Article

Siderophore-based immunization strategy to inhibit growth of enteric pathogens

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Abstract

Infections with Gram-negative pathogens pose a serious threat to public health. This scenario is exacerbated by increases in antibiotic resistance and the limited availability of vaccines and therapeutic tools to combat these infections. Here, we report an immunization approach that targets siderophores, which are small molecules exported by enteric Gram-negative pathogens to acquire iron, an essential nutrient, in the host. Because siderophores are nonimmunogenic, we designed and synthesized conjugates of a native siderophore and the immunogenic carrier protein cholera toxin subunit B (CTB). Mice immunized with the CTB-siderophore conjugate developed anti-siderophore antibodies in the gut mucosa, and when mice were infected with the enteric pathogen Salmonella, they exhibited reduced intestinal colonization and reduced systemic dissemination of the pathogen. Moreover, analysis of the gut microbiota revealed that reduction of Salmonella colonization in the inflamed gut was accompanied by expansion of Lactobacillus spp., which are beneficial commensal organisms that thrive in similar locales as Enterobacteriaceae. Collectively, our results demonstrate that anti-siderophore antibodies inhibit Salmonella colonization. Because siderophore-mediated iron acquisition is a virulence trait shared by many bacterial and fungal pathogens, blocking microbial iron acquisition by siderophore-based immunization or other siderophore-targeted approaches may represent a novel strategy to prevent and ameliorate a broad range of infections.

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... A promising strategy to target Enterobacteriaceae, including E. coli, is the development of siderophore-based vaccines (28,29). Siderophores are iron-chelating secondary metabolites that are biosynthesized under conditions of iron limitation (such as colitis), released by producers to scavenge iron from the environment, and then imported by dedicated uptake machinery to provide iron to the cell. ...
... We have previously demonstrated that immunization of mice with Ent conjugated to the mucosal adjuvant cholera toxin subunit B (CTB) elicits a strong mucosal immune response against microbe-derived Ent and GlcEnt and conferred protection against intestinal infection with Salmonella enterica serovar Typhimurium (29). Because Ent and GlcEnt are also key virulence factors for E. coli (30,39), and the genes for their synthesis and transport are expressed by AIEC in the inflamed gut (40), we hypothesized that immunization against Ent/GlcEnt may limit the AIEC bloom during colitis. ...
... During infection, Ent is captured by the host protein Lcn2, which limits Ent-mediated iron acquisition and hinders bacterial growth (37). We previously showed that intranasal immunization of mice with Ent conjugated to the mucosal adjuvant cholera toxin subunit B (CTB), hereafter CTB-Ent, elicited specific antibodies against both Ent and GlcEnt (29). Thus, we sought to investigate the role of CTB-Ent immunization in mice that lack a functional Lcn2. ...
Article
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Adherent-invasive Escherichia coli (AIEC) is abnormally prevalent in patients with ileal Crohn’s disease and exacerbates intestinal inflammation, but treatment strategies that selectively target AIEC are unavailable. Iron is an essential micronutrient for most living organisms, and bacterial pathogens have evolved sophisticated strategies to capture iron from the host environment.
... During evolution, host acute phase protein lipocalin, due to its potent binding ability to Ent, could counteract Ent-mediated iron acquisition and serve as a bacteriostatic innate immune effector against enteric bacterial pathogens (9). Recently, novel Ent conjugate vaccines have also been successfully developed to induce lipocalin-like Ent-specific antibodies in the host, which showed significant potential for broad applications to prevent and control various Gram-negative infections in humans and animals (10,11). In particular, we have developed an efficient method to prepare a new type of Ent conjugate vaccine that can induce high level of Ent-specific antibodies in rabbits (up to 4,096 fold increase) (10,12). ...
... More importantly, the Ent-specific antibodies have been demonstrated to function similarly as lipocalin to interfere with Ent-dependent growth of different Gram-negative pathogens (10,12). Notably, induction of Ent-specific antibodies appeared to be affected by the choice of animal hosts and vaccination regimens because another reported Ent conjugate vaccine only induced weak Entspecific immune response (< 4 fold increase) in the immunized mice (11). Given substantial differences in the genetic and immune systems in animal hosts (13,14), assessment of immunogenicity of the new Ent conjugate vaccine in different and important animal hosts, such as chickens, is highly desirable. ...
... Iron is a critical element for bacterial metabolism and essential for in vivo infections of pathogenic bacteria. Ent-mediated high affinity iron acquisition is a highly conserved and efficient system utilized by enteric bacteria for establishing successful colonization in host (11,36,37). In the past decades, several intervention strategies targeting the Ent utilization system have been explored, such as vaccines targeting surface-exposed receptors (38,39), specific inhibitors blocking Ent biosynthesis (40,41), and the "Trojan Horse" compounds in which specific antimicrobial was covalently coupled with Ent (42)(43)(44)(45)(46). ...
Article
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Passive immunization with specific egg yolk antibodies (immunoglobulin Y, IgY) is emerging as a promising alternative to antibiotics to control bacterial infections. Recently, we developed a novel conjugate vaccine that could trigger a strong immune response in rabbits directed against enterobactin (Ent), a highly conserved siderophore molecule utilized by different Gram-negative pathogens. However, induction of Ent-specific antibodies appeared to be affected by the choice of animal host and vaccination regimen. It is still unknown if the Ent conjugate vaccine can trigger a specific immune response in layers for the purpose of production of anti-Ent egg yolk IgY. In this study, three chicken vaccination trials with different regimens were performed to determine conditions for efficient production of anti-Ent egg yolk IgY. Purified Ent was conjugated to three carrier proteins, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) and CmeC (a subunit vaccine candidate), respectively. Intramuscular immunization of Barred Rock layers with KLH-Ent conjugate four times induced strong immune response against whole conjugate vaccine but the titer of Ent-specific IgY did not change in yolk with only a 4 fold increase detected in serum. In the second trial, three different Ent conjugate vaccines were evaluated in Rhode Island Red pullets with four subcutaneous injections. The KLH-Ent or CmeC-Ent conjugate consistently induced high level of Ent-specific IgY in both serum (up to 2,048 fold) and yolk (up to 1,024 fold) in each individual chicken. However, the Ent-specific immune response was only temporarily and moderately induced using a BSA-Ent vaccination. In the third trial, ten White Leghorn layers were subcutaneously immunized three times with KLH-Ent, leading to consistent and strong immune response against both whole conjugate and the Ent molecule in each chicken; the mean titer of Ent-specific IgY increased approximately 32 and 256 fold in serum and yolk, respectively. Consistent with its potent binding to various Ent derivatives, the Ent-specific egg yolk IgY also inhibited in vitro growth of a representative Escherichia coli strain. Together, this study demonstrated that the novel Ent conjugate vaccine could induce strong, specific, and robust immune response in chickens. The Ent-specific hyperimmune egg yolk IgY has potential for passive immune intervention against Gram-negative infections.
... Siderophore-mediated iron uptake system plays an important role in modulation of bacteria colonization and growth in the host [27]. Currently, three strategies were developed to inhibit siderophore biosynthesis or utilization: (1) screening inhibitors of siderophore biosynthetic enzymes as novel antimicrobials [28]; (2) design of siderophore-antimicrobial conjugates against antimicrobial resistance [29,30]; and (3) development of siderophore-based conjugate vaccines [23,31,32]. ...
... Siderophore-based immunization strategy is promising to inhibit growth of different Gram-negative pathogens, such as E. coli and Salmonella infections in mice [31,32]. In the study by Mike et al. [32], the siderophore conjugate vaccine failed to elicit any detectable siderophore-specific antibodies in mice. ...
... In the study by Mike et al. [32], the siderophore conjugate vaccine failed to elicit any detectable siderophore-specific antibodies in mice. Relevant to this work, Sassone-Corsi et al. [31] reported an Ent conjugate vaccine that was synthesized using a lengthy and complicated procedure; this Ent conjugate elicited weak mucosal response (i.e., <4-fold increase in Ent-specific antibodies) and no systemic response (IgG) in the immunized mice. In our recent study [23], we developed a new Ent conjugate vaccine using a simple and efficient protocol. ...
Article
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Campylobacter jejuni is the leading bacterial cause of human enteritis in developed countries. Chicken is the major animal reservoir of C. jejuni and a powerful infection model for human campylobacteriosis. No commercial vaccine against C. jejuni is available to date. The high affinity iron acquisition mediated through enterobactin (Ent), a small siderophore, plays a critical role in the colonization of C. jejuni in the intestine. Recently, an innovative Ent conjugate vaccine has been demonstrated to induce high-level of Ent-specific antibodies in rabbits; the Ent-specific antibodies displayed potent binding ability to Ent and inhibited Ent-dependent growth of C. jejuni. In this study, using specific-pathogen-free (SPF) chickens, we performed three trials to evaluate the immunogenicity of the Ent conjugate vaccine and its efficacy to control C. jejuni colonization in the intestine. The purified Ent was conjugated to the carrier keyhole limpet hemocyanin (KLH). Intramuscular immunization of chickens with the Ent–KLH conjugate for up to three times did not affect the body weight gain, the development of major immune organs and the gut microbiota. In the first two trials, immunizations of chickens with different regimens (two or three times of vaccination) consistently induced strong Ent-specific immune response when compared to control group. Consistent with the high-level of systemic anti-Ent IgG, C. jejuni colonization was significantly reduced by 3–4 log10 units in the cecum in two independent vaccination trials. The third trial demonstrated that single Ent–KLH vaccination is sufficient to elicit high level of systemic Ent-specific antibodies, which could persist for up to eight weeks in chickens. Taken together, the Ent–KLH conjugate vaccine could induce high-level of Ent-specific antibodies in chickens and confer host protection against C. jejuni colonization, which provides a novel strategy for Campylobacter control in poultry and humans.
... Recently, two independent studies using murine models of infection showed that immunization with siderophores conjugated to carrier proteins conferred protection in response to infections caused by enteric pathogens. [38,39] In one study, immunization of mice with the siderophores yersiniabactin and aerobactin, each conjugated to bovine serum albumin, conferred protection against uropathogenic E. coli. [38] In the other study, immunization of mice with enterobactin conjugated to the cholera toxin B subunit resulted in the production of mucosal IgA that binds enterobactin and salmochelin, and reduced intestinal colonization and systemic dissemination of S. Typhimurium. ...
... [38] In the other study, immunization of mice with enterobactin conjugated to the cholera toxin B subunit resulted in the production of mucosal IgA that binds enterobactin and salmochelin, and reduced intestinal colonization and systemic dissemination of S. Typhimurium. [39] Moreover, analysis of the gut microbiota revealed that reduction of S. Typhimurium colonization in the inflamed gut was accompanied by expansion of Lactobacillus spp., beneficial commensal organisms that thrive in similar locales as Enterobacteriaceae. [39] Outlook Siderophores have captivated interest at the interface of basic science and medicine for decades, resulting in consideration of these molecules and their derivatives for translational applications that include the prevention and treatment of microbial infections. ...
... [39] Moreover, analysis of the gut microbiota revealed that reduction of S. Typhimurium colonization in the inflamed gut was accompanied by expansion of Lactobacillus spp., beneficial commensal organisms that thrive in similar locales as Enterobacteriaceae. [39] Outlook Siderophores have captivated interest at the interface of basic science and medicine for decades, resulting in consideration of these molecules and their derivatives for translational applications that include the prevention and treatment of microbial infections. ...
Article
Infections caused by Gram-negative bacteria can be challenging to treat due to the outer membrane permeability barrier and the increasing emergence of antibiotic resistance. During infection, Gram-negative pathogens must acquire iron, an essential nutrient, in the host. Many Gram-negative bacteria utilize sophisticated iron acquisition machineries based on siderophores, small molecules that bind iron with high affinity. In this review, we provide an overview of siderophore-mediated iron acquisition in Enterobacteriaceae and how these systems provide a foundation for the conceptualization and development of approaches to prevent and/or treat bacterial infections. Differences between the siderophore-based iron uptake machineries of pathogenic Enterobacteriaceae and commensal microbes may lead to the development of selective “Trojan-horse” antimicrobials and immunization strategies that will not harm the host microbiota.
... Rather than focusing on iron acquisition-related large cellular targets (e.g., ironregulated outer membrane proteins), an innovative "out-of-the-box" strategy focusing on the extracellular Ent siderophore has been inspired by a unique antibacterial function of lipocalins (13)(14)(15)(16)(17) and further supported by a recent study on the evaluation of an Ent conjugate vaccine (18). Lipocalins are host acute-phase proteins secreted by neutrophils and epithelial cells during inflammation and rapid cell growth. ...
... Therefore, specific high-affinity antibodies directed against Ent, a hapten with a low molecular mass of 669 Da, could be theoretically obtained and such Ent-specific antibodies may display a bacteriostatic effect similar to that observed for lipocalins. This speculation has been partly supported by a recent publication showing that an Ent conjugate vaccine inhibited Salmonella colonization in mice (18). Nevertheless, in this recent study, the preparation of the Ent conjugate was lengthy and complicated and required the addition of a linker to Ent for its conjugation with carrier protein (18). ...
... This speculation has been partly supported by a recent publication showing that an Ent conjugate vaccine inhibited Salmonella colonization in mice (18). Nevertheless, in this recent study, the preparation of the Ent conjugate was lengthy and complicated and required the addition of a linker to Ent for its conjugation with carrier protein (18). In addition, the Ent-specific immune response was weak upon immunization of mice with the Ent conjugate (i.e., a Ͻ4-fold increase in Ent-specific antibodies) (18), highlighting the need to develop new type of Ent conjugate that is capable of inducing high levels of Ent-specific antibodies. ...
Article
Ent-mediated high-affinity iron acquisition is a universal and critical contributor for Gram-negative pathogens to survive and infect hosts. Published information has supported an innovative immune intervention strategy that directly targets Ent to starve pathogens by limiting the availability of iron to be utilized. Compared to a recently published Ent conjugate, there are three advantages of the vaccine described in this study: ease of preparation, induction of high titer of anti-Ent IgG, and the ability of Ent-specific antibodies to bind various Ent derivatives, including the salmochelins that help enteric pathogens evade sequestration of siderophores by host lipocalins. In addition, the Ent-specific antibodies were demonstrated to function similarly to lipocalin to interfere with the Ent-dependent growth of Campylobacter and E. coli under iron-restricted conditions. This study has significant potential for broader applications to prevent and control various Gram-negative infections in humans and animals.
... 13 As an example, immunogenic activity derived from cholera toxin-conjugated siderophores protected mice from Salmonella infection. 148 Furthermore, improved GI localization of metronidazole by conjugation to reutericyclin from Lactobacillus improved outcomes in a hamster model of CDI. 148,149 It is important to continue to improve targeted delivery mechanisms to the GI tract such that collateral damage to the human and microbiome are minimized. ...
... 148 Furthermore, improved GI localization of metronidazole by conjugation to reutericyclin from Lactobacillus improved outcomes in a hamster model of CDI. 148,149 It is important to continue to improve targeted delivery mechanisms to the GI tract such that collateral damage to the human and microbiome are minimized. Finally, combinations of existing antimicrobials have yielded surprisingly effective activity against recalcitrant pathogens such as heteroresistant bacteria. ...
Article
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Antimicrobial resistance is one of the largest threats to global health and imposes substantial burdens in terms of morbidity, mortality, and economic costs. The gut is a key conduit for the genesis and spread of antimicrobial resistance in enteric bacterial pathogens. Distinct bacterial species that cause enteric disease can exist as invasive enteropathogens that immediately evoke gastrointestinal distress, or pathobionts that can arise from established bacterial commensals to inflict dysbiosis and disease. Furthermore, various environmental reservoirs and stressors facilitate the evolution and transmission of resistance. In this review, we present a comprehensive discussion on circulating resistance profiles and gene mobilization strategies of the most problematic species of enteric bacterial pathogens. Importantly, we present emerging approaches toward surveillance of pathogens and their resistance elements as well as promising treatment strategies that can circumvent common resistance mechanisms.
... EIE89256.1) were obtained from the NCBI gene bank database (Table 1) (Carroll et al., 2017;Gebremariam et al., 2019;Sassone-Corsi et al., 2016;Sato et al., 2017;Sircar et al., 2012). All the antigens belong to Rhizopus delemar strain RA 99-880 except for hypothetical protein G6F43_000212 (Rhizopus delemar strain GL54). ...
... The hypothetical protein G6F43_000212 (KAG1057968.1) is also known as rhizoferrin biosynthesis protein which is a fungal NRPS-independent siderophore (NIS) enzyme secreted by R. delemar (Carroll et al., 2017). The infection of mice with CTB-siderophore results in antibody elicitation and reduction in intestinal colonization of salmonella (Sassone-Corsi et al., 2016). The endopeptidase (EIE89256.1) is associated with the allergen family of fungus and can induce IgE (Sircar et al., 2012). ...
Article
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Mucormycosis is a deadly fungal disease mainly caused by Rhizopus oryzae (strain 99–880), also known as Rhizopus delemar. Previously, mucormycosis occurs in immunocompromised patients of diabetes mellitus, cancer, organ transplant, etc. But there was a drastic increase in mucormycosis cases in the ongoing COVID-19 pandemic. Despite several available therapies and antifungal treatments, the mortality rate of mucormycosis is about more than 50%. Currently, there is no vaccine available in the market for mucormycosis that urgently needs to develop a potential vaccine against mucormycosis with high efficacy. In the present study, we have screened 4 genome-derived predicted antigens (GDPA) through sequential filtration of the whole proteome of R. delemar using different benchmarked bioinformatics tools. These 4 GDPA along with 4 randomly selected experimentally reported antigens (ERA) were sourced for prediction of B- and T- cell epitopes and utilized in designing of two potential multi-epitope vaccine candidates which can induce both innate and adaptive immunity against R. delemar. Besides these, comparative immune simulation studies and in silico cloning were performed using L. lactis as an expression system for their possible uses as oral vaccines. This is the first multi-epitope vaccine designed against R. delemar through systematic pipelined reverse vaccinology and immunoinformatic approaches. Although the wet-lab based experimental validation of designed vaccines is required before testing in the preclinical model, the current study will significantly help in reducing the cost of experimentation as well as improving the efficacy of vaccine therapy against mucormycosis and other pathogenic diseases.
... Mice immunised mice with siderophores conjugated to an immunogenic carrier protein were able to elicit a potent immune response and to protect against urinary tract infections. Mike et al. (2016) and Sassone-Corsi et al. (2016a) Mice immunised with a cholera toxin β-siderophore conjugate show a potent immune response and are able to protect against infection with Salmonella typhimurium. ...
... This will surely open the way for more targeted and personalised interventions to infectious diseases based on interference or corrections to the misbalances in the gut ecosystem and restoration of gut homeostasis. This could include siderophore-based immunisation strategies (Mike et al. 2016;Sassone-Corsi et al. 2016a), probiotic bacteria (e.g. E. coli strain Nissle) using similar iron-scavenging mechanisms than the invading pathogen (Deriu et al. 2013), probiotic strains consuming H 2 and hence restricting the use of this energy source for invading pathogens (Maier et al. 2013), the development of probiotic strains expressing bacteriocins or microcins targeting the pathogen (Kommineni et al. 2015;Hegarty et al. 2016;Sassone-Corsi et al. 2016b), expressing iron-sequestering mechanisms to inhibit invading pathogens (Vazquez-Gutierrez et al. 2016), siacylidase inhibitors (Huang et al. 2015) or inhibitors of anaerobic respiration (Winter and Bäumler 2014b) (see Table 1). ...
Article
Even though tremendous progress has been made in the last decades to elucidate the mechanisms of intestinal homeostasis, dysbiosis and disease, we are only at the beginning of understanding the complexity of the gut ecosystem and the underlying interaction networks. We are also only starting to unravel the mechanisms that pathogens have evolved to overcome the barriers imposed by the microbiota and host to exploit the system to their own benefit. Recent work in these domains clearly indicates that the 'traditional Koch's postulate', which state that a given pathogen leads to a distinct disease, are not valid for all 'infectious' diseases, but that a more complete and complex interpretation of the Koch's postulate is needed in order to understand and explain them.This review summarizes the current understanding of what defines a healthy gut ecosystem and highlights recent progress in uncovering the interplay between the host, its microbiota and invading intestinal pathogens. Based on these recent findings, we propose a new interpretation of the Koch's postulate, that we term 'ecological Koch's postulate'.
... [13][14][15][16][17] It seems logical that external interventions to disrupt iron acquisition and metabolism in pathogenic bacterial cells will shift the balance of power in the battle, and may constitute a novel approach to treating bacterial infections. [18][19][20][21] However, use of synthetic iron chelators for intracellular iron depletion to exploit this vulnerability has thus far met with limited success as the naturally occurring siderophores form iron chelates with higher thermodynamic stability than the synthetic iron-chelating ligands, [22][23][24][25][26] which makes it difficult for the latter to compete with siderophores and to displace iron from them. Furthermore, the typical synthetic iron chelators lack the ability to penetrate the bacterial cell membrane, which renders iron chelation therapy feasible only in lowering the systemic iron level in host vertebrates, but not in targeting the intracellular iron in bacteria for depletion. ...
Article
Biocompatible nanoparticles based on a calcium analogue of Prussian blue were designed and synthesized to take advantage of their ability to penetrate the cell membrane in Staphylococcus aureus and to undergo selective ion exchange with intracellular iron to disrupt iron metabolism in such pathogenic bacteria for antibacterial applications. KCa(H2O)2[FeIII(CN)6]⋅H2O nanoparticles penetrate the bacterial cell membrane and sequester intracellular iron by ion exchange to form insoluble Prussian blue, thus inhibiting bacterial growth.
... 31 The importance of this competition for trace metals is highlighted by observations demonstrating that immunization of mice to generate antibodies against iron-scavenging siderophores is sufficient to reduce the burden of Salmonella infection. 32 Bile salts. Bile salts are secreted into the small intestine by the host and function to increase fat solubilization and digestion. ...
Article
The communities of bacteria that reside in the intestinal tract are in constant competition within this dynamic and densely colonized environment. At homeostasis, the equilibrium that exists between these species and strains is shaped by their metabolism and also by pathways of active antagonism, which drive competition with related and unrelated strains. Importantly, these normal activities contribute to colonization resistance by the healthy microbiota, which includes the ability to prevent the expansion of potential pathogens. Disruption of the microbiota, resulting from, for example, inflammation or antibiotic use, can reduce colonization resistance. Pathogens that engraft following disruption of the microbiota are often adapted to expand into newly created niches and compete in an altered gut environment. In this review, we examine both the interbacterial mechanisms of colonization resistance and the strategies of pathogenic strains to exploit gaps in colonization resistance.
... Mice were then intragastrically inoculated with a Nissle 1917 DentB mutant as well as a S. Tm entB mutant (group 1). Because defects in siderophore production attenuate S. Tm virulence and gut colonization (Crouch et al., 2008;Raffatellu et al., 2009;Sassone-Corsi et al., 2016) (Figure 5C), we used lipocalin-2-deficient (Lcn2) mice on the C57BL/6 background. Lipocalin-2, a protein released by neutrophils and epithelial cells, sequesters enterobactin, thus impeding bacterial iron acquisition through enterobactin. ...
Article
During short-lived perturbations, such as inflammation, the gut microbiota exhibits resilience and reverts to its original configuration. Although microbial access to the micronutrient iron is decreased during colitis, pathogens can scavenge iron by using siderophores. How commensal bacteria acquire iron during gut inflammation is incompletely understood. Curiously, the human commensal Bacteroides thetaiotaomicron does not produce siderophores but grows under iron-limiting conditions using enterobacterial siderophores. Using RNA-seq, we identify B. thetaiotaomicron genes that were upregulated during Salmonella-induced gut inflammation and were predicted to be involved in iron uptake. Mutants in the xusABC locus (BT2063-2065) were defective for xenosiderophore-mediated iron uptake in vitro. In the normal mouse gut, the XusABC system was dispensable, while a xusA mutant colonized poorly during colitis. This work identifies xenosiderophore utilization as a critical mechanism for B. thetaiotaomicron to sustain colonization during inflammation and suggests a mechanism of how interphylum iron metabolism contributes to gut microbiota resilience.
... Anticalins: The Emerging Stealth Lipocalins Synthesizing a siderophore-based vaccine that is capable of inducing the adaptive immune system to generate anti-siderophore antibodies are interesting avenues for future study [91]. However, an emerging alternative to siderophore antibodies are anticalins, which are molecules "generated by combinatorial design from natural lipocalins" [92,93], including Lcn2 and tear lipocalin. ...
Article
Full-text available
Iron is necessary for the survival of almost all aerobic organisms. In the mammalian host, iron is a required cofactor for the assembly of functional iron-sulfur (Fe-S) cluster proteins, heme-binding proteins and ribonucleotide reductases that regulate various functions, including heme synthesis, oxygen transport and DNA synthesis. However, the bioavailability of iron is low due to its insolubility under aerobic conditions. Moreover, the host coordinates a nutritional immune response to restrict the accessibility of iron against potential pathogens. To counter nutritional immunity, most commensal and pathogenic bacteria synthesize and secrete small iron chelators termed siderophores. Siderophores have potent affinity for iron, which allows them to seize the essential metal from the host iron-binding proteins. To safeguard against iron thievery, the host relies upon the innate immune protein, lipocalin 2 (Lcn2), which could sequester catecholate-type siderophores and thus impede bacterial growth. However, certain bacteria are capable of outmaneuvering the host by either producing "stealth" siderophores or by expressing competitive antagonists that bind Lcn2 in lieu of siderophores. In this review, we summarize the mechanisms underlying the complex iron tug-of-war between host and bacteria with an emphasis on how host innate immunity responds to siderophores.
... Due to increased multidrug resistance, the WHO has included Salmonella in the list of the most serious infectious disease threats to human health. Salmonella has both siderophore-dependent and -independent strategies to acquire iron from the host (14). Salmonella synthesizes catecholate-type siderophores such as enterochelin and salmochelin, a C-glucosylated enterobactin, to capture and internalize ferric iron via siderophore receptors (15)(16)(17). ...
Article
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We have recently shown that the catecholamine dopamine regulates cellular iron homeostasis in macrophages. As iron is an essential nutrient for microbes, and intracellular iron availability affects the growth of intracellular bacteria, we studied whether dopamine administration impacts the course of Salmonella infections. Dopamine was found to promote the growth of Salmonella both in culture and within bone marrow-derived macrophages, which was dependent on increased bacterial iron acquisition. Dopamine administration to mice infected with Salmonella enterica serovar Typhimurium resulted in significantly increased bacterial burdens in liver and spleen, as well as reduced survival. The promotion of bacterial growth by dopamine was independent of the siderophore-binding host peptide lipocalin-2. Rather, dopamine enhancement of iron uptake requires both the histidine sensor kinase QseC and bacterial iron transporters, in particular SitABCD, and may also involve the increased expression of bacterial iron uptake genes. Deletion or pharmacological blockade of QseC reduced but did not abolish the growth-promoting effects of dopamine. Dopamine also modulated systemic iron homeostasis by increasing hepcidin expression and depleting macrophages of the iron exporter ferroportin, which enhanced intracellular bacterial growth. Salmonella lacking all central iron uptake pathways failed to benefit from dopamine treatment. These observations are potentially relevant to critically ill patients, in whom the pharmacological administration of catecholamines to improve circulatory performance may exacerbate the course of infection with siderophilic bacteria. IMPORTANCE Here we show that dopamine increases bacterial iron incorporation and promotes Salmonella Typhimurium growth both in vitro and in vivo. These observations suggest the potential hazards of pharmacological catecholamine administration in patients with bacterial sepsis but also suggest that the inhibition of bacterial iron acquisition might provide a useful approach to antimicrobial therapy.
... Siderophores have been used to develop vaccines and have been covered extensively (80)(81)(82)(83)(84). Bergeron and colleagues reported the use of a vibriobactin analogue linked to either ovalbumin (OVA) or bovine serum albumin (BSA) and their ability to promote antibody production in mice (85). ...
Article
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Different aspects of bacterial and fungal siderophore biotechnological applications will be discussed. Areas of application presented include, but are not limited to agriculture, medicine, pharmacology, bioremediation, biodegradation and food industry. In agriculture-related applications, siderophores could be employed to enhance plant growth due to their uptake by rhizobia. Siderophores hindered the presence of plant pathogens in biocontrol strategies. Bioremediation studies on siderophores discuss mostly the mobilization of heavy metals and radionuclides; the emulsifying effects of siderophore-producing microorganisms in oil-contaminated environments are also presented. The different applications found in literature based in medicine and pharmacological approaches range from iron overload to drug delivery systems and, more recently, vaccines. Additional research should be done in siderophore production and their metabolic relevance to have a deeper understanding for future biotechnological advances.
... S. Typhimurium-derived Omvs containing LPS and Omps known to serve as adjuvant are able to induce cross-protection against S. Choleraesuis and S. Enteritidis challenge (204,205). In the search of a multiprotective Ag, nasal immunization of mice with the enterobactin (a siderophore involved in iron acquisition by the bacteria and fungi) of Salmonella coupled with the immunogenic carrier protein cholera toxin subunit B induced a decrease of systemic invasion together with an increase of specific IgA (206). ...
Article
Salmonella enterica subspeciesentericaincludes several serovars infecting both humans and other animals and leading to typhoid fever or gastroenteritis. The high prevalence of associated morbidity and mortality, together with an increased emergence of multidrug-resistant strains, is a current global health issue that has prompted the development of vaccination strategies that confer protection against most serovars. Currently available systemic vaccine approaches have major limitations, including a reduced effectiveness in young children and a lack of cross-protection among different strains. Having studied host-pathogen interactions, microbiologists and immunologists argue in favor of topical gastrointestinal administration for improvement in vaccine efficacy. Here, recent advances in this field are summarized, including mechanisms of bacterial uptake at the intestinal epithelium, the assessment of protective host immunity, and improved animal models that closely mimic infection in humans. The pros and cons of existing vaccines are presented, along with recent progress made with novel formulations. Finally, new candidate antigens and their relevance in the refined design of anti-Salmonellavaccines are discussed, along with antigen vectorization strategies such as nanoparticles or secretory immunoglobulins, with a focus on potentiating mucosal vaccine efficacy.
... Strategies targeting siderophore acquisition of enteric pathogens have been tested as new therapeutic and preventive strategies (Sassone-Corsi et al., 2016). ...
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L’apport de fer est essentiel pour la plupart des organismes vivants, incluant la majorité des bactéries pathogènes. Cependant, le fer libre est toxique : il est lié à des protéines de stockage et de transport (e.g. ferritine, hémoprotéines…) et voit son homéostasie finement régulée. Afin d’extraire le fer de ces protéines, les bactéries utilisent divers systèmes tels que des protéines de surface ou encore des sidérophores. Bacillus cereus est une bactérie Gram-positive sporulante, pathogène opportuniste chez l’homme, 2ème cause en France de toxi-infection alimentaire collective. Chez B. cereus, la protéine de surface IlsA et le sidérophore bacillibactine (BB) sont impliqués dans l’acquisition du fer de la ferritine exogène et elles sont importantes pour l’infection de l’insecte modèle Galleria mellonella. Mes travaux présentaient deux parties : tout d’abord, l’étude de l’import du complexe BB-Fe3+ dans la cellule par FeuA, protéine de liaison de ce complexe à la surface de la bactérie, souligne le rôle central du couple BB-FeuA. La délétion des gènes codants pour ces deux molécules limite l’acquisition par B. cereus du fer de la ferritine, de l’hème, de l’hémoglobine et du fer inorganique in vitro. En revanche, elle présente un phénotype de virulence in vivo comparable à la souche de référence dans le cas d’injection intra-hémocœlique de larves de G. mellonella. Ce résultat surprenant suggère un probable rétrocontrôle sur l’expression de facteurs de virulence lorsque B. cereus ne produit ni BB ni FeuA, et se trouve par conséquent fortement carencé en fer. Le second volet de mes travaux s’intéresse à l’expression des gènes liés à l’homéostasie du fer in vivo, au cours de l’infection de l’intestin de larves de G. mellonella axéniques. Nous avons choisi une approche de type microgénomique, en prélevant les échantillons par microdissection laser, sur de façon à prélever de petits échantillons dans une zone définie, puis en analysant l’expression de quelques gènes ciblés par RT-qPCR et ddPCR à 3h et 16h post ingestion. Nos résultats montrent que : i) la colonisation intestinale de G. mellonella est impactée lorsque B. cereus est dépourvu du couple BB-FeuA ; ii) ilsA est exprimé lors de l’infection intestinale ; iii) les gènes ciblés impliqués dans l’homéostasie du fer sont activés dès le début de l’infection, suggérant un rôle dans l’adaptation et la pathogénicité ; iv) une faible modulation de l’expression est observée entre les deux temps. Ces travaux ouvrent de nouvelles connaissances fondamentales sur l’homéostasie du fer et des perspectives quant à l’utilisation de nouvelles techniques pour l’étude in situ des interactions hôte-pathogène.
... For example, Ent was conjugated to the cholera toxin subunit Ba sa ni mmunogenic carrier protein and applied in a mousem odel to elicit IgA antibodies both against the Ent hapten and the structurally relateds almochelins. [38] Indeed, such immunized mice contained fewer Salmonella in their in- testines when infectedw ith this bacterial pathogen, apparently due to the neutralization of the salmochelin stealth siderophore. Similarly,avaccine based on yersiniabactin and aerobactin was shown to protect against ap athogenic strain of E. coli in amouse model of urinary tract infection. ...
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Iron acquisition mediated by siderophores, high‐affinity chelators for which bacteria have evolved specific synthesis and uptake mechanisms, plays a crucial role in microbiology and in host‐pathogen interactions. In the ongoing fight against bacterial infections this area has attracted biomedical interest. Beyond several approaches to interfere with the siderophore‐mediated iron uptake from medicinal and immuno‐chemistry, the development of high‐affinity protein scavengers that tightly complex siderophores produced by pathogenic bacteria has appeared as a novel strategy. Such binding proteins were engineered based on siderocalin – also known as lipocalin 2 – an endogenous human scavenger of enterobactin and bacillibactin which controls the systemic spreading of commensal bacteria such as Escherichia coli . Using combinatorial protein design, siderocalin was reshaped to bind several siderophores from Pseudomonas aeruginosa and, in particular, petrobactin from Bacillus anthracis , all of which are not recognized by the natural protein. Such engineered versions of siderocalin effectively suppress growth of corresponding pathogenic bacteria by deprivation from iron supply and bear potential to complement antibiotic therapy in situations of acute or persistent infection.
... In a Gramnegative bacterium such as E. coli K-12, these three proteins are thought to function in concert to regulate the movement of ferrous iron into the cytosol to be incorporated into the intracellular labile iron pool (Fig. 1B). 7 Although ferric siderophore-and heme-transport systems have been historically recognized as important contributors to bacterial virulence, [11][12][13] emerging evidence demonstrates that ferrous iron contributes significantly to the establishment of infection by a wide array of pathogens within mammalian hosts. For example, FeoA and FeoB knockouts in model pathogens have decreased or abrogated growth of several strains. ...
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The acquisition of iron is essential to establishing virulence among most pathogens. Under acidic and/or anaerobic conditions, most bacteria utilize the widely-distributed ferrous iron (Fe²⁺) uptake (Feo) system to import metabolically-required iron. The Feo system is inadequately understood at the atomic, molecular, and mechanistic levels, but we do know it is composed of a main membrane component (FeoB) essential for iron translocation, as well as two small, cytosolic proteins (FeoA and FeoC) hypothesized to function as accessories to this process. FeoC has many hypothetical functions, including that of an iron-responsive transcriptional regulator. Here, we demonstrate for the first time that Escherichia coli FeoC (EcFeoC) binds an [Fe-S] cluster. Using electronic absorption, X-ray absorption, and electron paramagnetic resonance spectroscopies, we extensively characterize the nature of this cluster. Under strictly anaerobic conditions after chemical reconstitution, we demonstrate that EcFeoC binds a redox-active [4Fe-4S]2+/+ cluster that is rapidly oxygen-sensitive and decays to a [2Fe-2S]²⁺ cluster (t½ ≈ 20 s), similar to the [Fe-S] cluster in the fumarate and nitrate reductase (FNR) transcriptional regulator. We further show that this behavior is nearly identical to the homologous K. pneumoniae FeoC, suggesting a redox-active, oxygen-sensitive [4Fe-4S]²⁺ cofactor is a general phenomenon of cluster-binding FeoCs. Finally, in contrast to FNR, we show that [4Fe-4S]²⁺ cluster binding to FeoC is associated with modest conformational changes of the polypeptide, but not protein dimerization. We thus posit a working hypothesis in which the cluster-binding FeoCs may function as oxygen-sensitive iron sensors that fine-tune pathogenic ferrous iron acquisition.
... Interestingly, some Bacillus species produce petrobactin, a hybrid NRPS-NIS stealth siderophore. 1 The necessity of micromolar concentrations of iron to support microbial growth makes siderophore production an important phenotype of many bacteria. Targeting siderophore systems has therefore been examined from many angles for potential antivirulence treatment approaches, including obstructing ferric siderophore import systems, 5 blocking siderophore uptake and cycling with rigidified analogues and mimetics, 6 neutralization via siderophorebased immunization strategies, 7,8 increasing drug potency and specificity through siderophore-antibiotic "Trojan horse" conjugates, 9 and antagonizing siderophore biosynthetic enzymes. 10 Multiple studies support the strategy of targeting siderophore systems by demonstrating that genetic or chemical disruption can result in the attenuation of microbial growth and virulence under iron-limiting conditions. ...
Article
Acquiring sufficient quantities of iron to support survival is often a critical limitation for pathogenic bacteria. To meet this demand, bacteria have evolved unique strategies to scavenge iron and circumvent the nutritional immunity exerted by their hosts. One common strategy, which is often a key virulence factor for bacterial pathogens, involves the synthesis, secretion, and reuptake of iron chelators known as siderophores. In vitro and in vivo studies have demonstrated that the siderophore aerobactin is critical for virulence in the hypervirulent pathotype of Klebsiella pneumoniae (hvKP). Given the high rate of multidrug resistance in K. pneumoniae, and in light of the ever-increasing demand for novel Gram-negative therapeutic targets, we identified aerobactin production as a promising antivirulence target in hvKP. Herein, we describe the development of a high-throughput biochemical assay for identifying inhibitors of the aerobactin synthetase IucA. The assay was employed to screen ~110,000 compounds across several commercially available small-molecule libraries. IucA inhibitors with activity at micromolar concentrations were identified in our screening campaigns and confirmed using secondary orthogonal assays. However, the most potent compounds also exhibited some properties commonly observed with promiscuous/nonspecific inhibitors, including incubation time and target enzyme concentration dependence, as well as the potential to antagonize unrelated enzymes.
... Importantly, this approach supported, rather than undermined, the non-pathogenic role of commensal microbiota. 174 Alternatively, some investigators have proposed mirroring the mechanism of action of bacterially produced microcins and sideromycins, and exploiting siderophores as vehicles to facilitate the delivery of conjugated antibiotics to pathogens, overcoming resistance mechanisms such as enhanced drug efflux activity. 170,175 Finally, siderophore-based therapies extend to exploiting probiotic bacteria that can more effectively compete with Salmonella for iron. ...
Article
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Iron is an essential element for almost all living organisms, which can also be extremely toxic in high concentrations. All organisms must therefore employ homeostatic mechanisms to finely regulate iron uptake, usage and storage in the face of dynamic environmental conditions. The critical step in mammalian systemic iron homeostasis is the fine regulation of dietary iron absorption. However, since the gastrointestinal system is also home to >1014 bacteria, all of which engage in their own programs of iron homeostasis, the gut represents an anatomical location where the inter‐kingdom fight for iron is never‐ending. Here, we explore the molecular mechanisms of, and interactions between, host and bacterial iron homeostasis in the gastrointestinal tract. We first detail how mammalian systemic and cellular iron homeostasis influences gastrointestinal iron availability. We then focus on two important human pathogens, Salmonella and Clostridia; despite their differences, they exemplify how a bacterial pathogen must navigate and exploit this web of iron homeostasis interactions to avoid host nutritional immunity and replicate successfully. We then reciprocally explore how iron availability interacts with the gastrointestinal microbiota, and the consequences of this on mammalian physiology and pathogen iron acquisition. Finally, we address how understanding the battle for iron in the gastrointestinal tract might inform clinical practice and inspire new treatments for important diseases.
... Taking advantage of widely conserved Salmonella antigens may offer one potential solution to increase the cross-reactivity of vaccine candidates. Outer membrane proteins (eg, OmpC, OmpD, OmpF) [32,33], siderophores (enterobactin) [34], and type III secretion system proteins (eg, SipB, SipD, SseB, SseC, and PrgI) [35][36][37] have been explored as vaccine antigens in mice, demonstrating robust immunogenicity and protective efficacy against homologous strains. Alternatively, access to conserved membrane antigens can be enhanced for live-attenuated and OMV platforms. ...
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Nontyphoidal Salmonella and Salmonella Paratyphi are responsible for significant morbidity and mortality worldwide. To date, no vaccine has been licensed against these organisms. The development of effective vaccines remains an urgent priority. In this review, the rationale for and current status of various vaccine candidates against S. Paratyphi and nontyphoidal Salmonella are presented, with a focus on the research findings from the 2019 International Conference on Typhoid and Other Invasive Salmonelloses. Additionally, other vaccine candidates that are currently undergoing clinical development are highlighted. Future approaches, which may include antigens that are genetically conserved across Salmonella and confer broad, non-serotype-specific protection, are also discussed.
... To this end, immunization against yersiniabactin and aerobactin in the context of UTIs protected from systemic infection with UPEC (Fig. 4a) (Mike et al., 2016). Similarly, immunization with the siderophore enterobactin, which resulted in the production of antibodies against both enterobactin and salmochelin, protected against intestinal Salmonella infection (Fig. 4a) (Sassone-Corsi et al., 2016a). Intriguingly, the reduced Salmonella burden in enterobactin-immunized mice was paralleled by increased levels of Lactobacillus spp. ...
Article
Iron is an essential micronutrient for nearly all living organisms. In addition to facilitating redox reactions, iron is bound by metalloproteins that participate in a variety of biological processes. As the bioavailability of free iron in host environments is extremely low, iron lies at the center of a battle for nutrients between microbes and their host. Mucosal surfaces such as the respiratory and gastrointestinal tracts are constantly exposed to commensal and pathogenic microorganisms. Whereas a key strategy of mammalian antimicrobial defense is to deprive microbes of iron, pathogens and some commensals have evolved effective strategies to circumvent iron limitation. Here we provide an overview of mechanisms underpinning the tug-of-war for iron between microbes and their host, with a particular focus on mucosal surfaces.
... Likewise, gut microbiota and probiotic bacteria are reported to be involved in iron uptake in the gut, thereby preventing sustainable colonization of several bacterial species like Salmonella [28,29]. Strategies targeting siderophore acquisition of enteric pathogens have been tested as new therapeutic and preventive strategies [30]. ...
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Bacillus cereus is a Gram-positive opportunistic pathogen closely related to the entomopathogen, Bacillus thuringiensis, both of which are involved in intestinal infections. Iron is an essential micronutrient for full growth and virulence of pathogens during infection. However, little is known about iron homeostasis during gut infection. Therefore, we aimed to assess the expression of B. cereus genes related to bacterial iron homeostasis, virulence and oxidative stress. The hypothesis is that the expression of such genes would vary between early and later stage colonization in correlation to gut cell damage. To perform the study, a germ-free Galleria mellonella model was set up in order to adapt the use of Laser-capture microdissection (LCM), to select precise areas in the gut lumen from frozen whole larval cryo-sections. Analyses were performed from alive larvae and the expression of targeted genes was assessed byspecific pre-amplification of mRNA followed by quantitative PCR. Firstly, the results reinforce the reliability of LCM, despite a low amount of bacterial RNA recovered. Secondly, bacterial genes involved in iron homeostasis are expressed in the lumen at both 3 and 16 hours post force-feeding. Thirdly, iron gene expression is slightly modulated during gut infection, and lastly, the mRNA of G. mellonella encoding for ferritin and transferrin iron storage and transport are recovered too. Therefore, iron homeostasis should play a role in B. cereus gut colonization. Furthermore, we demonstrate for the first time the value of using LCM for specific in situ gene expression analysis of extracellular bacteria in a whole animal.
... Iron acquisition genes have a well-known and extensively studied association with uropathogenesis (116,121,122,124,(134)(135)(136)(137). Indeed, vaccines targeting siderophores have been proven as a concept in animal models, including mouse models of both E. coli urinary tract infection (UTI) and intestinal colonization by Salmonella (138)(139)(140)(141)(142)(143)(144)(145). While some of these studies showed that protective antibodies against siderophores can be generated, the results are not as efficacious as one would hope. ...
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Comparative genomics of bacterial pathogens has been useful for revealing potential virulence factors. Escherichia coli is a significant cause of human morbidity and mortality worldwide but can also exist as a commensal in the human gastrointestinal tract. With many sequenced genomes, it has served as a model organism for comparative genomic studies to understand the link between genetic content and potential for virulence. To date, however, no comprehensive analysis of its complete "virulome" has been performed for the purpose of identifying universal or pathotype-specific targets for vaccine development. Here, we describe the construction of a pathotype database of 107 well-characterized completely sequenced pathogenic and non-pathogenic E. coli strains, which we annotated for major virulence factors (VFs). Data are cross referenced for patterns against pathotype, phylogroup, and sequence type and results verified against all 1,348 complete E. coli chromosomes in the NCBI RefSeq database. Our results demonstrate that phylogroup drives many of the "pathotype-associated" VFs, and ExPEC-associated VFs are found predominantly within the B2/D/F/G phylogenetic clade, suggesting these phylogroups are more adapted to infect human hosts. Finally, we used this information to propose polyvalent vaccine targets with specificity towards extraintestinal strains, targeting key invasive strategies including immune evasion (group 2 capsule), iron acquisition (FyuA, IutA, Sit), adherence (SinH, Afa, Pap, Sfa, Iha), and toxins (Usp, Sat, Vat, Cdt, Cnf1, HlyA). While many of these targets have been proposed before, this work is the first to examine their pathotype and phylogroup distribution and how they may be targeted together to prevent disease.
... Through the many years since, considerable effort has been invested in understanding the biological metal uptake, efflux and regulatory elements in Salmonella. This ground-breaking body of work demonstrated the immense complexity of metal availabilities across mammalian tissues as well as its necessity for pathogenesis during infection (Diaz-Ochoa et al. 2016;Sassone-Corsi et al. 2016;Huang et al. 2017;Frawley et al. 2018;Cunrath and Bumann 2019). Despite these successes, however, experimentation to reveal the relative importance of each of the biological metals have utilized highly variable mouse models, genetically different Salmonella genotypes and various modes of infection, which have subsequently delivered occasional contradictory and confusing results. ...
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Nutritional immunity is a powerful strategy at the core of the battlefield between host survival and pathogen proliferation. A host can prevent pathogens from accessing biological metals such as Mg, Fe, Zn, Mn, Cu, Co or Ni, or actively intoxicate them with metal overload. While the importance of metal homeostasis for the enteric pathogen Salmonella enterica Typhimurium was demonstrated many decades ago, inconsistent results across various mouse models, diverse Salmonella genotypes, and differing infection routes challenge aspects of our understanding of this phenomenon. With expanding access to CRISPR-Cas9 for host genome manipulation, it is now pertinent to re-visit past results in the context of specific mouse models, identify gaps and incongruities in current knowledge landscape of Salmonella homeostasis, and recommend a straight path forward towards a more universal understanding of this historic host-microbe relationship.
Chapter
Many Trichoderma species can grow in soil and for long years; several species of this genus have been applied against many plant pathogens for eco-friendly biocontrol of fungal diseases in agrifields. The genus includes more than 300 species. Over the years, researchers revealed that more than 1000 bioactive compounds have been estimated to be produced by this genus. These metabolites are chemically diverse compounds, viz., non-ribosomal peptides (NRPs), such as antibiotic peptides known as peptaibols (peptaibiotics), siderophores and diketopiperazines-like gliotoxin and gliovirin, polyketides, terpenes, pyrones, and isocyanate metabolites; enzymes, fatty acids; etc. Due to climatic changes and anthropogenic activities, we are facing some fatal diseases and some human pathogenic bacteria, fungi, etc. are becoming multidrug resistant; similarly, diabetes and cancer diseases are on the way to become epidemic, threatening our existence. To combat these fatal diseases, scientists are searching new bioactive compounds for development of next-generation drugs. In this juncture, Trichoderma and its derived compounds can serve, or serving human society for combating those fatal ailments is now a major concern among scientists. In this book chapter, we will discuss briefly the utilization of Trichoderma-derived compounds in medical science for human welfare.
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Biocompatible nanoparticles based on a calcium analogue of Prussian blue were designed and synthesized to take advantage of their ability to penetrate the cell membrane in Staphylococcus aureus and to undergo selective ion exchange with intracellular iron to disrupt iron metabolism in such pathogenic bacteria for antibacterial applications. KCa(H2O)2[FeIII(CN)6]⋅H2O nanoparticles penetrate the bacterial cell membrane and sequester intracellular iron by ion exchange to form insoluble Prussian blue, thus inhibiting bacterial growth.
Chapter
The overuse of broad-spectrum antibiotics rapidly selects for dangerous multi-drug resistant bacterial pathogens. The landscape of antibiotic drug discovery is adapting to this wave of resistance with a movement towards narrow-spectrum, pathogen-targeted antibiotics that limit the emergence of new resistance. Sideromycins (siderophore-antibiotic conjugates) exploit essential iron acquisition pathways to achieve receptor-mediated cell entry where the spectrum of antibiotic activity is determined by highly selective cell surface siderophore receptors rather than the widely distributed and highly conserved antibacterial target. Sideromycins overwhelm traditional resistance mechanisms through high intracellular antibiotic concentrations and resistance adaptation renders pathogens avirulent. The timing is optimal to pursue sideromycins as pathogen-targeted antibiotics and chemical probes for rapid pathogen diagnostics.
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A direct and selective functionalization of aerobactin has been described. The selectivity was achieved by masking the functioning carboxylate through iron-chelation, leaving two remaining carboxylates for direct modification. Both mono-...
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Plantaricin EF (PlnEF) is a class IIb bacteriocin produced by Lactobacillus plantarum. We compared L. plantarum NCIMB8826 and LM0419, a plnEFI deletion mutant of that strain lacking plnEF and the gene for the cognate immunity protein plnI, in a 2,4,6-trinitrobenzenesulfonic acid (TNBS) induced mouse model of acute inflammatory bowel disease. Mice fed either L. plantarum NCIMB8826 or LM0419 were not protected against TNBS according to either disease activity or histology (Ameho) scores. Mice consuming NCIMB8826 exhibited intermediate (non-significant) levels of colonic tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) that ranged between the TNBS-treated animals and healthy controls. By comparison, TNF-α and IL-6 quantities were elevated in mice given L. plantarum LM0419 and equivalent to mice given TNBS alone. Both strains survived digestive tract transit in equal numbers and did not result in global changes to the bacterial composition in the intestine according to 16S rRNA gene sequencing either prior to or after TNBS administration. Examination of intestinal taxa showed that mice consuming wild-type L. plantarum, but not LM0419 contained lower proportions of Mucispirillum (Deferribacteres phylum) in the faeces prior to TNBS administration and Parabacteroides (Bacteroidetes phylum) in the caecum after disease induction. Parabacteroides also positively correlated with disease activity and histology scores. These findings suggest a role for PlnEFI production by L. plantarum in benefiting digestive tract health.
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Staphylopine (StP) and other nicotianamine-like metallophores are crucial for many pathogens to acquire the transition metals from hosts during invasion. CntL from Staphylococcus aureus (SaCntL) catalyzes the condensation of the 2-aminobutyrate (Ab) moiety of S-adenosylmethionine (SAM) with D-histidine in the biosynthesis of StP. Here, we report the crystal structures of SaCntL in complex with either SAM or two products. The structure of SaCntL consists of an N-terminal four-helix bundle (holding catalytic residue E84) and a C-terminal Rossmann fold (binding the substrates). The sequence connecting the N- and C-terminal domains (N-C linker) in SaCntL was found to undergo conformational alternation between open and closed states. Our structural and biochemical analyses suggested that this intrinsically dynamic interdomain linker forms an additional structural module that plays essential roles in ligand diffusion, recognition, and catalysis. We confirmed that SaCntL stereoselectively carries out the catalysis of D-His but not its enantiomer, L-His, and we found that the N-C linker and active site of SaCntL could accommodate both enantiomers. SaCntL is likely able to bind L-His without catalysis, and as a result, L-His could show inhibitory effects toward SaCntL. These findings provide critical structural and mechanistic insights into CntL, which facilitates a better understanding of the biosynthesis of nicotianamine-like metallophores and the discovery of inhibitors of this process.
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Siderophore natural products are characterized by an ability to tightly chelate metals. The origins of such compounds are often pathogenic microbes utilizing siderophores as virulence factors during host infection. The mechanism for siderophore formation typically involves the activity of nonribosomal peptide synthetases producing compounds across functional group classifications that include catecholate, phenolate, hydroxamate, and mixed categories. Though siderophore production has been a hallmark of pathogenicity, the evolutionarily-optimized binding abilities of siderophores suggest the possibility of re-directing the compounds towards alternative beneficial applications. In this mini-review, we will first describe siderophore formation origins before discussing alternative applications as pharmaceutical products. In so doing, we will cover examples and applications that include reducing metal overload, targeted antibiotic delivery, cancer treatment, vaccine development, and diagnostics. Included in this analysis will be a discussion on the native production hosts of siderophores and prospects for improvement in compound access through the adoption of heterologous biosynthesis.
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The pathogen Salmonella enterica is a leading cause of infection worldwide. Nontyphoidal Salmonella (NTS) serovars typically cause inflammatory diarrhea in healthy individuals, and can cause bacteremia in immunocompromised patients, children, and the elderly. Management of NTS infection poses a challenge because antibiotic treatment prolongs fecal shedding of the pathogen and is thus not recommended for most patients. In recent years, the emergence of antibiotic resistance in NTS has also become a major issue. Thus, new therapeutic strategies to target NTS are needed. Here, we evaluated whether six siderophore-β-lactam conjugates based on enterobactin (Ent) and salmochelin S4 (digulcosylated Ent, DGE) provide antimicrobial activity against the two highly prevalent NTS serovars Typhimurium and Enteritidis by targeting the siderophore receptors FepA and/or IroN. The conjugates showed 10- to 1000-fold lower minimum inhibitory concentrations against both serovars Typhimurium and Enteritidis compared to the parent antibiotics under iron limitation and were recognized and transported by FepA and/or IroN. NTS treated with the Ent/DGE-β-lactam conjugates exhibited aberrant cellular morphologies suggesting inhibition of penicillin-binding proteins, and the conjugates selectively killed NTS in coculture with Staphylococcus aureus. Lastly, the DGE-based conjugates proved to be effective at inhibiting growth of NTS in the presence of the Ent-sequestering protein lipocalin-2. This work describes the successful use of siderophore-antibiotic conjugates against NTS and highlights the opportunity for narrowing the activity spectrum of antibiotics by using Ent and DGE to target enteric bacterial pathogens.
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The defeat of the fruits of fungal diseases is currently an important issue of plant science and is also of great economic importance. With the help of microscopic methods the leaves and fruits surface tissues of plants of four genera of the Maloideae subfamily were screened: Malus Mill., Pyrus L., Cydonia Mill., Mespilus L. and attempts were made to explain the dependence of mycosis damage on micro structural features. The species composition of fungi that cause damage to the Maloideae leaves and fruits in the Russia southern regions is analyzed. It is established that among pathogens with different types of parasitism there are common excitants, as well as highly specialized responses as on Mespilus germanica L. Higher resistance to the complex of fungal diseases, in comparison with apple and pear, was found in quince and medlar. This stability at the initial stage of the pathological process is associated with structural features such as micro morphology of the fruits and stomata cuticle in the abaxial epidermis of leaves. The leaves stomatal openings of medlar are narrow with raised outgrowths, on the surface of the fruits-the layered structure of the cuticular layer. Quince has a continuous cuticular cover. In the species least affected by mycoses, a high content of very-long-chain fatty acids in the external tissues was revealed, which may be one of the factors of resistance to pathogens. In addition, the studied species revealed differences in the content of polyphenols, which can inhibit the development of pathogens at the stage of their penetration. Thus, during the study, using the example of the Maloideae subfamily, we identified several factors of passive immunity of plants. Conventionally, they can be divided into two groups: mechanical and chemical, working at various stages of pathogen penetration into plant organism.
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In recent years, the alarming increase of antibiotic resistance, compounded by the simultaneous decrease in development of new antibiotics, has created serious concerns for public health. Moreover, current antibiotics also target the beneficial commensal microbes (microbiota) that inhabit our body, sometimes with significant health consequences. The answer to the antibiotic crisis thus involves broad, creative efforts to develop new treatments for infectious agents. Here I discuss what can be learned from investigating microbial competition in vivo and how this knowledge can be utilized to devise new narrow-spectrum therapeutics that target bacterial pathogens while minimizing deleterious effects to the microbiota.
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Siderophores are low molecular weight organic molecules produced by microorganisms under iron-restricted conditions. These metabolites are responsible for the transport of iron into microorganisms and have a high affinity to bind to ferric iron (Fe3) and produce iron chelates. Siderophores can be divided into three main groups: catechols, hydroxamates, and carboxylates, depending on the parts that bind to iron. Of course, siderophores that have more than one type of iron ligand group are classified into a mixed category. Siderophores are used in ecology, agriculture, bioremediation and medicine. In this article, the synthesis pathway and function of siderophores, their role as antibiotics and their use as non-antibiotic drugs have been investigated. Other applications of siderophore in various industries are also briefly mentioned.
Chapter
Aquaculture is an important food source worldwide but one of its main problems is the spread of infectious diseases caused by Gram-negative pathogenic bacteria that results in considerable economic losses. Since siderophores, low molecular weight organic compounds involved in the iron-uptake mechanisms of the bacteria, are critical for the growth and virulence of the producer pathogens, the bacterial iron acquisition systems are promising targets for the design of new antimicrobial strategies. In this chapter, the current chemical knowledge of the siderophores involved in the iron-uptake mechanisms of Gram-negative pathogenic bacteria Vibrio anguillarum, Photobacterium damselae subsp. piscicida, and Aeromonas salmonicida subsp. salmonicida, responsible for the main fish infectious diseases Vibriosis, photobacteriosis, and furunculosis, respectively, is summarized. The isolation, structural elucidation, and the chemical synthesis of the siderophores biosynthesized from those bacteria are displayed. Their involvement in the iron-uptake mechanisms, virulence importance, and the application in the development of new strategies to fight against the infectious diseases will be also discussed.
Article
Siderophores are low-molecular-weight chelators produced by microorganisms to scavenge iron from the environment and deliver it to cells via specific receptors. Tremendous researches on the molecular basis of siderophore regulation, synthesis, secretion, and uptake have inspired their diverse applications in the medical field. Replacing iron with radionuclides in siderophores, such as the most prominent Ga-68 for positron emission tomography (PET), carves out ways for targeted imaging of infectious diseases and cancers. Additionally, the high affinity of siderophores for metal ions or microorganisms makes them a potent detecting moiety in sensors that can be used for diagnosis. As for therapeutics, the notable Trojan horse-inspired siderophore-antibiotic conjugates demonstrate enhanced toxicity against multi-drug resistant (MDR) pathogens. Besides, siderophores can tackle iron overload diseases and, when combined with moieties such as hydrogels and nanoparticles, a wide spectrum of iron-induced diseases and even cancers. In this review, we briefly outline the related mechanisms, before summarizing the siderophore-based applications in imaging, sensors, and therapeutics.
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Drug-resistant infections pose a significant risk to global health as pathogenic bacteria become increasingly difficult to treat. The rapid selection of resistant strains through poor antibiotic stewardship has reduced the number of viable treatments and increased morbidity of infections, especially among the immunocompromised. To circumvent such challenges, new strategies are required to stay ahead of emerging resistance trends, yet research and funding for antibiotic development lags other classes of therapeutics. Though the use of metals in therapeutics has been around for centuries, recent strategies have devoted a great deal of effort into the pathways through which bacteria acquire and utilize iron, which is critical for the establishment of infection. To target iron uptake systems, siderophore–drug conjugates have been developed that hijack siderophore-based iron uptake for delivery of antibiotics. While this strategy has produced several potential leads, the use of siderophores in infection is diminished over time when bacteria adapt to utilize heme as an iron source, leading to a need for the development of porphyrin mimetics as therapeutics. The use of such strategies as well as the inclusion of gallium, a redox-inert iron mimic, are herein reviewed.
Article
Enterobactin (Ent)-mediated high affinity iron acquisition is critically important for Gram-negative bacterial pathogens to survive and infect the host. Recently, we reported an efficient method to prepare novel Ent conjugate vaccines for inducing high level of Ent-specific antibodies, which displayed similar bacteriostatic feature as lipocalins, the host innate immune effectors with potent Ent-binding ability. The Ent-specific antibodies also showed a significant advantage over lipocalins by cross-reacting to various Ent derivatives including salmochelins, the glycosylated Ent that can help enteric pathogens evade the siderophore sequestration by host lipocalins. To demonstrate significant potential of the Ent conjugate vaccine for broader applications to prevent and control various Gram-negative infections in human and animal, in this study, we examined inhibitory effect of Ent-specific antibodies on the in vitro growth of three significant Gram-negative pathogens: Escherichia coli (n = 27), Salmonella enterica (n = 8), and Campylobacter spp. (n = 6). The tested strains were diverse with respect to hosts, geographical origins, serotypes, infection sites and siderophore productions. The Ent-specific antibodies significantly suppressed the growth of each tested strain under iron-restricted conditions. For example, the Ent-specific antibodies consistently exerted 2–5 log10 units of growth reduction on most tested avian pathogenic E. coli (9 of 10 strains) isolated in five countries. Despite various dynamic growth responses observed, notably, the Ent-specific antibodies displayed significantly higher magnitude of growth reduction than lipocalin-2 (up to 5 log10 units of difference) on majority of tested E. coli and S. enterica, which is likely due to sequestration of other siderophores (e.g., salmochelins) by the Ent-specific antibodies. Production of a variety of major siderophores by the tested E. coli and S. enterica strains was examined and confirmed by ultra high performance liquid chromatography-high resolution mass spectrometry analysis. Collectively, this study provides critical and compelling in vitro evidence supporting the feasibility of Ent-based immune interventions against several Gram-negative pathogens.
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In the gastrointestinal tract, the tug of war for iron may provide a new way to vaccinate. Recent work shows that immunizing mice with siderophores (small molecules that microbes produce to capture iron) foils pathogen colonization and may instead allow a commensal to expand.
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The gut microbiome is significantly altered in inflammatory bowel diseases, but the basis of these changes is not well understood. We have combined metagenomic and metatranscriptomic profiling of the gut microbiome to assess modifications to both bacterial community structure and transcriptional activity in a mouse model of colitis. By using transcriptomic analysis of colonic tissue and luminal RNA derived from the host, we have also characterised how host transcription relates to the microbial transcriptional response in inflammation. In colitis, increased abundance and transcription of diverse microbial gene families involved in responses to nutrient deprivation, antimicrobial peptide production and oxidative stress support an adaptation of multiple commensal genera to withstand a diverse set of environmental stressors in the inflammatory environment. These data are supported by a transcriptional signature of activated macrophages and granulocytes in the gut lumen during colitis, a signature that includes the transcription of the key antimicrobial genes S100a8 and S100a9 (calprotectin). Genes involved in microbial resistance to oxidative stress, including Dps/ferritin, Fe-dependent peroxidase and glutathione S-transferase were identified as changing to a greater extent at the level of transcription than would be predicted by DNA abundance changes, implicating a role for increased oxygen tension and/or host-derived reactive oxygen species in driving transcriptional changes in commensal microbes.The ISME Journal advance online publication, 22 March 2016; doi:10.1038/ismej.2016.40.
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Siderophores are low molecular weight, high affinity iron chelating molecules that are essential virulence factors in many Gram-negative bacterial pathogens. Whereas the chemical structure of siderophores is extremely variable, the function of siderophores has been narrowly defined as the chelation and delivery of iron to bacteria for proliferation. The discovery of the host protein Lipocalin 2, capable of specifically sequestering the siderophore Enterobactin but not its glycosylated-derivative Salmochelin, indicated that diversity in structure could be an immune evasion mechanism that provides functional redundancy during infection. However, there is growing evidence that siderophores are specialized in their iron-acquisition functions, can perturb iron homeostasis in their hosts, and even bind non-iron metals to promote bacterial fitness. The combination of siderophores produced by a pathogen can enable inter-bacterial competition, modulate host cellular pathways, and determine the bacterial "replicative niche" during infection. This review will examine both classical and novel functions of siderophores to address the concept that siderophores are non-redundant virulence factors used to enhance bacterial pathogenesis.
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Bacteria secrete small molecules known as siderophores to acquire iron from their surroundings. For over 60 years, investigations into the bioinorganic chemistry of these molecules, including fundamental coordination chemistry studies, have provided insight into the crucial role that siderophores play in bacterial iron homeostasis. The importance of understanding the fundamental chemistry underlying bacterial life has been highlighted evermore in recent years because of the emergence of antibiotic-resistant bacteria and need to prevent the global rise of these superbugs. Increasing reports of siderophores functioning in capacities other than iron transport have appeared recently, but reports of “non-classical” siderophore functions have long paralleled those of iron transport. One particular “non-classical” function of these iron chelators, namely antibiotic activity, was even documented before the role of siderophores in iron transport was established. In this Perspective, we present an exposition of past and current work into non-classical functions of siderophores and highlight the directions in which we anticipate that this research is headed. Examples include the ability of siderophores to function as zincophores, chalkophores, and metallophores for a variety of other metals, sequester heavy metal toxins, transport boron, act as signalling molecules, regulate oxidative stress, and provide antibacterial activity.
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Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including Enterobactin (Ent). However, Ent is bound by the host protein Lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent. Furthermore, the combination of Ent and Lcn2 (Ent+Lcn2) leads to enhanced secretion of Interleukin-8 (IL-8) compared to either stimulus alone. Modified or structurally distinct siderophores, including Yersiniabactin (Ybt) and Glycosylated Ent (GlyEnt or Salmochelin), deliver iron to bacteria despite Lcn2. We hypothesized that the robust immune response to Ent and Lcn2 requires iron chelation rather than the Ent+Lcn2 complex itself, and can also be stimulated by Lcn2-evasive siderophores. To test this hypothesis, cultured respiratory epithelial cells were stimulated with combinations of purified siderophores and Lcn2, and analyzed by gene-expression microarrays, quantitative PCR, and cytokine immunoassays. Ent caused HIF-1α protein stabilization, induced the expression of genes regulated by hypoxia-inducible factor 1 α (HIF-1α) and repressed genes involved in cell cycle and DNA replication, whereas Lcn2 induced expression of pro-inflammatory cytokines. Iron chelation by excess Ent or Ybt significantly increased Lcn2-induced secretion of IL-8, IL-6 and CCL20. Stabilization of HIF-1α was sufficient to enhance Lcn2-induced IL-6 secretion. These data indicate that respiratory epithelial cells can respond to bacterial siderophores that evade or overwhelm Lcn2 binding by increasing pro-inflammatory cytokine production.
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Numerous bacteria have evolved different iron uptake systems with the ability to make use of their own and heterologous siderophores. However, there is growing evidence attributing alternative roles for siderophores that might explain the potential adaptive advantages of microorganisms having multiple siderophore systems. In this work, we show the requirement of the siderophore enterobactin for Escherichia coli colony development in minimal media. We observed that a strain impaired in enterobactin production (entE mutant) was unable to form colonies on M9 agar medium meanwhile its growth was normal on LB agar medium. Given that, neither iron nor citrate supplementation restored colony growth, the role of enterobactin as an iron uptake-facilitator would not explain its requirement for colony development. The absence of colony development was reverted either by addition of enterobactin, the reducing agent ascorbic acid or by incubating in anaerobic culture conditions with no additives. Then, we associated the enterobactin requirement for colony development with its ability to reduce oxidative stress, which we found to be higher in media where the colony development was impaired (M9) compared with media where the strain was able to form colonies (LB). Since oxyR and soxS mutants (two major stress response regulators) formed colonies in M9 agar medium, we hypothesize that enterobactin could be an important piece in the oxidative stress response repertoire, particularly required in the context of colony formation. In addition, we show that enterobactin has to be hydrolyzed after reaching the cell cytoplasm in order to enable colony development. By favoring iron release, hydrolysis of the enterobactin-iron complex, not only would assure covering iron needs, but would also provide the cell with a molecule with exposed hydroxyl groups (hydrolyzed enterobactin). This molecule would be able to scavenge radicals and therefore reduce oxidative stress.
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Unlabelled: Treatment with streptomycin enhances the growth of human commensal Escherichia coli isolates in the mouse intestine, suggesting that the resident microbial community (microbiota) can inhibit the growth of invading microbes, a phenomenon known as "colonization resistance." However, the precise mechanisms by which streptomycin treatment lowers colonization resistance remain obscure. Here we show that streptomycin treatment rendered mice more susceptible to the development of chemically induced colitis, raising the possibility that the antibiotic might lower colonization resistance by changing mucosal immune responses rather than by preventing microbe-microbe interactions. Investigation of the underlying mechanism revealed a mild inflammatory infiltrate in the cecal mucosa of streptomycin-treated mice, which was accompanied by elevated expression of Nos2, the gene that encodes inducible nitric oxide synthase. In turn, this inflammatory response enhanced the luminal growth of E. coli by nitrate respiration in a Nos2-dependent fashion. These data identify low-level intestinal inflammation as one of the factors responsible for the loss of resistance to E. coli colonization after streptomycin treatment. Importance: Our intestine is host to a complex microbial community that confers benefits by educating the immune system and providing niche protection. Perturbation of intestinal communities by streptomycin treatment lowers "colonization resistance" through unknown mechanisms. Here we show that streptomycin increases the inflammatory tone of the intestinal mucosa, thereby making the bowel more susceptible to dextran sulfate sodium treatment and boosting the Nos2-dependent growth of commensal Escherichia coli by nitrate respiration. These data point to the generation of alternative electron acceptors as a by-product of the inflammatory host response as an important factor responsible for lowering resistance to colonization by facultative anaerobic bacteria such as E. coli.
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Probiotics are beneficial components of the microbiota that have been used for centuries because of the health benefits they confer to the host. Only recently, however, has the contribution of probiotics to modulation of immunological, respiratory, and gastrointestinal functions started to be fully appreciated and scientifically evaluated. Probiotics such as Escherichia coli Nissle 1917 and lactic acid bacteria are currently used to, or have been evaluated for use to, prevent or treat a range of intestinal maladies including inflammatory bowel disease, constipation, and colon cancer. Engineering these natural probiotics to produce immunomodulatory molecules may help to further increase the benefit to the host. In this article, we will discuss some of the mechanisms of action of probiotics as well as advances in the rational design of probiotics.
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Changes in the microbial community structure are observed in individuals with intestinal inflammatory disorders. These changes are often characterized by a depletion of obligate anaerobic bacteria, whereas the relative abundance of facultative anaerobic Enterobacteriaceae increases. The mechanisms by which the host response shapes the microbial community structure, however, remain unknown. We show that nitrate generated as a by-product of the inflammatory response conferred a growth advantage to the commensal bacterium Escherichia coli in the large intestine of mice. Mice deficient in inducible nitric oxide synthase did not support the growth of E. coli by nitrate respiration, suggesting that the nitrate generated during inflammation was host-derived. Thus, the inflammatory host response selectively enhances the growth of commensal Enterobacteriaceae by generating electron acceptors for anaerobic respiration.
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Host-to-host transmission in most Salmonella serovars occurs primarily via the fecal-oral route. Salmonella enterica serovar Typhi is a human host-adapted pathogen and some S. Typhi patients become asymptomatic carriers. These individuals excrete large numbers of the bacteria in their feces and transmit the pathogen by contaminating water or food sources. The carrier state has also been described in livestock animals and is responsible for food-borne epidemics. Identification and treatment of carriers are crucial for the control of disease outbreaks. In this review, we describe recent advances in molecular profiling of human carriers and the use of animal models to identify potential host and bacterial genes involved in the establishment of the carrier state.
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Conventional wisdom holds that microbes support their growth in vertebrate hosts by exploiting a large variety of nutrients. We show here that use of a specific nutrient (ethanolamine) confers a marked growth advantage on Salmonella enterica serovar Typhimurium (S. Typhimurium) in the lumen of the inflamed intestine. In the anaerobic environment of the gut, ethanolamine supports little or no growth by fermentation. However, S. Typhimurium is able to use this carbon source by inducing the gut to produce a respiratory electron acceptor (tetrathionate), which supports anaerobic growth on ethanolamine. The gut normally converts ambient hydrogen sulfide to thiosulfate, which it then oxidizes further to tetrathionate during inflammation. Evidence is provided that S. Typhimurium's growth advantage in an inflamed gut is because of its ability to respire ethanolamine, which is released from host tissue, but is not utilizable by competing bacteria. By inducing intestinal inflammation, S. Typhimurium sidesteps nutritional competition and gains the ability to use an abundant simple substrate, ethanolamine, which is provided by the host.
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Most Salmonella enterica serotypes are associated with acute intestinal inflammation and diarrhea in humans. While the mechanisms triggering intestinal inflammation are well studied, relatively little is known about how the pathogen benefits from causing disease. Recent work has provided first insights into the genetic design that enables S. enterica to benefit from the host response by outgrowing the microbiota in the gut. The pathogen gained an edge over its competitors by acquiring genes conferring resistance against antimicrobials, such as lipocalin-2, that are encountered in the intestinal lumen only during inflammation. This strategy enables the pathogen to exploit host responses to gain a competitive advantage over other microbes during its growth in the inflamed gut.
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Members of a family of catecholate siderophores, called salmochelins, were isolated by reversed-phase HPLC from Salmonella enterica serotype Typhimurium and structurally characterized by Fourier transform ion cyclotron resonance-MSMS and GC-MS. The tentative structure of salmochelin 1 contained two 2,3- dihydroxybenzoylserine moieties bridged by a glucose residue, bound to the serine hydroxyl group of one moiety and the carboxylate of the second moiety. Salmochelin 2 contained in addition a second glucose residue linked to a third 2,3-dihydroxybenzoylserine moiety. Salmochelins were not produced by an iroBC mutant, which indicated that the IroB protein might be responsible for the glucosyl transfer predicted by sequence similarities to known glycosyltransferases. Uptake experiments with radiolabeled (55)Fe-salmochelin and growth promotion tests with salmochelins showed that the IroN outer membrane receptor, encoded in the iroA locus of S. enterica and uropathogenic Escherichia coli strains, was the main receptor for ferric salmochelin transport.
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Although iron is required to sustain life, its free concentration and metabolism have to be tightly regulated. This is achieved through a variety of iron-binding proteins including transferrin and ferritin. During infection, bacteria acquire much of their iron from the host by synthesizing siderophores that scavenge iron and transport it into the pathogen. We recently demonstrated that enterochelin, a bacterial catecholate siderophore, binds to the host protein lipocalin 2 (ref. 5). Here, we show that this event is pivotal in the innate immune response to bacterial infection. Upon encountering invading bacteria the Toll-like receptors on immune cells stimulate the transcription, translation and secretion of lipocalin 2; secreted lipocalin 2 then limits bacterial growth by sequestrating the iron-laden siderophore. Our finding represents a new component of the innate immune system and the acute phase response to infection.
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Pathogenic strains of Escherichia coli and Salmonella enterica modify the tricatecholic siderophore enterobactin (Ent) by glucosylation of three aryl carbon atoms, a process controlled by the iroA locus [Hantke, K., Nicholson, G., Rabsch, W. & Winkelmann, G. (2003) Proc. Natl. Acad. Sci. USA 100, 3677–3682]. Here, we report the purification of the IroB protein and its characterization as the Ent C-glucosyltransferase. IroB transfers glucosyl groups from uridine-5′-diphosphoglucose to C5 of one, two, or three of the 2,3-dihydroxybenzoyl units of Ent to yield monoglucosyl-C-Ent (MGE), diglucosyl-C-Ent (DGE), and triglucosyl-C-Ent (TGE). DGE, also known as salmochelin S4, and macrolactone-opened derivatives have been isolated from the culture broths of S. enterica and uropathogenic E. coli [Bister, B., Bischoff, D., Nicholson, G. J., Valdebenito, M., Schneider, K., Winkelmann, G., Hantke, K. & Sussmuth, R. D. (2004) Biometals 17, 471–481], but MGE and TGE have not been reported previously. IroB has a k cat of ≈10 min⁻¹ for the first C-glucosylation and is distributive, with sequential conversion and buildup of MGE and then DGE. The C5 to C1′ regio-selectivity of the 2,3-dihydroxybenzoyl-glucose linkage at all three rings of TGE suggests a C5 carbanion, para to the C2 phenolate oxygen, as the carbon nucleophile in this novel enzymatic C-glucosylation. • glycosyltransferase • pathogenicity • siderophore
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There is currently great interest in developing mucosal vaccines against a variety of microbial pathogens. Mucosally induced tolerance also seems to be a promising form of immunomodulation for treating certain autoimmune diseases and allergies. Here we review the properties of the mucosal immune system and discuss advances in the development of mucosal vaccines for protection against infections and for treatment of various inflammatory disorders.
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Salmonella enterica subspecies 1 serovar Typhimurium is a principal cause of human enterocolitis. For unknown reasons, in mice serovar Typhimurium does not provoke intestinal inflammation but rather targets the gut-associated lymphatic tissues and causes a systemic typhoid-like infection. The lack of a suitable murine model has limited the analysis of the pathogenetic mechanisms of intestinal salmonellosis. We describe here how streptomycin-pretreated mice provide a mouse model for serovar Typhimurium colitis. Serovar Typhimurium colitis in streptomycin-pretreated mice resembles many aspects of the human infection, including epithelial ulceration, edema, induction of intercellular adhesion molecule 1, and massive infiltration of PMN/CD18+ cells. This pathology is strongly dependent on protein translocation via the serovar Typhimurium SPI1 type III secretion system. Using a lymphotoxin β-receptor knockout mouse strain that lacks all lymph nodes and organized gut-associated lymphatic tissues, we demonstrate that Peyer's patches and mesenteric lymph nodes are dispensable for the initiation of murine serovar Typhimurium colitis. Our results demonstrate that streptomycin-pretreated mice offer a unique infection model that allows for the first time to use mutants of both the pathogen and the host to study the molecular mechanisms of enteric salmonellosis.
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Many bacteria, including numerous human pathogens, synthesize small molecules known as siderophores to scavenge iron. Enterobactin, a siderophore produced by enteric bacteria, is surprisingly ineffective as an iron-scavenging agent for bacteria growing in animals because of its hydrophobicity and its sequestration by the mammalian protein siderocalin, a component of the innate immune system. However, pathogenic strains of Escherichia coli and Salmonella use enzymes encoded by the iroA gene cluster to tailor enterobactin by glycosylation and linearization. The resulting modified forms of enterobactin, known as salmochelins, can evade siderocalin and are less hydrophobic than enterobactin, restoring this siderophore's iron-scavenging ability in mammals.
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Numerous bacteria cope with the scarcity of iron in their microenvironment by synthesizing small iron-scavenging molecules known as siderophores. Mammals have evolved countermeasures to block siderophore-mediated iron acquisition as part of their innate immune response. Secreted lipocalin 2 (Lcn2) sequesters the Escherichia coli siderophore enterobactin (Ent), preventing E. coli from acquiring iron and protecting mammals from infection by E. coli. Here, we show that the iroA gene cluster, found in many pathogenic strains of Gram-negative enteric bacteria, including E. coli, Salmonella spp., and Klebsiella pneumoniae, allows bacteria to evade sequestration of Ent by Lcn2. We demonstrate that C-glucosylated derivatives of Ent produced by iroA-encoded enzymes do not bind purified Lcn2, and an iroA-harboring strain of E. coli is insensitive to the growth inhibitory effects of Lcn2 in vitro. Furthermore, we show that mice rapidly succumb to infection by an iroA-harboring strain of E. coli but not its wild-type counterpart, and that this increased virulence depends on evasion of host Lcn2. Our findings indicate that the iroA gene cluster allows bacteria to evade this component of the innate immune system, rejuvenating their Ent-mediated iron-acquisition pathway and playing an important role in their virulence. • bacterial pathogens • host defense • innate immunity • iron • siderophores
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Author Summary A dense microbial community colonizes the intestinal tract of mammals, contributing to health and nutrition and conferring efficient protection against most pathogenic intruders. Intestinal pathogens can overcome this colonization resistance and cause disease; however, the mechanisms used to do this are still elusive. In this study we analyzed intestinal infection by the model pathogen Salmonella enterica subspecies 1 serovar Typhimurium (S. Tm). We show that the virulent wild-type pathogen overcomes colonization resistance by inducing the host's inflammatory immune response and exploiting it for its purpose. In contrast, an avirulent Salmonella mutant defective in triggering inflammation was unable to overcome colonization resistance by itself. Colonization by this mutant was restored if inflammation was provided concomitantly, in mice with inflammatory bowel disease (genetic and inducible) or by co-infection with wild-type S. Tm. These findings reveal a previously unrecognized strategy by which pathogenic bacteria overcome colonization resistance: abusing the host's inflammatory immune response to gain an edge against the normal microbial community of the gut. This represents a first step towards unravelling the molecular mechanisms underlying this three-way interaction of host, microbiota, and pathogens.
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The design, synthesis, and characterization of enterobactin-antibiotic conjugates, hereafter Ent-Amp/Amx, where the β-lactam antibiotics ampicillin (Amp) and amoxicillin (Amx) are linked to a monofunctionalized enterobactin scaffold via a stable polyethyleneglycol linker are reported. Under conditions of iron limitation, these siderophore-modified antibiotics provide enhanced antibacterial activity against Escherichia coli strains, including uropathogenic E. coli CFT073 and UTI89, enterohemorrhagic E. coli O157:H7, and enterotoxigenic E. coli O78:H11, compared to the parent β-lactams. Studies with E. coli K-12 derivatives defective in ferric enterobactin transport reveal that the enhanced antibacterial activity observed for this strain requires the outer membrane ferric enterobactin transporter FepA. A remarkable 1000-fold decrease in minimum inhibitory concentration (MIC) value is observed for uropathogenic E. coli CFT073 relative to Amp/Amx, and time-kill kinetic studies demonstrate that Ent-Amp/Amx kill this strain more rapidly at 10-fold lower concentrations than the parent antibiotics. Moreover, Ent-Amp and Ent-Amx selectively kill E. coli CFT073 co-cultured with other bacterial species such as Staphylococcus aureus, and Ent-Amp exhibits low cytotoxicity against human T84 intestinal cells in both the apo and iron-bound forms. These studies demonstrate that the native enterobactin platform provides a means to effectively deliver antibacterial cargo across the outer membrane permeability barrier of Gram-negative pathogens utilizing enterobactin for iron acquisition.
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Interleukin-22 (IL-22) is highly induced in response to infections with a variety of pathogens, and its main functions are considered to be tissue repair and host defense at mucosal surfaces. Here we showed that IL-22 has a unique role during infection in that its expression suppressed the intestinal microbiota and enhanced the colonization of a pathogen. IL-22 induced the expression of antimicrobial proteins, including lipocalin-2 and calprotectin, which sequester essential metal ions from microbes. Because Salmonella enterica ser. Typhimurium can overcome metal ion starvation mediated by lipocalin-2 and calprotectin via alternative pathways, IL-22 boosted its colonization of the inflamed intestine by suppressing commensal Enterobacteriaceae, which are susceptible to the antimicrobial proteins. Thus, IL-22 tipped the balance between pathogenic and commensal bacteria in favor of a pathogen. Taken together, IL-22 induction can be exploited by pathogens to suppress the growth of their closest competitors, thereby enhancing pathogen colonization of mucosal surfaces.
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The large intestine is host to a complex ecological community composed predominantly of obligate anaerobic bacteria belonging to the classes Bacteroidia and Clostridia. This community confers benefit through its metabolic activities and host interactions. However, a microbial imbalance (dysbiosis) characterized by a decreased abundance of Clostridia and a bloom of facultative anaerobic Proteobacteria is commonly observed during inflammation in the large bowel. Here we review recent insights into the principles that favor simultaneous increases in the abundance of closely related species belonging to the Proteobacteria during inflammation, which provides important clues for the rational design of strategies to treat dysbiosis.
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Host inflammation alters the availability of nutrients such as iron to limit microbial growth. However, Salmonella enterica serovar Typhimurium thrives in the inflamed gut by scavenging for iron with siderophores. By administering Escherichia coli strain Nissle 1917, which assimilates iron by similar mechanisms, we show that this nonpathogenic bacterium can outcompete and reduce S. Typhimurium colonization in mouse models of acute colitis and chronic persistent infection. This probiotic activity depends on E. coli Nissle iron acquisition, given that mutants deficient in iron uptake colonize the intestine but do not reduce S. Typhimurium colonization. Additionally, the ability of E. coli Nissle to overcome iron restriction by the host protein lipocalin 2, which counteracts some siderophores, is essential, given that S. Typhimurium is unaffected by E. coli Nissle in lipocalin 2-deficient mice. Thus, iron availability impacts S. Typhimurium growth, and E. coli Nissle reduces S. Typhimurium intestinal colonization by competing for this limiting nutrient.
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Iron is an essential nutrient for both humans and pathogenic microbes. Because of its ability to exist in one of two oxidation states, iron is an ideal redox catalyst for diverse cellular processes including respiration and DNA replication. However, the redox potential of iron also contributes to its toxicity; thus, iron concentration and distribution must be carefully controlled. Given the absolute requirement for iron by virtually all human pathogens, an important facet of the innate immune system is to limit iron availability to invading microbes in a process termed nutritional immunity. Successful human pathogens must therefore possess mechanisms to circumvent nutritional immunity in order to cause disease. In this review, we discuss regulation of iron metabolism in the setting of infection and delineate strategies used by human pathogens to overcome iron-withholding defenses.
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The healthy intestine is characterized by a low level of oxygen and by the presence of large bacterial communities of obligate anaerobes. Dysbiosis of the gut microbiota has been reported in patients suffering from inflammatory bowel diseases (IBDs), but the mechanisms causing this imbalance remain unknown. Observations have included a decrease in obligate anaerobes of the phylum Firmicutes and an increase in facultative anaerobes, including members of the family Enterobacteriaceae. The shift of bacterial communities from obligate to facultative anaerobes strongly suggests a disruption in anaerobiosis and points to a role for oxygen in intestinal dysbiosis. Proposals to evaluate this hypothesis of a role for oxygen in IBD dysbiosis are provided. If this hypothesis is confirmed, decreasing oxygen in the intestine could open novel means to rebalance the microbiota and could provide novel preventative or therapeutic strategies for IBD patients in whom current treatments are ineffective.
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WITHIN the past half century, the doctrine of the biochemical unity of living matter has become well established. Metabolic pathways as well as methods of storage, transfer, and use of energy and of genetic information are found to have strong commonality in the cells of microorganisms, plants, and animals. As a consequence, similar kinds of organic and inorganic components are used in the building of protoplasm by the three forms of life. Moreover, the quantities of inorganic nutrilites needed for cell replication are quite similar. For example, to achieve maximal growth, cultures of cells of microorganisms, plants, and animals each require approximately 1μM solution of iron, 1 mM solution of phosphorus, and 1 mM solution of magnesium.Immunity In view of these similarities, it is remarkable that plants and animals have been able to develop, during evolution, methods for keeping many of their tissues and organs free of microbial invaders.
Article
Our intestine is host to a large microbial community (microbiota) that educates the immune system and confers niche protection. Profiling of the gut-associated microbial community reveals a dominance of obligate anaerobic bacteria in healthy individuals. However, intestinal inflammation is associated with a disturbance of the microbiota-known as dysbiosis-that often includes an increased prevalence of facultative anaerobic bacteria. This group contains potentially harmful bacterial species, the bloom of which can further exacerbate inflammation. Here, we review the mechanisms that generate changes in the microbial community structure during inflammation. One emerging concept is that electron acceptors generated as by-products of the host inflammatory response feed facultative anaerobic bacteria selectively, thereby increasing their prevalence within the community. This new paradigm has broad implications for understanding dysbiosis during gut inflammation and identifies potential targets for intervention strategies.
Article
The design and syntheses of monofunctionalized enterobactin (Ent, l- and d-isomers) scaffolds where one catecholate moiety of enterobactin houses an alkene, aldehyde, or carboxylic acid at the C5 position are described. These molecules are key precursors to a family of 10 enterobactin-cargo conjugates presented in this work, which were designed to probe the extent to which the Gram-negative ferric enterobactin uptake and processing machinery recognizes, transports, and utilizes derivatized enterobactin scaffolds. A series of growth recovery assays employing enterobactin-deficient E. coli ATCC 33475 (ent-) revealed that six conjugates based on l-Ent having relatively small cargos promoted E. coli growth under iron-limiting conditions whereas negligible-to-no growth recovery was observed for four conjugates with relatively large cargos. No growth recovery was observed for the enterobactin receptor-deficient strain of E. coli H1187 (fepA-) or the enterobactin esterase-deficient derivative of E. coli K-12 JW0576 (fes-), or when the d-isomer of enterobactin was employed. These results demonstrate that the E. coli ferric enterobactin transport machinery identifies and delivers select cargo-modified scaffolds to the E. coli cytoplasm. Pseudomonas aeruginosa PAO1 K648 (pvd-, pch-) exhibited greater promiscuity than that of E. coli for the uptake and utilization of the enterobactin-cargo conjugates, and growth promotion was observed for eight conjugates under iron-limiting conditions. Enterobactin may be utilized for delivering molecular cargos via its transport machinery to the cytoplasm of E. coli and P. aeruginosa thereby providing a means to overcome the Gram-negative outer membrane permeability barrier.
Article
Siderophores are chelators synthesized by bacteria and fungi to sequester iron, which is essential for virulence and pathogenicity. Since the process involves active transport, which is highly regulated, remarkably efficient and often microbially selective, it has been exploited as a Trojan Horse method for development of microbe-selective antibiotics. Siderophores also have significant potential for the development of imaging contrast agents and diagnostics for pathogen-selective detection. These promising results demonstrate the versatility of natural and synthetic microbial iron chelators and their potential therapeutic applications.
Article
It is interesting to speculate that the evolutionary drive for microbes to develop pathogenic characteristics was to access the nutrient resources that animals provided. Animal environments that pathogens colonize have likely driven the evolution of new bacterial characteristics to maximize these new nutritional opportunities. This review focuses on genomic and functional aspects of pathogen metabolism that allow efficient utilization of nutrient resources provided by animals. Similar to genes encoding specific virulence traits, genes encoding metabolic functions have been horizontally acquired by pathogens to provide a selective advantage in host tissues. Selective advantage in host tissues can also be gained by loss of function mutations that alter metabolic capabilities. Greater understanding of bacterial metabolism within host tissues should be important for increased understanding of host-pathogen interactions and the development of future therapeutic strategies.
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The lower gastrointestinal tract is densely populated with resident microbial communities (microbiota), which do not elicit overt host responses but rather provide benefit to the host, including niche protection from pathogens. However, introduction of bacteria into the underlying tissue evokes acute inflammation. Non-typhoidal Salmonella serotypes (NTS) elicit this stereotypic host response by actively penetrating the intestinal epithelium and surviving in tissue macrophages. Initial responses generated by bacterial host cell interaction are amplified in tissue through the interleukin (IL)-18/interferon-gamma and IL-23/IL-17 axes, resulting in the activation of mucosal barrier functions against NTS dissemination. However, the pathogen is adapted to survive antimicrobial defenses encountered in the lumen of the inflamed intestine. This strategy enables NTS to exploit inflammation to outcompete the intestinal microbiota, and promotes the Salmonella transmission by the fecal/oral route.
Article
A new target strategy in the development of bacterial vaccines, the induction of antibodies to microbial outer membrane ferrisiderophore complexes, is explored. A vibriobactin (VIB) analogue, with a thiol tether, 1-(2,3-dihydroxybenzoyl)-5,9-bis[[(4S,5R)-2-(2,3-dihydroxyphenyl)-4,5-dihydro-5-methyl-4-oxazolyl]carbonyl]-14-(3-mercaptopropanoyl)-1,5,9,14-tetraazatetradecane, was synthesized and linked to ovalbumin (OVA) and bovine serum albumin (BSA). The antigenicity of the VIB microbial iron chelator conjugates and their iron complexes was evaluated. When mice were immunized with the resulting OVA-VIB conjugate, a selective and unequivocal antigenic response to the VIB hapten was observed; IgG monoclonal antibodies specific to the vibriobactin fragment of the BSA and OVA conjugates were isolated. The results are consistent with the idea that the isolated adducts of siderophores covalently linked to their bacterial outer membrane receptors represent a credible target for vaccine development.
Article
In response to enteric pathogens, the inflamed intestine produces antimicrobial proteins, a process mediated by the cytokines IL-17 and IL-22. Salmonella enterica serotype Typhimurium thrives in the inflamed intestinal environment, suggesting that the pathogen is resistant to antimicrobials it encounters in the intestinal lumen. However, the identity of these antimicrobials and corresponding bacterial resistance mechanisms remain unknown. Here, we report that enteric infection of rhesus macaques and mice with S. Typhimurium resulted in marked Il-17- and IL-22-dependent intestinal epithelial induction and luminal accumulation of lipocalin-2, an antimicrobial protein that prevents bacterial iron acquisition. Resistance to lipocalin-2, mediated by the iroBCDE iroN locus, conferred a competitive advantage to the bacterium in colonizing the inflamed intestine of wild-type but not of lipocalin-2-deficient mice. Thus, resistance to lipocalin-2 defines a specific adaptation of S. Typhimurium for growth in the inflamed intestine.
Article
Uptake of 55Fe- and 3H-labeled siderophores and their chronic analogues have been studied in Salmonella typhimurium LT-2 and Escherichia coli K-12. In S. typhimurium LT-2, at least two different mechanisms for siderophore iron transport may be operative. Uptake of 55Fe- and 3H-labeled ferrichrome and kinetically inert lambda-cis-chromic [3H]deferriferrichrome by the S. typhimurium LT-2 enb7 mutant, which is defective in the production of its native siderophore, enterobactin, appears to occur by two concurrent mechanisms. The first mechanism is postulated to involve either rapid uptake of iron released from the ferric complex by cellular reduction without penetration of the complex or ligand or dissociation of the complex and simultaneous uptake of both ligand and iron coupled with simultaneous expulsion of the ligand. The second mechanism appears to consist of slower uptake of the intact ferric complex.
Article
Within the past half century, the doctrine of the biochemical unity of living matter has become well established. Metabolic pathways as well as methods of storage, microorganisms, plants and animals are similar kinds of organic and inorganic components in the building of protoplasm. The quantities of inorganic nutrilites needed for cell replication are quite similar. For example, to achieve maximal growth, cultures of cells of microorganisms, plants, and animals each require approximately 1 μM solution of iron, 1 mM solution of phosphorus, and 1 mM solution of magnesium. In view of these similarities, it is remarkable that plants and animals have been able to develop, during evolution, methods for keeping many of their tissues and organs free of microbial invaders. In animal systems, the methods involving humoral and cellular immunity are well known. Much less attention has been given to a possible third method: that of nutritional immunity. In this method, the invaded host would attempt to withhold an essential nutrilite from the pathogenic microorganisms. However, because of the qualitative and quantitative similarities between the essential nutrilites of animal hosts and invading microorganisms, such a process appears at first glance to be highly impractical. Nevertheless, hosts go to considerable lengths to deprive invaders of at least one nutrilite (iron), and much evidence exists that this example of nutritional immunity is an important defense component in the never ending war with potential pathogens.
Article
Escherichia coli septicemia is a common disease of young poultry and several species of mammals. Rabbit antiserum was prepared against iron-regulated outer membrane proteins of E. coli. Eighteen-day-old turkeys were passively immunized with antiserum and challenged by air sac inoculation of 1 X 10(6) to 2 X 10(6) CFU of E. coli O78:K80:H9. Turkeys injected with normal rabbit serum or saline solution before challenge served as controls. Fatalities (8 of 51 turkeys inoculated) occurred only in groups given saline solution or normal rabbit serum. The remaining turkeys were necropsied 96 h after challenge. Passive immunization with antiserum significantly (P less than 0.05) reduced the frequency of bacteremia at 96 h after challenge, the frequency of recovery of E. coli from air sacs, and the severity of gross lesions in inoculated birds as compared with birds given normal rabbit serum or saline solution.
Article
The regulatory logic of siderophore biosynthetic genes in bacteria involves the universal repressor Fur, which acts together with iron as a negative regulator. However in other bacteria, in addition to the Fur-mediated mechanism of regulation, there is a concurrent positive regulation of iron transport and siderophore biosynthetic genes that occurs under conditions of iron deprivation. Despite these regulatory differences the mechanisms of siderophore biosynthesis follow the same fundamental enzymatic logic, which involves a series of elongating acyl-S-enzyme intermediates on multimodular protein assembly lines: nonribosomal peptide synthetases (NRPS). A substantial variety of siderophore structures are produced from similar NRPS assembly lines, and variation can come in the choice of the phenolic acid selected as the N-cap, the tailoring of amino acid residues during chain elongation, the mode of chain termination, and the nature of the capturing nucleophile of the siderophore acyl chain being released. Of course the specific parts that get assembled in a given bacterium may reflect a combination of the inventory of biosynthetic and tailoring gene clusters available. This modular assembly logic can account for all known siderophores. The ability to mix and match domains within modules and to swap modules themselves is likely to be an ongoing process in combinatorial biosynthesis. NRPS evolution will try out new combinations of chain initiation, elongation and tailoring, and termination steps, possibly by genetic exchange with other microorganisms and/or within the same bacterium, to create new variants of iron-chelating siderophores that can fit a particular niche for the producer bacterium.
Article
A commercial inactivated iron restricted Salmonella Typhimurium and Salmonella Enteritidis vaccine was used to vaccinate chicks at 1 day and again at 4 weeks of age, with challenge by a high and a low dose of S. Typhimurium given either orally or by contact with seeder birds inoculated orally with a high dose of S. Typhimurium. In all three challenge regimes, the shedding of challenge strain was reduced significantly (p < 0.05) in vaccinated birds compared with unvaccinated controls. Vaccination reduced colonisation of internal organs after challenge by contact seeder birds. However, no effect of vaccination upon colonisation of internal organs after either high or low oral challenge was apparent. In conclusion, the data indicate that the vaccine should be a useful tool in the control of S. Typhimurium infection in chickens.
Article
First identified as a neutrophil granule component, neutrophil gelatinase-associated lipocalin (NGAL; also called human neutrophil lipocalin, 24p3, uterocalin, or neu-related lipocalin) is a member of the lipocalin family of binding proteins. Putative NGAL ligands, including neutrophil chemotactic agents such as N-formylated tripeptides, have all been refuted by recent biochemical and structural results. NGAL has subsequently been implicated in diverse cellular processes, but without a characterized ligand, the molecular basis of these functions remained mysterious. Here we report that NGAL tightly binds bacterial catecholate-type ferric siderophores through a cyclically permuted, hybrid electrostatic/cation-pi interaction and is a potent bacteriostatic agent in iron-limiting conditions. We therefore propose that NGAL participates in the antibacterial iron depletion strategy of the innate immune system.
Article
Bacteria have aggressive acquisition processes for iron, an essential nutrient. Siderophores are small iron chelators that facilitate cellular iron transport. The siderophore enterobactin is a triscatechol derivative of a cyclic triserine lactone. Studies of the chemistry, regulation, synthesis, recognition, and transport of enterobactin make it perhaps the best understood of the siderophore-mediated iron uptake systems, displa