement compared to those treated by fenestration decompression ( p < 0.05). C-reactive protein and erythrocyte sedimentation rate in both groups were significantly lower than before treatment, but the improvement effect of vancomycin calcium sulfate implantation was better ( p < 0.05).
Conclusion
Both treatment methods can relieve pain effectively. Compared with fenestration decompression, vancomycin calcium sulfate implantation can shorten the treatment time effectively, control the infection better.
A limited number of Methicillin-resistant Staphylococcus aureus (MRSA) clones are responsible for MRSA infections worldwide, and those of different lineages carry unique Type I restriction-modification
(RM) variants. We have identified the specific DNA sequence targets for the dominant MRSA lineages CC1, CC5, CC8 and ST239.
We experimentally demonstrate that this RM system is sufficient to block horizontal gene transfer between clinically important
MRSA, confirming the bioinformatic evidence that each lineage is evolving independently. Target sites are distributed randomly
in S. aureus genomes, except in a set of large conjugative plasmids encoding resistance genes that show evidence of spreading between
two successful MRSA lineages. This analysis of the identification and distribution of target sites explains evolutionary patterns
in a pathogenic bacterium. We show that a lack of specific target sites enables plasmids to evade the Type I RM system thereby
contributing to the evolution of increasingly resistant community and hospital MRSA.
Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here,
we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses
of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r
with C1q. As a result, C1r2C1s2 was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential
immune evasion strategy that could be used by numerous Gram-positive pathogens.
Healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) form biofilm in vitro that is dependent on the surface located fibronectin binding proteins A and B (FnBPA, FnBPB). Here we provide new insights into the requirements for FnBP-dependent biofilm formation by MRSA. We show that expression of FnBPs is sustained at high levels throughout the growth cycle in the HA-MRSA strain BH1CC in contrast to laboratory strain SH1000 where expression could only be detected in exponential phase. We found that FnBP-mediated biofilm accumulation required Zn(2+) while the removal of Zn(2+) had no effect on the ability of FnBPA to mediate bacterial adherence to fibrinogen.We also investigated the role of FnBPA expressed on the surface of S. aureus in promoting biofilm formation and bacterial adhesion to fibrinogen. The minimum part of FnBPA required for ligand binding has so far only been defined with recombinant proteins. Here we found that the N1 subdomain was not required for biofilm formation or for FnBPA to promote bacterial adherence to fibrinogen. Residues at the C-terminus of subdomain N3 required for FnBPA to bind to ligands using the 'dock, lock, and latch' mechanism were necessary for FnBPA to promote bacterial adherence to fibrinogen. However these residues were not necessary to form biofilm allowing us to localise the region of FnBPA required for biofilm accumulation to residues 166-498. Thus, FnBPA mediates biofilm formation and bacterial adhesion to fibrinogen using two distinct mechanisms. Finally, we identified a hitherto unrecognised thrombin cleavage site close to the boundary between subdomains N1 and N2 of FnBPA.
Infections due to Staphylococcus aureus are serious complications of cardiothoracic surgery. A novel vaccine candidate (V710) containing the highly conserved S. aureus iron surface determinant B is immunogenic and generally well tolerated in volunteers.
To evaluate the efficacy and safety of preoperative vaccination in preventing serious postoperative S. aureus infection in patients undergoing cardiothoracic surgery.
Double-blind, randomized, event-driven trial conducted between December 2007 and August 2011 among 8031 patients aged 18 years or older who were scheduled for full median sternotomy within 14 to 60 days of vaccination at 165 sites in 26 countries.
Participants were randomly assigned to receive a single 0.5-mL intramuscular injection of either V710 vaccine, 60 μg (n = 4015), or placebo (n = 4016).
The primary efficacy end point was prevention of S. aureus bacteremia and/or deep sternal wound infection (including mediastinitis) through postoperative day 90. Secondary end points included all S. aureus surgical site and invasive infections through postoperative day 90. Three interim analyses with futility assessments were planned.
The independent data monitoring committee recommended termination of the study after the second interim analysis because of safety concerns and low efficacy. At the end of the study, the V710 vaccine was not significantly more efficacious than placebo in preventing either the primary end points (22/3528 V710 vaccine recipients [2.6 per 100 person-years] vs 27/3517 placebo recipients [3.2 per 100 person-years]; relative risk, 0.81; 95% CI, 0.44-1.48; P = .58) or secondary end points despite eliciting robust antibody responses. Compared with placebo, the V710 vaccine was associated with more adverse experiences during the first 14 days after vaccination (1219/3958 vaccine recipients [30.8%; 95% CI, 29.4%-32.3%] and 866/3967 placebo recipients [21.8%; 95% CI, 20.6%-23.1%], including 797 [20.1%; 95% CI, 18.9%-21.4%] and 378 [9.5%; 95% CI, 8.6%-10.5%] with injection site reactions and 66 [1.7%; 95% CI, 1.3%-2.1%] and 51 [1.3%; 95% CI, 1.0%-1.7%] with serious adverse events, respectively) and a significantly higher rate of multiorgan failure during the entire study (31 vs 17 events; 0.9 [95% CI, 0.6-1.2] vs 0.5 [95% CI, 0.3-0.8] events per 100 person-years; P = .04). Although the overall incidence of vaccine-related serious adverse events (1 in each group) and the all-cause mortality rate (201/3958 vs 177/3967; 5.7 [95% CI, 4.9-6.5] vs 5.0 [95% CI, 4.3-5.7] deaths per 100 person-years; P = .20) were not statistically different between groups, the mortality rate in patients with staphylococcal infections was significantly higher among V710 vaccine than placebo recipients (15/73 vs 4/96; 23.0 [95% CI, 12.9-37.9] vs 4.2 [95% CI, 1.2-10.8] per 100 person-years; difference, 18.8 [95% CI, 8.0-34.1] per 100 person-years).
Among patients undergoing cardiothoracic surgery with median sternotomy, the use of a vaccine against S. aureus compared with placebo did not reduce the rate of serious postoperative S. aureus infections and was associated with increased mortality among patients who developed S. aureus infections. These findings do not support the use of the V710 vaccine for patients undergoing surgical interventions.
clinicaltrials.gov Identifier: NCT00518687.
Staphylococcus aureus asymptomatically colonises the anterior nares, but the host and bacterial factors that facilitate colonisation remain incompletely understood. The S. aureus surface protein ClfB has been shown to mediate adherence to squamous epithelial cells in vitro and to promote nasal colonisation in both mice and humans. Here, we demonstrate that the squamous epithelial cell envelope protein loricrin represents the major target ligand for ClfB during S. aureus nasal colonisation. In vitro adherence assays indicated that bacteria expressing ClfB bound loricrin most likely by the “dock, lock and latch” mechanism. Using surface plasmon resonance we showed that ClfB bound cytokeratin 10 (K10), a structural protein of squamous epithelial cells, and loricrin with similar affinities that were in the low µM range. Loricrin is composed of three separate regions comprising GS-rich omega loops. Each loop was expressed separately and found to bind ClfB, However region 2 bound with highest affinity. To investigate if the specific interaction between ClfB and loricrin was sufficient to facilitate S. aureus nasal colonisation, we compared the ability of ClfB+
S. aureus to colonise the nares of wild-type and loricrin-deficient (Lor−/−) mice. In the absence of loricrin, S. aureus nasal colonisation was significantly impaired. Furthermore a ClfB− mutant colonised wild-type mice less efficiently than the parental ClfB+ strain whereas a similar lower level of colonisation was observed with both the parental strain and the ClfB− mutant in the Lor−/− mice. The ability of ClfB to support nasal colonisation by binding loricrin in vivo was confirmed by the ability of Lactococcus lactis expressing ClfB to be retained in the nares of WT mice but not in the Lor−/− mice. By combining in vitro biochemical analysis with animal model studies we have identified the squamous epithelial cell envelope protein loricrin as the target ligand for ClfB during nasal colonisation by S. aureus.
Fibrinogen can support host antimicrobial containment/clearance mechanisms, yet selected pathogens appear to benefit from host procoagulants to drive bacterial virulence. Here, we explored the hypothesis that host fibrin(ogen), on balance, supports Staphylococcus aureus infection in the context of septicemia. Survival studies following intravenous infection in control and fibrinogen-deficient mice established the overall utility of host fibrin(ogen) to S. aureus virulence. Complementary studies in mice expressing mutant forms of fibrinogen-retaining clotting function, but lacking either the bacterial ClfA (Fibγ(Δ5)) binding motif or the host leukocyte integrin receptor αMβ2 (Fibγ(390-396A)) binding motif, revealed the preeminent importance of the bacterial ClfA-fibrin(ogen) interaction in determining host survival. Studies of mice lacking platelets or the platelet integrin receptor subunit αIIb established that the survival benefits observed in Fibγ(Δ5) mice were largely independent of platelet αIIbβ3-mediated engagement of fibrinogen. Fibγ(Δ5) mice exhibited reduced bacterial burdens in the hearts and kidneys, a blunted host proinflammatory cytokine response, diminished microscopic tissue damage, and significantly diminished plasma markers of cardiac and other organ damage. These findings indicate that host fibrin(ogen) and bacterial ClfA are dual determinants of virulence and that therapeutic interventions at the level of fibrinogen could be advantageous in S. aureus septicemia.
Staphylococcus aureus is a human pathogen that causes invasive and recurring infections. The ability to internalize into and persist within host cells is thought to contribute to infection. Here we report a novel role for the well characterized iron regulated surface determinant B (IsdB) protein which we have shown can promote adhesion of 293T, HeLa cells and platelets to immobilized bacteria independently of its ability to bind haemoglobin. IsdB bound to the active form of the platelet integrin α(IIb) β(3) , both on platelets and when the integrin was expressed ectopically in CHO cells. IsdB also promoted bacterial invasion into human cells. This was clearly demonstrated with bacteria lacking fibronectin-binding proteins (FnBPs), which are known to promote invasion in the presence of fibronectin, However, IsdB also contributed significantly to invasion by cells expressing FnBPs in the presence of serum. Thus IsdB appears to be able to interact with the broader family of integrins that bind ligands with the RGD motif and to act as a back up mechanism to promote interactions with mammalian cells.
The serine-rich repeat glycoprotein Srr1 of Streptococcus agalactiae (GBS) is thought to be an important adhesin for the pathogenesis of meningitis. Although expression of Srr1 is associated with increased binding to human brain microvascular endothelial cells (hBMEC), the molecular basis for this interaction is not well defined. We now demonstrate that Srr1 contributes to GBS attachment to hBMEC via the direct interaction of its binding region (BR) with human fibrinogen. When assessed by Far Western blotting, Srr1 was the only protein in GBS extracts that bound fibrinogen. Studies using recombinant Srr1-BR and purified fibrinogen in vitro confirmed a direct protein-protein interaction. Srr1-BR binding was localized to amino acids 283-410 of the fibrinogen Aα chain. Structural predictions indicated that the conformation of Srr1-BR is likely to resemble that of SdrG and other related staphylococcal proteins that bind to fibrinogen through a "dock, lock, and latch" mechanism (DLL). Deletion of the predicted latch domain of Srr1-BR abolished the interaction of the BR with fibrinogen. In addition, a mutant GBS strain lacking the latch domain exhibited reduced binding to hBMEC, and was significantly attenuated in an in vivo model of meningitis. These results indicate that Srr1 can bind fibrinogen directly likely through a DLL mechanism, which has not been described for other streptococcal adhesins. This interaction was important for the pathogenesis of GBS central nervous system invasion and subsequent disease progression.
Staphylococcus aureus is a major cause of healthcare-associated infections and is responsible for a substantial burden of disease in hospitalized patients. Despite increasingly rigorous infection control guidelines, the prevalence and corresponding negative impact of S. aureus infections remain considerable. Difficulties in controlling S. aureus infections as well as the associated treatment costs are exacerbated by increasing rates of resistance to available antibiotics. Despite ongoing efforts over the past 20 y, no licensed S. aureus vaccine is currently available. However, learnings from past clinical failures of vaccine candidates and a better understanding of the immunopathology of S. aureus colonization and infection have aided in the design of new vaccine candidates based on multiple important bacterial pathogenesis mechanisms. This review outlines important considerations in designing a vaccine for the prevention of S. aureus disease in healthcare settings.
Staphylococcus aureus may cause serious skin and soft tissue infections, deep abscesses, endocarditis, osteomyelitis, pneumonia, and sepsis. S. aureus persistently colonizes 25-30% of the adult human population, and S. aureus carriers have an increased risk for infections caused by the bacterium. The major site of colonization is the nose, i.e., the vestibulum nasi, which is covered with ordinary skin and hair follicles. Several host and microbe determinants are assumed to be associated with colonization. These include the presence and expression level of bacterial adhesins, which can adhere to various proteins in the extracellular matrix or on the cellular surface of human skin. The host expresses several antimicrobial peptides and lipids. The level of β-defensin 3, free sphingosine, and cis-6-hexadecenoic acid are found to be associated with nasal carriage of S. aureus. Other host factors are certain polymorphisms in Toll-like receptor 2, mannose-binding lectin, C-reactive protein, glucocorticoid-, and vitamin D receptor. Additional putative determinants for carriage include genetic variation and expression of microbial surface components recognizing adhesive matrix molecules and their interaction partners, as well as variation among humans in the ability of recognizing and responding appropriately to the bacteria. Moreover, the available microflora may influence the success of S. aureus colonization. In conclusion, colonization is a complex interplay between the bacteria and its host. Several bacterial and host factors are involved, and an increased molecular understanding of these are needed.
Most strains of Staphylococcus aureus and Staphylococcus epidermidis possess a strong restriction barrier that hinders exchange of DNA. Recently, major advances have been made in identifying and characterizing the restriction-modification (RM) systems involved. In particular a novel type IV restriction enzyme that recognizes cytosine methylated DNA has been shown to be the major barrier to transfer of plasmid DNA from Escherichia coli into S. aureus and S. epidermidis. While the conserved type I RM system provides a further barrier. Here we review the recent advances in understanding of restriction systems in staphylococci and highlight how this has been exploited to improve our ability to manipulate genetically previously untransformable strains.
Multidrug resistant Staphylococcus aureus strains are emerging and current antibiotics are not efficacious against such strains. Therefore, there is an urgent need to develop vaccines to target this pathogen. However, both active and passive immunization strategies have thus far failed to show efficacy in humans. There are several potential reasons behind the disappointing results of clinical trials, however, we believe that just a few of them, which are common to all the trials, determined their downfall. First of all, preclinical results obtained with antigens tested in clinical trials were likely overestimated by vaccine manufacturers. Furthermore, vaccines tested in humans to date, since they all targeted single antigens, were probably disproportionate to the complex pathogenic mechanisms of the bacterium. In addition, the lack of known correlates of protection in humans has severely limited the ability to interpret both preclinical and clinical data. Finally, the vaccines did not contain new generation adjuvants, which may be critical in augmenting antibody production and steering the T-cell response toward the proper profile of cytokine production.
The biofilm matrix, composed of exopolysaccharides, proteins, nucleic acids and lipids, plays a well-known role as a defence structure, protecting bacteria from the host immune system and antimicrobial therapy. However, little is known about its responsibility in the interaction of biofilm cells with host tissues. Staphylococcus aureus, a leading cause of biofilm-associated chronic infections, is able to develop a biofilm built on a proteinaceous Bap-mediated matrix. Here, we used the Bap protein as a model to investigate the role that components of the biofilm matrix play in the interaction of S. aureus with host cells. The results show that Bap promotes the adhesion but prevents the entry of S. aureus into epithelial cells. A broad analysis of potential interaction partners for Bap using ligand overlayer immunoblotting, immunoprecipitation with purified Bap and pull down with intact bacteria, identified a direct binding between Bap and Gp96/GRP94/Hsp90 protein. The interaction of Bap with Gp96 provokes a significant reduction in the capacity of S. aureus to invade epithelial cells by interfering with the fibronectin binding protein invasion pathway. Consistent with these results, Bap deficient bacteria displayed an enhanced capacity to invade mammary gland epithelial cells in a lactating mice mastitis model. Our observations begin to elucidate the mechanisms by which components of the biofilm matrix can facilitate the colonization of host tissues and the establishment of persistent infections.
The collagen-binding bacterial proteins, Ace and Cna, are well characterized on the biochemical and structural level. Despite
overall structural similarity, recombinant forms of the Ace and Cna ligand-binding domains exhibit significantly different
affinities and binding kinetics for collagen type I (CI) in vitro. In this study, we sought to understand, in submolecular detail, the bases for these differences. Using a structure-based
approach, we engineered Cna and Ace variants by altering specific structural elements within the ligand-binding domains. Surface
plasmon resonance-based binding analysis demonstrated that mutations that are predicted to alter the orientation of the Ace
and Cna N1 and N2 subdomains significantly affect the interaction between the MSCRAMM (microbial surface components recognizing adhesive matrix molecule) and CI in vitro, including affinity, association/dissociation rates and binding ratio. Moreover, we utilized this information to engineer
an Ace variant with an 11,000-fold higher CI affinity than the parent protein. Finally, we noted that several engineered proteins
that exhibited a weak interaction with CI recognized more sites on CI, suggesting an inverse correlation between affinity
and specificity.
Staphylococcus aureus (S. aureus) pathogenesis is a complex process involving a diverse array of extracellular and cell wall components. ClfB, an MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules) family surface protein, described as a fibrinogen-binding clumping factor, is a key determinant of S. aureus nasal colonization, but the molecular basis for ClfB-ligand recognition remains unknown. In this study, we solved the crystal structures of apo-ClfB and its complexes with fibrinogen α (Fg α) and cytokeratin 10 (CK10) peptides. Structural comparison revealed a conserved glycine-serine-rich (GSR) ClfB binding motif (GSSGXGXXG) within the ligands, which was also found in other human proteins such as Engrailed protein, TCF20 and Dermokine proteins. Interaction between Dermokine and ClfB was confirmed by subsequent binding assays. The crystal structure of ClfB complexed with a 15-residue peptide derived from Dermokine revealed the same peptide binding mode of ClfB as identified in the crystal structures of ClfB-Fg α and ClfB-CK10. The results presented here highlight the multi-ligand binding property of ClfB, which is very distinct from other characterized MSCRAMMs to-date. The adherence of multiple peptides carrying the GSR motif into the same pocket in ClfB is reminiscent of MHC molecules. Our results provide a template for the identification of other molecules targeted by S. aureus during its colonization and infection. We propose that other MSCRAMMs like ClfA and SdrG also possess multi-ligand binding properties.
Similar to other highly successful invasive bacterial pathogens, Staphylococcus aureus recruits the complement regulatory protein factor H (fH) to its surface to inhibit the alternative pathway of complement. Here, we report the identification of the surface-associated protein SdrE as a fH-binding protein using purified fH overlay of S. aureus fractionated cell wall proteins and fH cross-linking to S. aureus followed by mass spectrometry. Studies using recombinant SdrE revealed that rSdrE bound significant fH whether from serum or as a purified form, in both a time- and dose-dependent manner. Furthermore, rSdrE-bound fH exhibited cofactor functionality for factor I (fI)-mediated cleavage of C3b to iC3b which correlated positively with increasing amounts of fH. Expression of SdrE on the surface of the surrogate bacterium Lactococcus lactis enhanced recruitment of fH which resulted in increased iC3b generation. Moreover, surface expression of SdrE led to a reduction in C3-fragment deposition, less C5a generation, and reduced killing by polymorphonuclear cells. Thus, we report the first identification of a S. aureus protein associated with the staphylococcal surface that binds factor H as an immune evasion mechanism.
The molecular processes underlying epidemic waves of methicillin-resistant Staphylococcus aureus (MRSA) infection are poorly understood(1). Although a major role has been attributed to the acquisition of virulence determinants by horizontal gene transfer(2), there are insufficient epidemiological and functional data supporting that concept. We here report the spread of clones containing a previously extremely rare(3,4) mobile genetic element–encoded gene, sasX. We demonstrate that sasX has a key role in MRSA colonization and pathogenesis, substantially enhancing nasal colonization, lung disease and abscess formation and promoting mechanisms of immune evasion. Moreover, we observed the recent spread of sasX from sequence type 239 (ST239) to invasive clones belonging to other sequence types. Our study identifies sasX as a quickly spreading crucial determinant of MRSA pathogenic success and a promising target for therapeutic interference. Our results provide proof of principle that horizontal gene transfer of key virulence determinants drives MRSA epidemic waves.
Staphylococcus aureus and Staphylococcus epidermidis form communities (called biofilms) on inserted medical devices, leading to infections that affect many millions of patients worldwide and cause substantial morbidity and mortality. As biofilms are resistant to antibiotics, device removal is often required to resolve the infection. Thus, there is a need for new therapeutic strategies and molecular data that might assist their development. Surface proteins S. aureus surface protein G (SasG) and accumulation-associated protein (S. epidermidis) promote biofilm formation through their "B" regions. B regions contain tandemly arrayed G5 domains interspersed with approximately 50 residue sequences (herein called E) and have been proposed to mediate intercellular accumulation through Zn(2+)-mediated homodimerization. Although E regions are predicted to be unstructured, SasG and accumulation-associated protein form extended fibrils on the bacterial surface. Here we report structures of E-G5 and G5-E-G5 from SasG and biophysical characteristics of single and multidomain fragments. E sequences fold cooperatively and form interlocking interfaces with G5 domains in a head-to-tail fashion, resulting in a contiguous, elongated, monomeric structure. E and G5 domains lack a compact hydrophobic core, and yet G5 domain and multidomain constructs have thermodynamic stabilities only slightly lower than globular proteins of similar size. Zn(2+) does not cause SasG domains to form dimers. The work reveals a paradigm for formation of fibrils on the 100-nm scale and suggests that biofilm accumulation occurs through a mechanism distinct from the "zinc zipper." Finally, formation of two domains by each repeat (as in SasG) might reduce misfolding in proteins when the tandem arrangement of highly similar sequences is advantageous.
Staphylococcus aureus is the leading cause of nosocomial infections and a major cause of community-acquired infections. Biofilm formation is a key virulence determinant in certain types of S. aureus infection, especially those involving inserted medical devices. We found in a previous study that the calcium chelators sodium citrate and EGTA inhibit biofilm formation in certain strains of S. aureus but actually augment biofilm formation in other strains. Even two closely related strains, Newman and 10833, exhibited strikingly different biofilm phenotypes in the presence of calcium chelators, in that biofilm formation was inhibited in Newman but augmented in 10833. We also found that the surface protein clumping factor B (ClfB) plays a role in this phenomenon. In this study, we confirm that ClfB is required for biofilm formation under calcium-depleted conditions. We investigated the post-translational regulation of ClfB-mediated biofilm formation and found evidence that both calcium and the protease aureolysin disrupt established ClfB-dependent biofilms. Finally, we investigated the genetic basis for the biofilm-negative phenotype in strain Newman versus the biofilm-positive phenotype in strain 10833 under calcium-depleted conditions and found that strain 10833 contains a deletion that results in a stop codon within the aureolysin gene (aur). When 10833 expressed Newman aur, surface-associated ClfB and the ability to form a biofilm in chelating conditions was lost. Thus, the positive effect of chelating agents on biofilm formation in certain strains can be explained by increased ClfB activity in the absence of calcium and the discrepancy in the response of strains 10833 and Newman can be explained by point mutations in aur. This study reveals a previously unknown, to our knowledge, role for ClfB in biofilm formation and underscores the potential for striking phenotypic variability resulting from minor differences in strain background.
The strong restriction barrier present in Staphylococcus aureus and Staphylococcus epidermidis has limited functional genomic analysis to a small subset of strains that are amenable to genetic manipulation. Recently, a conserved type IV restriction system termed SauUSI (which specifically recognizes cytosine methylated DNA) was identified as the major barrier to transformation with foreign DNA. Here we have independently corroborated these findings in a widely used laboratory strain of S. aureus. Additionally, we have constructed a DNA cytosine methyltransferase mutant in the high-efficiency Escherichia coli cloning strain DH10B (called DC10B). Plasmids isolated from DC10B can be directly transformed into clinical isolates of S. aureus and S. epidermidis. We also show that the loss of restriction (both type I and IV) in an S. aureus USA300 strain does not have an impact on virulence. Circumventing the SauUSI restriction barrier, combined with an improved deletion and transformation protocol, has allowed the genetic manipulation of previously untransformable strains of these important opportunistic pathogens.
IMPORTANCE Staphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding of Staphylococcus aureus and Staphylococcus epidermidis infections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated.
The Sbi protein of Staphylococcus aureus comprises two IgG-binding domains similar to those of protein A and a region that triggers the activation of complement C3. Sbi is expressed on the cell surface but its C-terminal domain lacks motifs associated with wall or membrane anchoring of proteins in Gram-positive bacteria. Cell-associated Sbi fractionates with the cytoplasmic membrane and is not solubilized during protoplast formation. S. aureus expressing Sbi truncates of the C-terminal Y domain allowed identification of residues that are required for association of Sbi with the membrane. Recombinant Sbi bound to purified cytoplasmic membrane material in vitro and to purified lipoteichoic acid. This explains how Sbi partitions with the membrane in fractionation experiments yet is partially exposed on the cell surface. An LTA-defective mutant of S. aureus had reduced levels of Sbi in the cytoplasmic membrane.
Staphylococcus aureus is a human pathogen that produces extracellular adenosine to evade clearance by the host immune system, an activity attributed to the 5'-nucleotidase activity of adenosine synthase (AdsA). In mammals, conversion of adenosine triphosphate to adenosine is catalyzed in a two-step process: ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTDPases) hydrolyze ATP and ADP to AMP, whereas 5'-nucleotidases hydrolyze AMP to adenosine. NTPDases harbor apyrase conserved regions (ACRs) that are critical for activity.
NTPDase ACR motifs are absent in AdsA, yet we report here that recombinant AdsA hydrolyzes ADP and ATP in addition to AMP. Competition assays suggest that hydrolysis occurs following binding of all three substrates at a unique site. Alanine substitution of two amino acids, aspartic acid 127 and histidine 196 within the 5'-nucleotidase signature sequence, leads to reduced AMP or ADP hydrolysis but does not affect the binding of these substrates.
Collectively, these results provide insight into the unique ability of AdsA to produce adenosine through the consecutive hydrolysis of ATP, ADP and AMP, thereby endowing S. aureus with the ability to modulate host immune responses.
Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a biofilm, a structured community of bacterial cells adherent to the surface of a solid substrate. Every biofilm begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated from humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct binding-force signature and had specific single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension.
Staphylococcus aureus is a major cause of human disease, responsible for half a million infections and approximately 20,000 deaths per year in the United States alone. This pathogen secretes α-hemolysin, a pore-forming cytotoxin that contributes to the pathogenesis of pneumonia. α-hemolysin injures epithelial cells in vitro by interacting with its receptor, the zinc-dependent metalloprotease ADAM10 (ref. 6). We show here that mice harboring a conditional disruption of the Adam10 gene in lung epithelium are resistant to lethal pneumonia. Investigation of the molecular mechanism of toxin-receptor function revealed that α-hemolysin upregulates ADAM10 metalloprotease activity in alveolar epithelial cells, resulting in cleavage of the adherens junction protein E-cadherin. Cleavage is associated with disruption of epithelial barrier function, contributing to the pathogenesis of lethal acute lung injury. A metalloprotease inhibitor of ADAM10 prevents E-cadherin cleavage in response to Hla; similarly, toxin-dependent E-cadherin proteolysis and barrier disruption is attenuated in ADAM10-knockout mice. Together, these data attest to the function of ADAM10 as the cellular receptor for α-hemolysin. The observation that α-hemolysin can usurp the metalloprotease activity of its receptor reveals a previously unknown mechanism of pore-forming cytotoxin action in which pathologic insults are not solely the result of irreversible membrane injury and defines ADAM10 inhibition as a strategy to attenuate α-hemolysin-induced disease.
The second immunoglobulin-binding protein (Sbi) of Staphylococcus aureus has two N-terminal domains that bind the Fc region of IgG in a fashion similar to that of protein A and two domains that
can bind to the complement protein C3 and promote its futile consumption in the fluid phase. It has been proposed that Sbi
helps bacteria to avoid innate immune defenses. By comparing a mutant defective in Sbi with mutants defective in protein A,
clumping factor A, iron-regulated surface determinant H, and capsular polysaccharide, it was shown that Sbi is indeed an immune
evasion factor that promotes bacterial survival in whole human blood and the avoidance of neutrophil-mediated opsonophagocytosis.
Sbi is present in the culture supernatant and is also associated with the cell envelope. S. aureus strains that expressed truncates of Sbi lacking N-terminal domains D1 and D2 (D1D2) or D3 and D4 (D3D4) or a C-terminal truncate
that was no longer retained in the cell envelope were analyzed. Both the secreted and envelope-associated forms of Sbi contributed
to immune evasion. The IgG-binding domains contributed only when Sbi was attached to the cell, while only the secreted C3-binding
domains were biologically active.
Staphylococcus lugdunensis is an opportunistic pathogen related to Staphylococcus aureus and Staphylococcus epidermidis. The genome sequence of S. lugdunensis strain N920143 has been compared with other staphylococci, and genes were identified that could promote survival of S. lugdunensis on human skin and pathogenesis of infections. Staphylococcus lugdunensis lacks virulence factors that characterize S. aureus and harbours a smaller number of genes encoding surface proteins. It is the only staphylococcal species other than S. aureus that possesses a locus encoding iron-regulated surface determinant (Isd) proteins involved in iron acquisition from haemoglobin.
Microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) are bacterial surface proteins mediating adherence of the microbes to components of the extracellular matrix of the host. On Staphylococci, the MSCRAMMs often have multiple ligands. Consequently, we hypothesized that the Staphylococcus aureus MSCRAMM bone sialoprotein-binding protein (Bbp) might recognize host molecules other than the identified bone protein. A ligand screen revealed that Bbp binds human fibrinogen (Fg) but not Fg from other mammals. We have characterized the interaction between Bbp and Fg. The binding site for Bbp was mapped to residues 561-575 in the Fg Aα chain using recombinant Fg chains and truncation mutants in Far Western blots and solid-phase binding assays. Surface plasmon resonance was used to determine the affinity of Bbp for Fg. The interaction of Bbp with Fg peptides corresponding to the mapped residues was further characterized using isothermal titration calorimetry. In addition, Bbp expressed on the surface of bacteria mediated adherence to immobilized Fg Aα. Also, Bbp interferes with thrombin-induced Fg coagulation. Together these data demonstrate that human Fg is a ligand for Bbp and that Bbp can manipulate the biology of the Fg ligand in the host.
A large range of debilitating medical conditions is linked to protein misfolding, which may compete with productive folding particularly in proteins containing multiple domains. Seventy-five per cent of the eukaryotic proteome consists of multidomain proteins, yet it is not understood how interdomain misfolding is avoided. It has been proposed that maintaining low sequence identity between covalently linked domains is a mechanism to avoid misfolding. Here we use single-molecule Förster resonance energy transfer to detect and quantify rare misfolding events in tandem immunoglobulin domains from the I band of titin under native conditions. About 5.5 per cent of molecules with identical domains misfold during refolding in vitro and form an unexpectedly stable state with an unfolding half-time of several days. Tandem arrays of immunoglobulin-like domains in humans show significantly lower sequence identity between neighbouring domains than between non-adjacent domains. In particular, the sequence identity of neighbouring domains has been found to be preferentially below 40 per cent. We observe no misfolding for a tandem of naturally neighbouring domains with low sequence identity (24 per cent), whereas misfolding occurs between domains that are 42 per cent identical. Coarse-grained molecular simulations predict the formation of domain-swapped structures that are in excellent agreement with the observed transfer efficiency of the misfolded species. We infer that the interactions underlying misfolding are very specific and result in a sequence-specific domain-swapping mechanism. Diversifying the sequence between neighbouring domains seems to be a successful evolutionary strategy to avoid misfolding in multidomain proteins.
Cell wall-associated (CWA) proteins made by Gram-positive pathogens play a fundamental role in pathogenesis. Staphylococcus pseudintermedius is a major animal pathogen responsible for the canine skin disease bacterial pyoderma. Here, we describe the bioinformatic
analysis of the family of 18 predicted CWA proteins encoded in the genome of S. pseudintermedius strain ED99 and determine their distribution among a phylogenetically diverse panel of S. pseudintermedius clinical isolates and closely related species of the Staphylococcus intermedius group. In parallel, we employed a proteomic approach to identify proteins presented on the surface of strain ED99 in vitro, revealing a total of 60 surface-localized proteins in one or more phases of growth, including 6 of the 18 genome-predicted
CWA proteins. Based on these analyses, we selected two CWA proteins (SpsD and SpsL) encoded by all strains examined and investigated
their capacity to mediate adherence to extracellular matrix proteins. We discovered that SpsD and SpsL mediated binding of
a heterologous host, Lactococcus lactis, to fibrinogen and fibronectin and that SpsD mediated binding to cytokeratin 10, a major constituent of mammalian skin. Of
note, the interaction with fibrinogen was host-species dependent, suggestive of a role for SpsD and SpsL in the host tropism
of S. pseudintermedius. Finally, we identified IgG specific for SpsD and SpsL in sera from dogs with bacterial pyoderma, implying that both proteins
are expressed during infection. The combined genomic and proteomic approach employed in the current study has revealed novel
host-pathogen interactions which represent candidate therapeutic targets for the control of bacterial pyoderma.
Clumping factor B (ClfB) from Staphylococcus aureus is a bifunctional protein that binds to human cytokeratin 10 (K10) and fibrinogen (Fg). ClfB has been implicated in S. aureus colonization of nasal epithelium and is therefore a key virulence factor. People colonized with S. aureus are at an increased risk for invasive staphylococcal disease. In this study, we have determined the crystal structures of the ligand-binding region of ClfB in an apo-form and in complex with human K10 and Fg α-chain-derived peptides, respectively. We have determined the structures of MSCRAMM binding to two ligands with different sequences in the same site showing the versatile nature of the ligand recognition mode of microbial surface components recognizing adhesive matrix molecules. Both ligands bind ClfB by parallel β-sheet complementation as observed for the clumping factor A·γ-chain peptide complex. The β-sheet complementation is shorter in the ClfB·Fg α-chain peptide complex. The structures show that several residues in ClfB are important for binding to both ligands, whereas others only make contact with one of the ligands. A common motif GSSGXG found in both ligands is part of the ClfB-binding site. This motif is found in many human proteins thus raising the possibility that ClfB recognizes additional ligands.
The SasG surface protein of Staphylococcus aureus has been shown to promote the formation of biofilm. SasG comprises an N-terminal A domain and repeated B domains. Here we
demonstrate that SasG is involved in the accumulation phase of biofilm, a process that requires a physiological concentration
of Zn2+. The B domains, but not the A domain, are required. Purified recombinant B domain protein can form dimers in vitro in a Zn2+-dependent fashion. Furthermore, the protein can bind to cells that have B domains anchored to their surface and block biofilm
formation. The full-length SasG protein exposed on the cell surface is processed within the B domains to a limited degree,
resulting in cleaved proteins of various lengths being released into the supernatant. Some of the released molecules associate
with the surface-exposed B domains that remain attached to the cell. Studies using inhibitors and mutants failed to identify
any protease that could cause the observed cleavage within the B domains. Extensively purified recombinant B domain protein
is very labile, and we propose that cleavage occurs spontaneously at labile peptide bonds and that this is necessary for biofilm
formation.
The current epidemic of hospital- and community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections has caused significant human morbidity, but a protective vaccine is not yet available. Prior infection with S. aureus is not associated with protective immunity. This phenomenon involves staphylococcal protein A (SpA), an S. aureus surface molecule that binds to Fcgamma of immunoglobulin (Ig) and to the Fab portion of V(H)3-type B cell receptors, thereby interfering with opsonophagocytic clearance of the pathogen and ablating adaptive immune responses. We show that mutation of each of the five Ig-binding domains of SpA with amino acid substitutions abolished the ability of the resulting variant SpA(KKAA) to bind Fcgamma or Fab V(H)3 and promote B cell apoptosis. Immunization of mice with SpA(KKAA) raised antibodies that blocked the virulence of staphylococci, promoted opsonophagocytic clearance, and protected mice against challenge with highly virulent MRSA strains. Furthermore, SpA(KKAA) immunization enabled MRSA-challenged mice to mount antibody responses to many different staphylococcal antigens.
Staphylococcus aureus and Streptococcus pyogenes, two important human pathogens, target host fibronectin (Fn) in their adhesion to and invasion of host cells1, 2. Fibronectin-binding proteins (FnBPs), anchored in the bacterial cell wall, have multiple Fn-binding repeats3 in an unfolded4, 5 region of the protein. The bacterium-binding site in the amino-terminal domain (1–5F1) of Fn contains five sequential Fn type 1 (F1) modules. Here we show the structure of a streptococcal (S. dysgalactiae) FnBP peptide (B3)6, 7 in complex with the module pair 1F12F1. This identifies 1F1- and 2F1-binding motifs in B3 that form additional antiparallel -strands on sequential F1 modules—the first example of a tandem -zipper. Sequence analyses of larger regions of FnBPs from S. pyogenes and S. aureus reveal a repeating pattern of F1-binding motifs that match the pattern of F1 modules in 1–5F1 of Fn. In the process of Fn-mediated invasion of host cells, therefore, the bacterial proteins seem to exploit the modular structure of Fn by forming extended tandem -zippers. This work is a vital step forward in explaining the full mechanism of the integrin-dependent2, 8 FnBP-mediated invasion of host cells.
Staphylococcus aureus binds to human desquamated nasal epithelial cells, a phenomenon likely to be important in nasal colonization. ClfB was identified previously as one staphylococcal adhesin that promoted binding to nasal epithelia. In this study, it is shown that the S. aureus surface protein SasG, identified previously by in silico analysis of genome sequences, and two homologous proteins, Pls of S. aureus and AAP of Staphylococcus epidermidis, also promote bacterial adherence to nasal epithelial cells. Conditions for in vitro expression of SasG by S. aureus were not found. Adherence assays were therefore performed with S. aureus and Lactococcus lactis expressing SasG from an expression plasmid. These studies showed that SasG did not bind several ligands typically bound by S. aureus. Significantly, SasG and Pls did promote bacterial adherence to nasal epithelial cells. Furthermore, pre-incubation of epithelial cells with purified recombinant proteins revealed that the N-terminal A regions of SasG, Pls and AAP, but not the B repeats of SasG, inhibited adherence of L. lactis expressing SasG in a dose-dependent fashion. These results suggest that SasG, Pls and AAP bind to the same as-yet-unidentified receptor on the surface of nasal epithelial cells. In addition, expression of SasG, like Pls, reduced adherence of S. aureus to fibronectin and fibrinogen.
Twenty-one genes encoding surface proteins belonging to the LPXTG family have been identified by in silico analysis of six Staphylococcus aureus genome sequences. Eleven genes encode previously described proteins, while 10 have not yet been characterized. Of these, eight contain the cell-wall sorting signal LPXTG responsible for covalently anchoring proteins to the cell-wall peptidoglycan. The remaining two, SasF and SasD, harbour a single residue variation in the fourth position of the LPXTG motif (LPXAG). Western blotting of lysostaphin-solubilized S. aureus cell-wall proteins demonstrated the release of SasF in the cell-wall fraction, indicating that proteins carrying LPXAG are sorted normally. Analysis of primary sequences of the Staphylococcus aureus surface (Sas) proteins indicated that several share a similar structural organization and a common signal sequence with previously characterized LPXTG proteins of S. aureus and other Gram-positive cocci. Protein SasG has 128 residue B repeats that are almost identical at the DNA level. PCR analysis indicated that recombinants with repeat length variations are present in the bacterial population whereas they are not detectable in the B-repeat-encoding region of sdrD. The sasG and sasH genes are significantly associated with invasive disease isolates compared to nasal carriage isolates. Several IgG samples purified from patients recovering from S. aureus infections had higher titres against Sas proteins than control IgG, suggesting that expression occurred during infection in some patients.
The interactions have been studied between an IgG-binding domain derivative based on domain B of staphylococcal protein A (designated Z) and human immunoglobulin G class 1 (IgG1) and its Fc fragment (Fc1) respectively. Five single amino acid substituted mutant forms of Z were constructed at the gene level, produced intracellularly in Escherichia coli, purified to homogeneity and characterized. Four of these Z variants, designated Z(L17D), Z(N28A), Z(I31A) and Z(K35A), were mutated in residues suggested to be involved in binding, based on the three-dimensional structure of the complex between a one domain protein A molecule and Fc1 [Deisenhofer, J. (1981) Biochemistry, 20, 2361-2370]. The fifth mutant protein, Z(F30A), had a mutation in a phenylalanine residue which was not expected to be involved in the interaction. Analysis by far UV circular dichroism spectroscopy suggests that all Z mutant proteins have similar folds. Their respective binding to human monoclonal IgG1 and to human recombinant Fc1 were studied in a competitive binding assay using radioactively labeled Z as a tracer, demonstrating that the mutant proteins with a substitution in the postulated binding surface showed a weakened binding to both the full-length antibody and the recombinant Fc1. The affinity constants of the interactions as well as relative binding free energies from the parent Z molecule were calculated. These values were similar for each Z variant to both IgG1 and Fc1, suggesting that Fc and not Fab binding was measured also for IgG1. However, the binding strengths differ significantly, and these binding properties were used to compare the contribution of each mutated amino acid residue in the Fc interaction. Z(F30A) was shown to have binding properties similar to the parent Z molecule. However, the substitution was shown to result in a dramatic effect on conformational stability of the mutant protein.
Staphylococcus aureus is a major human pathogen that can colonize the nasal cavity, skin, intestine, and oral cavity as a commensal bacterium.
gp340, also known as DMBT1 (deleted in malignant brain tumors 1), is associated with epithelial differentiation and innate
immunity. In the oral cavity, gp340 induces salivary aggregation with several oral bacteria and promotes bacterial adhesion
to tissues such as the teeth and mucosa. S. aureus is often isolated from the oral cavity, but the mechanism underlying its persistence in the oral cavity remains unclear.
In this study, we investigated the interaction between S. aureus and gp340 and found that S. aureus interacts with saliva- and gp340-coated resin. We then identified the S. aureus factor(s) responsible for binding to gp340. The cell surface protein SasA, which is rich in basic amino acids (BR domain)
at the N terminus, was responsible for binding to gp340. Inactivation of the sasA gene resulted in a significant decrease in S. aureus binding to gp340-coated resin. Also, recombinant SasA protein (rSasA) showed binding affinity to gp340, which was inhibited
by the addition of N-acetylneuraminic acid. Surface plasmon resonance analysis showed that rSasA significantly bound to the NeuAcα(2-3)Galβ(1-4)GlcNAc
structure. These results indicate that SasA is responsible for binding to gp340 via the N-acetylneuraminic acid moiety.
SignificanceUnder adverse environmental conditions, bacteria can form specialized antibiotic-resistant colonies called "biofilms." In Staphylococcus epidermidis biofilms, a protein, Aap, links bacterial cells together but does so only in the presence of zinc ions. We have determined the atomic structure of an adhesive portion of Aap bound to zinc. The protein adopts an elongated, flexible fold with zinc ions bridging two protein chains. The mode of assembly indicates that Aap is likely to form twisted rope-like structures between bacterial cells. These data provide clues about regions of the protein that could be targeted to prevent intercellular adhesion in the developing biofilm.
Staphylococcal immunoglobulin-binding protein, Sbi, is a 436-residue protein produced by many strains of Staphylococcus aureus. It was previously characterized as being cell surface-associated and having binding capacity for human IgG and β2-glycoprotein I. Here we show using small angle x-ray scattering that the proposed extracellular region of Sbi (Sbi-E) is
an elongated molecule consisting of four globular domains, two immunoglobulin-binding domains (I and II) and two novel domains
(III and IV). We further show that together domains III and IV (Sbi-III-IV), as well as domain IV on its own (Sbi-IV), bind
complement component C3 via contacts involving both the C3dg fragment and the C3a anaphylatoxin domain. Preincubation of human
serum with either Sbi-E or Sbi-III-IV is inhibitory to all complement pathways, whereas domain IV specifically inhibits the
alternative pathway. Monitoring C3 activation in serum incubated with Sbi fragments reveals that Sbi-E and Sbi-III-IV both
activate the alternative pathway, leading to consumption of C3. By contrast, inhibition of this pathway by Sbi-IV does not
involve C3 consumption. The observation that Sbi-E activates the alternative pathway is counterintuitive to intact Sbi being
cell wall-associated, as recruiting complement to the surface of S. aureus would be deleterious to the bacterium. Upon re-examination of this issue, we found that Sbi was not associated with the cell
wall fraction, but rather was found in the growth medium, consistent with it being an excreted protein. As such, our data
suggest that Sbi helps mediate bacterial evasion of complement via a novel mechanism, namely futile fluid-phase consumption.
Gram-positive pathogens such as staphylococci contain multiple cell wall-anchored proteins that serve as an interface between the microbe and its environment. Some of these proteins act as adhesins and mediate bacterial attachment to host tissues. SdrG is a cell wall-anchored adhesin from Staphylococcus epidermidis that binds to the Bbeta chain of human fibrinogen (Fg) and is necessary and sufficient for bacterial attachment to Fg-coated biomaterials. Here, we present the crystal structures of the ligand binding region of SdrG as an apoprotein and in complex with a synthetic peptide analogous to its binding site in Fg. Analysis of the crystal structures, along with mutational studies of both the protein and of the peptide, reveals that SdrG binds to its ligand with a dynamic "dock, lock, and latch" mechanism. We propose that this mechanism represents a general mode of ligand binding for structurally related cell wall-anchored proteins of gram-positive bacteria.
The ability of Staphylococcus aureus to invade mammalian cells may explain its capacity to colonize mucosa and to persist in tissues after bacteraemia. To date, the underlying molecular mechanisms of cellular invasion by S. aureus are unknown, despite its high prevalence and difficulties in treatment. Here, we show cellular invasion as a novel function for an S. aureus adhesin, previously implicated solely in attachment. S. aureus, but not S. epidermidis, invaded epithelial 293 cells in a temperature- and F-actin-dependent manner. Formaldehyde-fixed and live bacteria were equally invasive, suggesting that no active bacterial process was involved. All clinical S. aureus isolates analysed, but only a subset of laboratory strains, were invasive. Fibronectin-binding proteins (FnBPs) acted as S. aureus invasins, because: (i) FnBP deletion mutants of invasive laboratory strains lost invasiveness; (ii) expression of FnBPs in non-invasive strains conferred invasiveness; and (iii) the soluble isolated fibronectin-binding domain of FnBP (D1–D4) completely blocked invasion. Integrin α5β1 served as host cell receptor, which interacted with staphylococcal FnBPs through cellular or soluble fibronectin. FnBP-deficient mutants lost invasiveness for epithelial cells, endothelial cells and fibroblasts. Thus, fibronectin-dependent bridging between S. aureus FnBPs and host cell integrin α5β1 is a conserved mechanism for S. aureus invasion of human cells. This may prove useful in developing new therapeutic and vaccine strategies for S. aureus infections.
Staphylococcus aureus is responsible for a wide range of different infections ranging in severity from mild to fatal. However, it primarily exists as a commensal organism in a number of different anatomical sites including the nasopharynx. Although colonization itself is a harmless state, colonized individuals are at risk of endogenous infection when S. aureus enters otherwise sterile sites via wounds or indwelling medical devices. As such, studies of colonization may identify important targets for vaccines or other prophylactic approaches. Colonization is a dynamic process; S. aureus must attach to host surfaces, overcome immune components and compete with other commensal microbes. This occurs via a number of surface-attached and secreted proteins and other factors such as wall teichoic acid. In addition, colonizing S. aureus must constantly replicate to maintain its niche and exclude other strains. These myriad interactions provide a strong selective pressure for the maintenance or enhancement of mechanisms of adhesion, invasion and immune evasion. The evolutionary implications of this may explain why S. aureus is such a capable pathogen because many of the proteins involved in colonization have also been identified as virulence factors. This review describes the diverse molecular mechanisms used by S. aureus to colonize the host and discusses how the pressures that have selected for these may have led to its virulence.
Transition metals are essential nutrients to virtually all forms of life, including bacterial pathogens. In Staphylococcus aureus, metal ions participate in diverse biochemical processes such as metabolism, DNA synthesis, regulation of virulence factors, and defense against oxidative stress. As an innate immune response to bacterial infection, vertebrate hosts sequester transition metals in a process that has been termed "nutritional immunity." To successfully infect vertebrates, S. aureus must overcome host sequestration of these critical nutrients. The objective of this review is to outline the current knowledge of staphylococcal metal ion acquisition systems, as well as to define the host mechanisms of nutritional immunity during staphylococcal infection.
The unique redox potential of iron makes it an ideal cofactor in diverse biochemical reactions. Iron is therefore vital for the growth and proliferation of nearly all organisms, including pathogenic bacteria. Vertebrates sequester excess iron within proteins in order to alleviate toxicity and restrict the amount of free iron available for invading pathogens. Restricting the growth of infectious microorganisms by sequestering essential nutrients is referred to as nutritional immunity. In order to circumvent nutritional immunity, bacterial pathogens have evolved elegant systems that allow for the acquisition of iron during infection. The gram-positive extracellular pathogen Staphylococcus aureus is a commensal organism that can cause severe disease when it gains access to underlying tissues. Iron acquisition is required for S. aureus colonization and subsequent pathogenesis. Herein we review the strategies S. aureus employs to obtain iron through the production of siderophores and the consumption of host heme.
The fibronectin-binding proteins FnBPA and FnBPB are multifunctional adhesins than can also bind to fibrinogen and elastin. In this study, the N2N3 subdomains of region A of FnBPB were shown to bind fibrinogen with a similar affinity to those of FnBPA (2 μm). The binding site for FnBPB in fibrinogen was localized to the C-terminus of the γ-chain. Like clumping factor A, region A of FnBPB bound to the γ-chain of fibrinogen in a Ca2+-inhibitable manner. The deletion of 17 residues from the C-terminus of domain N3 and the substitution of two residues in equivalent positions for crucial residues for fibrinogen binding in clumping factor A and FnBPA eliminated fibrinogen binding by FnBPB. This indicates that FnBPB binds fibrinogen by the dock–lock–latch mechanism. In contrast, the A domain of FnBPB bound fibronectin with KD = 2.5 μm despite lacking any of the known fibronectin-binding tandem repeats. A truncate lacking the C-terminal 17 residues (latching peptide) bound fibronectin with the same affinity, suggesting that the FnBPB A domain binds fibronectin by a novel mechanism. The substitution of the two residues required for fibrinogen binding also resulted in a loss of fibronectin binding. This, combined with the observation that purified subdomain N3 bound fibronectin with a measurable, but reduced, KD of 20 μm, indicates that the type I modules of fibronectin bind to both the N2 and N3 subdomains. The fibronectin-binding ability of the FnBPB A domain was also functional when the protein was expressed on and anchored to the surface of staphylococcal cells, showing that it is not an artifact of recombinant protein expression.
Structured digital abstract
Staphylococcus aureus bacteraemia remains very difficult to treat, and a large proportion of cases result in potentially lethal metastatic infection. Unpredictable and persistent bacteraemia in the face of highly active, usually bactericidal antibiotics is the strongest predictor of death or disseminated disease. Although S. aureus has conventionally been considered an extracellular pathogen, much evidence demonstrates that it can survive intracellularly. In this Opinion article, we propose that phagocytes, and specifically neutrophils, represent a privileged site for S. aureus in the bloodstream, offering protection from most antibiotics and providing a mechanism by which the bacterium can travel to and infect distant sites. Furthermore, we suggest how this can be experimentally confirmed and how it may prompt a change in the current paradigm of S. aureus bacteraemia and identify better treatment options for improved clinical outcomes.
Iron is required for bacterial proliferation, and Staphylococcus aureus steals this metal from host hemoglobin during invasive infections. This process involves hemoglobin binding to the cell wall of S. aureus, heme extraction, passage through the cell envelope, and degradation to release free iron. Herein, we demonstrate an enhanced ability of S. aureus to bind hemoglobin derived from humans as compared to other mammals. Increased specificity for human hemoglobin (hHb) translates into an improved ability to acquire iron and is entirely dependent on the staphylococcal hemoglobin receptor IsdB. This feature affects host-pathogen interaction as demonstrated by the increased susceptibility of hHb-expressing mice to systemic staphylococcal infection. Interestingly, enhanced utilization of human hemoglobin is not a uniform property of all bacterial pathogens. These results suggest a step in the evolution of S. aureus to better colonize the human host and establish hHb-expressing mice as a model of S. aureus pathogenesis.
Shotgun phage display was used to identify a homolog of the IgG-binding protein staphylococcal protein A in Staphylococcus hyicus type strain CCUG 15602/ATCC 11249. This bacterium is the causative agent of exudative epidermitis in pigs and can also cause mastitis in cattle. A protein with similar features as the originally identified protein A in Staphylococcus aureus was described; an YSIRK-type signal peptide, four IgG-binding domains, a putative peptidoglycan-binding domain, and a cell wall anchoring motif (LPXTG) was present. The highest degree of similarity was to a protein A homolog in Staphylococcus pseudintermedius. However, typical Xr polypeptide repeats present in the protein A of S. aureus and S. pseudintermedius could not be identified in the protein A of S. hyicus. The presence of the spa gene in ten porcine and eight bovine clinical isolates of S. hyicus was investigated by PCR. In all isolates, the spa gene could be detected but the amplicons were of two sizes. Sequence analysis of four selected PCR amplicons showed that only three IgG-binding domains were present in the protein A of clinical isolates generating a smaller spa fragment. The finding of spa in S. hyicus contributes to an increased understanding of potential virulence factors in this species.
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains are causing a severe pandemic of mainly skin and soft tissue and occasionally fatal infections. The basis of their success is the combination of methicillin resistance at low fitness cost and high virulence. Investigation of the virulence potential of CA-MRSA, a key prerequisite for the development of anti-CA-MRSA therapeutics, has focused on strain USA300, which is responsible for the most serious CA-MRSA epidemic seen in the United States. Current data indicate that in this strain virulence evolved via increased expression of core-genome-encoded virulence determinants, such as alpha-toxin and phenol-soluble modulins, and acquisition of the phage-encoded Panton-Valentine leukocidin (PVL) genes. All these toxins impact disease progression in animal models of USA300 infection. In contrast, the basis of virulence in other CA-MRSA epidemics, which also include PVL-negative strains, is poorly understood.