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Pili of Mycobacterium tuberculosis: current knowledge and future prospects

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Abstract

Many pathogenic bacteria express filamentous appendages, termed pili, on their surface. These organelles function in several important bacterial processes, including mediating bacterial interaction with, and colonization of the host, signalling events, locomotion, DNA uptake, electric conductance, and biofilm formation. In the last decade, it has been established that the tuberculosis-causing bacterium, Mycobacterium tuberculosis, produces two pili types: curli and type IV pili. In this paper, we review studies on M. tuberculosis pili, highlighting their structure and biological significance to M. tuberculosis pathogenesis, and discuss their potential as targets for therapeutic intervention and diagnostic test development.

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... It has been shown that pili are recognized by IgG antibodies contained in sera of patients with active tuberculosis, indicating that the bacilli produce pili or pili-associated antigen during human infection. 81 M. tuberculosis produces two pili types: curli and type IV pili. 43,81,82 Purified M. tuberculosis pili are composed of low molecularweight protein subunits, designated as Rv3312A. ...
... 81 M. tuberculosis produces two pili types: curli and type IV pili. 43,81,82 Purified M. tuberculosis pili are composed of low molecularweight protein subunits, designated as Rv3312A. 81 These pili bind to the extracellular matrix protein laminin in vitro and share with other bacterial pili morphological, biochemical, and functional properties, more similar to curli amyloid fibers. ...
... 43,81,82 Purified M. tuberculosis pili are composed of low molecularweight protein subunits, designated as Rv3312A. 81 These pili bind to the extracellular matrix protein laminin in vitro and share with other bacterial pili morphological, biochemical, and functional properties, more similar to curli amyloid fibers. 83 Their role as adherence factors is further confirmed by their involvement in bacterial aggregation and biofilm formation. ...
Article
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Adherence, colonization, and survival of mycobacteria in host cells require surface adhesins, which are attractive pharmacotherapeutic targets. A large arsenal of pilus and non-pilus adhesins have been identified in mycobacteria. These adhesins are capable of interacting with host cells, including macrophages and epithelial cells and are essential to microbial pathogenesis. In the last decade, several structures of mycobacterial adhesins responsible for adhesion to either macrophages or extra cellular matrix proteins have been elucidated. In addition, key structural and functional information have emerged for the process of mycobacterial adhesion to epithelial cells, mediated by the Heparin Binding Hemagglutinin HBHA. In this review, we provide an overview of the structural and functional features of mycobacterial adhesins and discuss their role as important biomarkers for diagnostics and therapeutics. Based on the reported data, it appears clear that adhesins are endowed with a variety of different structures and functions. Most adhesins play important roles in the cell life of mycobacteria and are key virulence factors. However, they have adapted to an extracellular life to exert a role in host-pathogen interaction. The type of interactions they form with the host and the adhesin regions involved in binding is partly known and is described in this review. This article is protected by copyright. All rights reserved.
... MTP binds laminin, can be recognized by immune sera from active TB patients (Alteri et al., 2007) and resembles curli, which are extracellular proteinaceous fibres produced by many Enterobacteriaceae that share biochemical properties with amyloid (Barnhart & Chapman, 2006;Blanco et al., 2012). The potential importance of MTP as a Mtb virulence factor and biomarker (Naidoo et al., 2014) has spurred research from multiple groups and has led to a number of reviews on mycobacterial adhesins (Govender et al., 2014;Hosseini et al., 2014;Ramsugit & Pillay, 2015). Recent studies using a clinical isolate of Mtb, V9124, have shown that an mtp deletion mutant is defective in in vitro pellicle biofilm formation (Ramsugit et al., 2013) and in adherence to and invasion of A549 epithelial cells (Ramsugit et al., 2016), but it has no defect in adhering to and invading THP-1 macrophages . ...
... However, the proteins encoded by these genes are missing the conserved N-terminal G/XXXXEF motif found in other pseudopilins (Kachlany et al., 2000) and, therefore, have been disregarded as true members of the tad system in Mtb. It has been speculated that this predicted Flp pilus could contribute to Mtb pathogenesis (Alteri, 2005;Ramsugit & Pillay, 2015), but this has not been investigated. ...
... In the case of MTP, this suggestion has been primarily supported by in vitro biofilm, attachment and invasion assays (Alteri et al., 2007;Ramsugit et al., 2013Ramsugit et al., , 2016. Despite the uncertain contribution of MTP to virulence, these studies have spurred other groups to study MTP and MTP has been included as important for Mtb adherence in reviews on the subject (Govender et al., 2014;Hosseini et al., 2014;Ramsugit & Pillay, 2015). In addition to MTP, a putative pilus encoded by the widely conserved tad locus has also been suggested to be expressed and important in virulence (Alteri, 2005;Govender et al., 2014). ...
Article
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Mycobacterium tuberculosis (Mtb) is one of the world's most successful pathogens. Millions of new cases of tuberculosis (TB) occur each year, emphasizing the need for better methods of treatment. The design of novel therapeutics is dependent on our understanding of factors that are essential for pathogenesis. Many bacterial pathogens use pili and other adhesins to mediate pathogenesis. The recently identified Mycobacterium tuberculosis pilus (MTP) and the hypothetical, widely conserved Flp pilus have been speculated to be important for Mtb virulence based on in vitro studies and homology to other pili, respectively. However, the roles for these pili during infection had yet to be tested. We addressed this gap in knowledge and found that although MTP can contribute to biofilm formation and subsequent isoniazid tolerance, neither MTP nor the hypothetical Flp pilus is required for Mtb survival in mouse models of infection. However, differences in mtp expression did affect lesion architecture in infected lungs. Deletion of mtp did not correlate with loss of cell-associated extracellular structures visualized by transmission electron microscopy in Mtb Erdman and HN878 strains, suggesting that the phenotypes of the mtp mutants were not due to defects in production of extracellular structures. These findings highlight the importance of testing the virulence of adhesion mutants in animal models to assess the contribution of the adhesin to infection. This study also underscores the need for further investigation into additional strategies Mtb may use to adhere to its host so that we may understand how this pathogen invades, colonizes, and disseminates.
... However, agglutination-inhibition and adhesion assays, genome analyses, and immunological studies have provided the first indications that glycan-binding proteins might be important mediators of TB infections and Mtb pathogenesis. Detection of appendages on the mycobacterial surface, as extensively reviewed by Ramsugit et al. [107], further supports the possible existence of carbohydrate-binding proteins on the cell surface of Mtb, since bacterial lectins are often located at the terminal end of fimbriae or pili. ...
... The importance of these interactions in patients, however, has yet to be confirmed, as the association of mycobacterial biofilms with bacterial pathogenesis has not yet been conclusively shown in vivo. Besides mediating interactions among mycobacterial cells, MTP has been shown to play a role in Mtb adhesion and invasion of A549 pulmonary epithelial cells and THP-1 macrophages [107,119]. Furthermore, an impact of MTP on histopathology in a mouse model of infection has previously been described [113]. Elsewhere, using purified proteins, Alteri et al. detected laminin as a ligand for MTP [108]. ...
Article
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The importance of bacterial lectins for adhesion, pathogenicity, and biofilm formation is well established for many Gram-positive and Gram-negative bacteria. However, there is very little information available about lectins of the tuberculosis-causing bacterium, Mycobacterium tuberculosis (Mtb). In this paper we review previous studies on the carbohydrate-binding characteristics of mycobacteria and related Mtb proteins, discussing their potential relevance to Mtb infection and pathogenesis.
... Flp pili is also required for adherence to human fibroblasts and virulence in humans by Haemophilus ducreyi, the causative agent of human chancroid disease (Spinola et al., 2003). The presence in the Mtb genome of Flp type IV pili gene homologs (tadZ, tadA, tadB, tadC, flp, tadE, and tadF) has been reported (Tomich et al., 2007;Imam et al., 2011;Angelov et al., 2015;Ramsugit and Pillay, 2015). In this study, we report the outcome of our investigation on the Flp pili of Mtb H37RV directed towards understanding the expression of the pili at different growth phases, during biofilm formation, and during interaction with phagocytic and host epithelial cells. ...
Article
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The genome of Mycobacterium tuberculosis (Mtb) harbors the genetic machinery for assembly of the Fimbrial low-molecular-weight protein (Flp) type IV pilus. Presumably, the Flp pilus is essential for pathogenesis. However, it remains unclear whether the pili genes are transcribed in culture or during infection of host cells. This study aimed to shed light on the expression of the Flp pili-assembly genes (tadZ, tadA, tadB, tadC, flp, tadE, and tadF) in Mtb growing under different growth conditions (exponential phase, stationary phase, and dormancy NRP1 and NRP2 phases induced by hypoxia), during biofilm formation, and in contact with macrophages and alveolar epithelial cells. We found that expression of tad/flp genes was significantly higher in the stationary phase than in exponential or NRP1 or NRP2 phases suggesting that the bacteria do not require type IV pili during dormancy. Elevated gene expression levels were recorded when the bacilli were in contact for 4 h with macrophages or epithelial cells, compared to mycobacteria propagated alone in the cultured medium. An antibody raised against a 12-mer peptide derived from the Flp pilin subunit detected the presence of Flp pili on intra- and extracellular bacteria infecting eukaryotic cells. Altogether, these are compelling data showing that the Flp pili genes are expressed during the interaction of Mtb with host cells and highlight a role for Flp pili in colonization and invasion of the host, subsequently promoting bacterial survival during dormancy.
... In addition, curli pili can bind to the extracellular matrix (ECM) proteins fibronectin and laminin (Olsen et al., 1989(Olsen et al., , 1993. For example, laminin-binding pili resembling curli amyloid fibers have been identified in Mycobacterium tuberculosis, which can adhere to damaged regions of the human respiratory mucosa where ECM proteins have become exposed (Alteri et al., 2007;Ramsugit and Pillay, 2015). Worth mentioning, the various roles of functional amyloids in bacteria (e.g., curli in E. coli and fap in P. aeruginosa), fungi, plants, and animals have recently been discussed (Balistreri et al., 2020). ...
Article
Surface pili (or fimbriae) are an important but conspicuous adaptation of several genera and species of Gram-negative and Gram-positive bacteria. These long and non-flagellar multi-subunit adhesins mediate the initial contact that a bacterium has with a host or environment, and thus have come to be regarded as a key colonization factor for virulence activity in pathogens or niche adaptation in commensals. Pili in pathogenic bacteria are well recognized for their roles in the adhesion to host cells, colonization of tissues, and establishment of infection. As an ‘anti-adhesive’ ploy, targeting pilus-mediated attachment for disruption has become a potentially effective alternative to using antibiotics. In this review, we give a description of the several structurally distinct bacterial pilus types thus far characterized, and as well offer details about the intricacy of their individual structure, assembly, and function. With a molecular understanding of pilus biogenesis and pilus-mediated host interactions also provided, we go on to describe some of the emerging new approaches and compounds that have been recently developed to prevent the adhesion, colonization, and infection of piliated bacterial pathogens.
... The amyloids being present within Gordonia, Corynebacterium, and Mycobacterium spp. are characterized functionally, yet their roles in the mechanism of pathogenesis have not been fully studied (Ramsugit and Pillay 2015). The amyloid type of protein subunits being observed in Salmonella sp. and E. coli is transported to the periplasm via sec translocon in association with the membrane protein CsgG. ...
Chapter
Biofilm-forming microbes are the root cause of various almost incurable chronic, nosocomial, and medical device-associated infections that are of serious concern in present-day condition. With the advent of science, a number of diseases, disorders, and abnormalities can be effectively managed by the use of various medical devices including pacemakers, vascular catheters, chronic hemodialysis catheters, prosthetic heart valves, and prosthetic joints. But the effectiveness of these medical devices is seriously hampered by the biofilm grown aggressively on these devices. The interactions existing between the microbial cells, host, and the biomaterials result in the development, persistence, and failure in treating these device-associated infections. The present chapter would focus on various medical devices-associated biofilm infections that are affecting the host immune system leading to chronic infections and failure of the objective of this implant operation.
... The flp locus of M. tuberculosis is homologous to flp-tad locus of Aggregatibacter actinomycetemcomitans and was presumably acquired by horizontal gene transfer. In many pathogens Flp/Tad pili serve as colonization factors and promote biofilm formation (Ramsugit and Pillay 2015). As M. tuberculosis is a non-motile organisms, its type IV pilus potentially functions as adherent factor, which was also initially proved by Alteri (2005). ...
Chapter
To interact with the external environments, bacteria often display long proteinaceous appendages on their cell surface, called pili or fimbriae. These non-flagellar thread-like structures are polymers composed of covalently or non-covalently interacting repeated pilin subunits. Distinct pilus classes can be identified on basis of their assembly pathways, including chaperone-usher pili, type V pili, type IV pili, curli and fap fibers, conjugative and type IV secretion pili, as well as sortase-mediated pili. Pili play versatile roles in bacterial physiology, and can be involved in adhesion and host cell invasion, DNA and protein secretion and uptake, biofilm formation, cell motility and more. Recent advances in structure determination of components involved in the various pilus systems has enabled a better molecular understanding of their mechanisms of assembly and function. In this chapter we describe the diversity in structure, biogenesis and function of the different pilus systems found in Gram-positive and Gram-negative bacteria, and review their potential as anti-microbial targets.
... Mycobacterium tuberculosis curli pilus (MTP), a cell surface appendage and adhesin, has recently been highlighted as an important virulence factor and biomarker for the design of such interventions 2 . The potential accuracy of MTP for use in a diagnostic test was also recently demonstrated, as this adhesin is unique to M. tuberculosis complex (MTBC) pathogens 2,3 . Consequently, the availability of the MTP antigen is essential for studies on TB diagnostics, pathogenesis and immunological response to M. tuberculosis infection and disease. ...
... Congo red, TEM. [24] Fungi: Podospora anserine HET-s Not reported Regulation of heterokaryon formation. ...
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Misfolded β-sheet structures of proteins leading to neurodegenerative diseases like Alzheimer's (AD) and Parkinson's diseases (PD) are in the spotlight since long. However, not much was known about the functional amyloids till the last decade. Researchers have become increasingly more concerned with the degree of involvement of these functional amyloids in human physiology. Interestingly, it has been found that the human body is exposed to a tremendous systemic amyloid burden, especially, during aging. Though many findings regarding these functional amyloids come up every day, some questions still remain unanswered like do these functional amyloids directly involve in the fibrillization of Amyloid beta (Aβ) 42 peptide or enhance the Aβ42 aggregation rate; whether functional bacterial amyloids (FuBA) co-localize with the senile plaques of AD or not. A detailed review of the latest status regarding the interrelationship between functional amyloids, pathogenic amyloids and misfolded prions and therapeutic assessment of functional amyloids for the treatment of neurodegenerative diseases can help identify an alternative medication for neurodegeneration. A unique mathematical model is proposed here for alteration of Aβ42 aggregation kinetics in AD to carve out the future direction of therapeutic consideration.
... The MT3760 gene of M. tuberculosis CDC1551, homologue of the Rv3660c gene of M. tuberculosis H37Rv, codes for the septum site determining protein (Ssd) that promotes bacterial filamentation by inhibition of septum synthesis, leading to elongated cells as a response to a stressful environment (England et al., 2011). In addition, Rv3660c is the first component of a genomic island that codes for the Mtb type IV pili (Ramsugit et al., 2015), which is involved in the assembly and secretion of such cellular structure (Tomich et al., 2007;O´Connell et al., 2011;Perez-Cheeks et al., 2012). In order to investigate whether SigD affects ssd regulation in a stressful envi-Key words: SigD, Septum site determining protein, Type IV pili, Stressful environments, M. tuberculosis. ...
Article
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This work examined the expression of the septum site determining gene (ssd) of Mycobacterium tuberculosis CDC1551 and its ΔsigD mutant under different growing conditions. The results showed an up-regulation of ssd during stationary phase and starvation conditions, but not during in vitro dormancy, suggesting a putative role for SigD in the control of ssd expression mainly under lack-of-nutrients environments. Furthermore, we elucidated a putative link between ssd expression and cell elongation of bacilli at stationary phase. In addition, a -35 sigD consensus sequence was found for the ssd promoter region, reinforcing the putative regulation of ssd by SigD, and in turn, supporting this protein role during the adaptation of M. tuberculosis to some stressful environments.
... The location of the pilin subunit gene in the mycobacterial genome is unique, as it is not part of an operon like all other pili types that have been discovered to date (Ramsugit & Pillay, 2015) (Table 2). It would be interesting to postulate the unique MTP assembly pathway based on the unique properties of the mycobacterial cell wall and protein secretion system components. ...
Article
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Context: Novel biomarkers are essential for developing rapid diagnostics and therapeutic interventions Objective: This review aimed to highlight biomarker characterization and assessment of unique bacterial pili. Methods: A PubMed search for bacterial pili, diagnostics, vaccine and therapeutics was performed, with emphasis on the well characterized pili. Results: In total, 46 papers were identified and reviewed. Conclusion: Extensive analyses of pili enabled by advanced nanotechnology and whole genome sequencing provide evidence that they are strong biomarker candidates. Mycobacterium tuberculosis curli pili are emphasized as important epitopes for development of much needed point-of-care diagnostics and therapeutics.
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Neisseria gonorrhoeae andNeisseria meningitidis are Gram-negative bacterial pathogens that infect human mucosal epithelia. Type IV pilus-mediated adherence of these bacteria is a crucial early event for establishment of infection. In this work, we show that the type IV pili transduce a signal into the eucaryotic host cell. Purified adherent pili, but not pili from a low binding mutant, trigger an increase in the cytosolic free calcium ([Ca2+]i) in target epithelial cells, a signal known to control many cellular responses. The [Ca2+]i increase was blocked by antibodies against CD46, a putative pilus receptor, suggesting a role for this protein in signal transduction. Pilus-mediated attachment was inhibited by depletion of host cell intracellular Ca2+ stores but not by removal of extracellular Ca2+. Further, kinase inhibition studies showed that pilus-mediated adherence is dependent on casein kinase II. In summary, these data reveal a novel function of the type IV pili, namely induction of signal transduction pathways in host cells.
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The gram-negative coccobacillus, Actinobacillus actinomycetemcomitans, is the putative agent for localized juvenile periodontitis, a particularly destructive form of periodontal disease in adolescents. This bacterium has also been isolated from a variety of other infections, notably endocarditis. Fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms, a property likely to be critical for colonization of teeth and other surfaces. Here we report the identification of a locus of seven genes required for nonspecific adherence of A. actinomycetemcomitans to surfaces. The recently developed transposon IS903φkan was used to isolate mutants of the rough clinical isolate CU1000 that are defective in tight adherence to surfaces (Tad−). Unlike wild-type cells, Tad−mutant cells adhere poorly to surfaces, fail to form large autoaggregates, and lack long, bundled fibrils. Nucleotide sequencing and genetic complementation analysis revealed a 6.7-kb region of the genome with seven adjacent genes (tadABCDEFG) required for tight adherence. The predicted TadA polypeptide is similar to VirB11, an ATPase involved in macromolecular transport. The predicted amino acid sequences of the other Tad polypeptides indicate membrane localization but no obvious functions. We suggest that thetad genes are involved in secretion of factors required for tight adherence of A. actinomycetemcomitans. Remarkably, complete and highly conserved tad gene clusters are present in the genomes of the bubonic plague bacillus Yersinia pestis and the human and animal pathogen Pasteurella multocida. Partial tad loci also occur in strikingly diverse Bacteria and Archaea. Our results show that the tad genes are required for tight adherence ofA. actinomycetemcomitans to surfaces and are therefore likely to be essential for colonization and pathogenesis. The occurrence of similar genes in a wide array of microorganisms indicates that they have important functions. We propose thattad-like genes have a significant role in microbial colonization.
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Amyloid is associated with debilitating human ailments including Alzheimer's and prion diseases. Biochemical, biophysical, and imaging analyses revealed that fibers produced by Escherichia coli called curli were amyloid. The CsgA curlin subunit, purified in the absence of the CsgB nucleator, adopted a soluble, unstructured form that upon prolonged incubation assembled into fibers that were indistinguishable from curli. In vivo, curli biogenesis was dependent on the nucleation-precipitation machinery requiring the CsgE and CsgF chaperone-like and nucleator proteins, respectively. Unlike eukaryotic amyloid formation, curli biogenesis is a productive pathway requiring a specific assembly machinery.
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Haemophilus ducreyi, the etiologic agent of chancroid, has been shown to form microcolonies when cultured in the presence of human foreskin fibroblasts. We identified a 15-gene cluster in H. ducreyi that encoded predicted protein products with significant homology to those encoded by the tad (for tight adhesion) locus in Actinobacillus actinomycetemcomitans that is involved in the production of fimbriae by this periodontal pathogen. The first three open reading frames in this H. ducreyi gene cluster encoded predicted proteins with a high degree of identity to the Flp (fimbria-like protein) encoded by the first open reading frame of the tad locus; this 15-gene cluster in H. ducreyi was designated flp. RT-PCR analysis indicated that the H. ducreyi flp gene cluster was likely to be a polycistronic operon. Mutations within the flp gene cluster resulted in an inability to form microcolonies in the presence of human foreskin fibroblasts. In addition, the same mutants were defective in the ability to attach to both plastic and human foreskin fibroblasts in vitro. An H. ducreyi mutant with an inactivated tadA gene exhibited a small decrease in virulence in the temperature-dependent rabbit model for experimental chancroid, whereas another H. ducreyi mutant with inactivated flp-1 and flp-2 genes was as virulent as the wild-type parent strain. These results indicate that the flp gene cluster is essential for microcolony formation by H. ducreyi, whereas this phenotypic trait is not linked to the virulence potential of the pathogen, at least in this animal model of infection.
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Twitching motility is a flagella-independent form of bacterial translocation over moist surfaces. It occurs by the extension, tethering, and then retraction of polar type IV pili, which operate in a manner similar to a grappling hook. Twitching motility is equivalent to social gliding motility in Myxococcus xanthus and is important in host colonization by a wide range of plant and animal pathogens, as well as in the formation of biofilms and fruiting bodies. The biogenesis and function of type IV pili is controlled by a large number of genes, almost 40 of which have been identified in Pseudomonas aeruginosa. A number of genes required for pili assembly are homologous to genes involved in type II protein secretion and competence for DNA uptake, suggesting that these systems share a common architecture. Twitching motility is also controlled by a range of signal transduction systems, including two-component sensor-regulators and a complex chemosensory system.
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Streptomycetes exhibit a complex morphological differentiation. After a submerged mycelium has been formed, filaments grow into the air to septate into spores. A class of eight hydrophobic secreted proteins, ChpA-H, was shown to be instrumental in the development of Streptomyces coelicolor. Mature forms of ChpD-H are up to 63 amino acids in length, and those of ChpA-C are larger (+/-225 amino acids). ChpA-C contain two domains similar to ChpD-H, as well as a cell-wall sorting signal. The chp genes were expressed in submerged mycelium (chpE and chpH) as well as in aerial hyphae (chpA-H). Formation of aerial hyphae was strongly affected in a strain in which six chp genes were deleted (DeltachpABCDEH). A mixture of ChpD-H purified from cell walls of aerial hyphae complemented the DeltachpABCDEH strain extracellularly, and it accelerated development in the wild-type strain. The protein mixture was highly surface active, and it self-assembled into amyloid-like fibrils at the water-air interface. The fibrils resembled those of a surface layer of aerial hyphae. We thus conclude that the amyloid-like fibrils of ChpD-H lower the water surface tension to allow aerial growth and cover aerial structures, rendering them hydrophobic. ChpA-C possibly bind ChpD-H to the cell wall.
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Adhesion to host cells is a precursor to host colonization and evasion of the host immune response. Conversely, it triggers the induction of the immune response, a process vital to the host's defence against infection. Adhesins are microbial cell surface molecules or structures that mediate the attachment of the microbe to host cells and thus the host-pathogen interaction. They also play a crucial role in bacterial aggregation and biofilm formation. In this review, we discuss the role of adhesins in the pathogenesis of the aetiological agent of tuberculosis, Mycobacterium tuberculosis. We also provide insight into the structure and characteristics of some of the characterized and putative M. tuberculosis adhesins. Finally, we examine the potential of adhesins as targets for the development of tuberculosis control strategies.
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Central to the paradigm of the pathogenesis of Mycobacterium tuberculosis is its ability to attach to, enter, and subsequently survive in host macrophages. However, little is known regarding the bacterial adhesins and invasins involved in this interaction with host macrophages. Pili are cell-surface structures produced by certain bacteria and have been implicated in adhesion to and invasion of phagocytes in several species. M. tuberculosis pili (MTP) are encoded by the Rv3312A (mtp) gene. In the present study, we assessed the ability of a Δmtp mutant and an mtp-complemented clinical strain to adhere to and invade THP-1 macrophages in comparison with the parental strain by determining colony-forming units. Both adhesion to and invasion of macrophages, although not reaching significance, were markedly reduced by 42.16% (P = 0.107) and 69.02% (P = 0.052), respectively, in the pili-deficient Δmtp mutant as compared with the wild-type. The pili-overexpressing complemented strain showed significantly higher levels of THP-1 macrophage adhesion (P = 0.000) and invasion (P = 0.040) than the mutant. We, thus, identified a novel adhesin and invasin of M. tuberculosis involved in adhesion to and invasion of macrophages.
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Many bacteria assemble extracellular amyloid fibers on their cell surface. Secretion of proteins across membranes and the assembly of complex macromolecular structures must be highly coordinated to avoid the accumulation of potentially toxic intracellular protein aggregates. Extracellular amyloid fiber assembly poses an even greater threat to cellular health due to the highly aggregative nature of amyloids and the inherent toxicity of amyloid assembly intermediates. Therefore, temporal and spatial control of amyloid protein secretion is paramount. The biogenesis and assembly of the extracellular bacterial amyloid curli is an ideal system for studying how bacteria cope with the many challenges of controlled and ordered amyloid assembly. Here, we review the recent progress in the curli field that has made curli biogenesis one of the best-understood functional amyloid assembly pathways. This article is part of a Special Issue entitled: Protein trafficking & Secretion.
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Organized bacterial communities, or biofilms, provide an important reservoir for persistent cells that are inaccessible or tolerant to antibiotics. Curli pili are cell-surface structures produced by certain bacteria and have been implicated in biofilm formation in these species. In order to determine whether these structures, which were suggested to be encoded by the Rv3312A (mtp) gene, have a similar role in Mycobacterium tuberculosis, we generated a Δmtp mutant and a mtp-complemented strain of a clinical isolate of M. tuberculosis and analyzed these strains for their ability to produce pili in comparison to the wild-type strain. Phenotypic analysis by transmission electron microscopy proved the essentiality of mtp for piliation in M. tuberculosis. We then compared biofilm formation of the derived strains in detergent-free Sauton's media. Biofilm mass was quantified spectrophotometrically using crystal violet. Furthermore, we examined mtp gene expression by quantitative real-time PCR in wild-type cells grown under biofilm versus planktonic growth conditions. We found a 68.4 % reduction in biofilm mass in the mutant compared to the wild-type strain (P = 0.002). Complementation of the mutant resulted in a restoration of the wild-type biofilm phenotype (P = 0.022). We, however, found no significant difference between mtp expression in cells of the biofilm to those growing planktonically. Our findings highlight a crucial, but non-specific, role of pili in the biofilm lifestyle of M. tuberculosis and indicate that they may represent an important target for the development of therapeutics to attenuate biofilm formation, thereby potentially reducing persistence.
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Galabiose (Galα1-4Gal) derivatives were coupled to carrier molecules having mono-, di-, tri-, and tetrameric functionalities, thus creating mono- to tetravalent dendritic galabiosides (1−9). The di- to tetravalent galabiosides were several hundred times more efficient than the monomeric galabiosides in inhibiting hemagglutination by the Gram-positive bacterium Streptococcus suis, resulting in complete inhibition at low nanomolar concentrations. This is unprecedented in the field of inhibition of bacterial adhesion by soluble compounds.
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Amyloid is a distinct β-sheet-rich fold that many proteins can acquire. Frequently associated with neurodegenerative diseases in humans, including Alzheimer's, Parkinson's and Huntington's diseases, amyloids are traditionally considered the product of protein misfolding. However, the amyloid fold is now recognized as a ubiquitous part of normal cellular biology. Functional amyloids have been identified in nearly all facets of cellular life, with microbial functional amyloids leading the way. Unlike disease-associated amyloids, functional amyloids are assembled by dedicated, directed pathways and ultimately perform a physiological function that benefits the organism. The evolved amyloid assembly and disassembly pathways of microbes have provided novel insights into how cells have harnessed the amyloid assembly process for productive means. An understanding of functional amyloid biogenesis promises to provide a fresh perspective on the molecular events that underlie disease-associated amyloidogenesis. Here, we review functional microbial amyloids with an emphasis on curli fibers and their role in promoting biofilm formation and other community behaviors.
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Listeriolysin O (LLO) is the major factor implicated in the escape of Listeria monocytogenes from the phagolysosome. It is the only representative of cholesterol-dependent cytolysins that exhibits pH-dependent activity. Despite intense studies of LLO pH-dependence, this feature of the toxin still remains incompletely explained. Here we used fluorescence and CD spectroscopy to show that the structure of LLO is not detectably affected by pH at room temperature. We observed slightly altered haemolytic and permeabilizing activities at different pH values, which we relate to reduced binding of LLO to the lipid membranes. However, alkaline pH and elevated temperatures caused rapid denaturation of LLO. Aggregates of the toxin were able to bind Congo red and Thioflavin T dyes and were visible under transmission electron microscopy as large, amorphous, micrometer-sized assemblies. The aggregates had the biophysical properties of amyloid. Analytical ultracentrifugation indicated dimerization of the protein in acidic conditions, which protects the protein against premature denaturation in the phagolysosome, where toxin activity takes place. We therefore suggest that LLO spontaneously aggregates at the neutral pH found in the host cell cytosol and that this is a major mechanism of LLO inactivation. Structured digital abstract
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Clostridium perfringens is an anaerobic, Gram-positive bacterium that causes a range of diseases in humans, including lethal gas gangrene. We have recently shown that strains of C. perfringens move across the surface of agar plates by a unique type IV pilus (TFP)-mediated social motility that had not been previously described. Based on sequence homology to pilins in Gram-negative bacteria, C. perfringens appears to have two pilin subunits, PilA1 and PilA2. Structural prediction analysis indicated PilA1 is similar to the pseudopilin found in Klebsiella oxytoca, while PilA2 is more similar to true pilins found in the Gram-negative pathogens Pseudomonas aeruginosa and Neisseria gonorrhoeae. Strains of N. gonorrhoeae that were genetically deficient in the native pilin, PilE, but supplemented with inducible expression of PilA1 and PilA2 of C. perfringens were constructed. Genetic competence, wild-type twitching motility, and attachment to human urogenital epithelial cells were not restored by expression of either pilin. However, attachment to mouse and rat myoblast (muscle) cell lines was observed with the N. gonorrhoeae strain expressing PilA2. Significantly, wild-type C. perfringens cells adhered to mouse myoblasts under anaerobic conditions, and adherence was 10-fold lower in a pilT mutant that lacked functional TFP. These findings implicate C. perfringens TFP in the ability of C. perfringens to adhere to and move along muscle fibers in vivo, which may provide a therapeutic approach to limiting this rapidly spreading and highly lethal infection.
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Summary Amyloids are highly abundant in many microbial biofilms and may play an important role in their architecture. Nevertheless, little is known of the amyloid proteins. We report the discovery of a novel functional amyloid expressed by a Pseudomonas strain of the P. fluorescens group. The amyloid protein was purified and the amyloid-like structure verified. Partial sequencing by MS/MS combined with full genomic sequencing of the Pseudomonas strain identified the gene coding for the major subunit of the amyloid fibril, termed fapC. FapC contains a thrice repeated motif that differs from those previously found in curli fimbrins and prion proteins. The lack of aromatic residues in the repeat shows that aromatic side chains are not needed for efficient amyloid formation. In contrast, glutamine and asparagine residues seem to play a major role in amyloid formation as these are highly conserved in curli, prion proteins and FapC. fapC is conserved in many Pseudomonas strains including the opportunistic pathogen P. aeruginosa and is situated in a conserved operon containing six genes, of which one encodes a fapC homologue. Heterologous expression of the fapA-F operon in Escherichia coli BL21(DE3) resulted in a highly aggregative phenotype, showing that the operon is involved in biofilm formation.
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Bacterial pili have long been recognized as mediators of initial host-pathogen interactions important for the progression of Gram-negative bacterial diseases. An appreciation of the role of pili on virulence in Gram-positive bacteria and the unique properties of their biogenesis is a rapidly emerging area of research. In this review, we focus on recent advances in one of the longest-studied Gram-negative pilus systems, the chaperone/usher assembled pili, along with the newcomer to the field, the sortase-assembled pili of Gram-positive bacteria. In both systems, a wealth of new structural and molecular details has emerged recently. In light of this, we explore similarities between chaperone/usher and sortase-assembled pilus biogenesis and highlight paradigms unique to each, with the goal of using knowledge of each system to raise new questions and inform future studies of the other.
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The increased prevalence of antibiotic resistant bacteria heralds a need for new drugs and novel strategies for identifying better drug targets. One such strategy is to target microbial virulence factors, which are important for causing pathology but are not required for the microbe to survive in vitro. This strategy avoids targeting essential gene functions, which may result in strong evolutionary selection for resistant strains. While this idea remains theoretical, efforts have been increased to develop new antibiotics based on this principle. One virulence process of particular interest to target is biofilm formation because of the associated antibiotic insensitivity of bacteria surviving within biofilms. Cegelski et al. (2009) have recently generated tools to allow researchers to address the relative importance of different bacterial attachment strategies during biofilm formation in vivo in a model for urinary tract infection.
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Most commensal and pathogenic bacteria interacting with eukaryotic hosts express adhesive molecules on their surfaces that promote interaction with host cell receptors or with soluble macromolecules. Even though bacterial attachment to epithelial cells may be beneficial for bacterial colonization, adhesion may come at a cost because bacterial attachment to immune cells can facilitate phagocytosis and clearing. Many pathogenic bacteria have solved this dilemma by producing an antiphagocytic surface layer usually consisting of polysaccharide and by expressing their adhesins on polymeric structures that extend out from the cell surface. In this review, we will focus on the interaction between bacterial adhesins and the host, with an emphasis on pilus-like structures.
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Gram-negative bacteria are known to produce two types of surface organelles: flagella, which are required for motility and chemotaxis, and pili (fimbriae), which play a part in the interaction of bacteria with other bacteria and with eukaryotic host cells. Here we report a third class of E. coli surface organelles for which we propose the name curli. Curli are coiled surface structures composed of a single type of subunit, the curlin, which differs from all known pilin proteins and is synthesized in the absence of a cleavable signal peptide. Although the gene encoding this structural subunit, crl, is present and transcribed in most natural isolates of E. coli, only certain strains are able to assemble the subunit protein into curli. This assembly process occurs preferentially at growth temperatures below 37 degrees C. The ability of curli to mediate binding to fibronectin may be a virulence-associated property for wound colonization and for the colonization of fibronectin-coated surfaces.
Article
Bacterial adhesion to other bacteria, to eukaryotic cells, and to extracellular matrix proteins is frequently mediated by cell surface-associated polymers (fimbriae) consisting of one or more subunit proteins. We have found that polymerization of curlin to fimbriae-like structures (curli) on the surface of Escherichia coli markedly differs from the prevailing model for fimbrial assembly in that it occurs extracellularly through a self-assembly process depending on a specific nucleator protein. The cell surface-bound nucleator primes the polymerization of curlin secreted by the nucleator-presenting cell or by adjacent cells. The addition of monomers to the growing filament seems to be driven by mass action and guided only by the diffusion gradient between the source of secreted monomer and the surface of monomer condensation.
Article
Two divergently transcribed operons in Escherichia coli required for the expression of fibronectin- and Congo red-binding curli polymers were identified and characterized by transposon mutagenesis, sequencing and transcriptional analyses, as well as for their ability to produce the curli subunit protein. The csgBA operon encodes CsgA, the major subunit protein of the fibre, and CsgB, a protein with sequence homology to CsgA. A non-polar csgB mutant is unaffected in its production of CsgA, but the subunit protein is not assembled into insoluble fibre polymers. A third open reading frame, orfC, positioned downstream of csgA may affect some functional property of curli since an insertion in this putative gene abolishes the autoagglutinating ability typical of curliated cells without affecting the production of the fibre. The promoter for the oppositely transcribed csgDEFG operon was identified by primer extension and shown, like the csgBA promoter, to be dependent upon the alternate stationary phase-specific sigma factor sigma s in wild-type cells, but not in mutants lacking the nucleoid associated protein H-NS. Insertions in csgD abolish completely trancription from the csgBA promoter. Therefore, any regulatory effect on the csgBA promoter might be secondary to events controlling the csgDEFG promoter and/or activation of CsgD. Insertions in csgE, csgF and csgG abolish curli formation but allow CsgA expression suggesting that one or more of these gene products are involved in secretion/assembly of the CsgA subunit protein. No amino acid sequence homologies were found between the CsgE, CsgF and CsgG proteins and secretion/assembly proteins for other known bacterial fibres, suggesting that the formation of curli follows a novel pathway.
Article
Curli, an adhesive surface fibre produced by Escherichia coli and salmonellae, was proposed on the basis of genetic evidence to follow a distinct assembly pathway involving an extracellular intermediate of the fibre subunit CsgA, the polymerization of which can be induced at the cell surface by a 'nucleator' protein (CsgB). Here we show biochemically that CsgA is actively secreted to the extracellular milieu and that CsgB is surface located. We demonstrate that the putative curli assembly factor CsgG is an outer membrane-located lipoprotein. CsgG is highly resistant to protease digestion both in vivo and in vitro. During curli assembly, CsgG is required to maintain the stability of CsgA and CsgB. In line with this, it is possible to modulate the steady-state levels of CsgA and CsgB by varying intracellular levels of CsgG. This suggests that, in the absence of CsgG, CsgA and CsgB are proteolytically degraded. Moreover, curli production and steady-state levels of CsgA and CsgB can be increased above wild-type levels by overexpression of CsgG, meaning that the quantity of assembled curli fibres can be controlled by this lipoprotein.
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Specific adhesion to host tissue cells is an essential virulence factor of most bacterial pathogens. The fundamental processes that determine bacterial attachment to host tissue surfaces are mediated by microbial adhesins. Host specificity and tissue tropism are characteristics exhibited by different bacteria and are determined (at least in part) by the interaction between adhesins and their complementary receptors on host cell surfaces. A detailed picture of how bacteria are able to target to various receptors is emerging. A large number of bacterial adhesins with individual receptor specificities have been identified. Furthermore, recent research has shown that individual adhesins are prone to rapid microevolution that results in changes in the receptor specificity of individual adhesins. Microbial adhesins are often assembled into complex polymeric organelle structures, however non-organelle adhesins linked to the cell surface as monomers or simple oligomers also exist. This review gives an overview of bacterial adhesins and focuses on some general aspects of their biogenesis and role in bacterial colonization of host cell surfaces and as virulence factors.
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The Gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans forms an extremely tenacious biofilm on solid surfaces such as glass, plastic and hydroxyapatite. This characteristic is likely to be important for colonization of the oral cavity and initiation of a potentially devastating form of periodontal disease. Genetic analysis has revealed a cluster of tad genes responsible for tight adherence to surfaces. Evidence indicates that the tad genes are part of a locus encoding a novel secretion system for the assembly and release of long, bundled Flp pili. Remarkably similar tad loci appear in the genomes of a wide variety of Gram-negative and Gram-positive bacteria, including many significant pathogens, and in Archaea. We propose that the tad loci are important for microbial colonization in a variety of environmental niches.
Article
This study was aimed at characterizing a cell-surface 25 kDa glycoprotein (GP25) that was previously shown to be underproduced by a spontaneous adhesion-defective mutant D5 of Ruminococcus albus 20. An antiserum against wild-type strain 20 was adsorbed with the mutant D5 to enrich it in antibodies 'specific' to adhesion structures of R. albus 20. The resulting antiserum, called anti-Adh serum, blocked adhesion of R. albus 20 and reacted mainly with GP25 in bacterial and extracellular protein fractions of R. albus 20. The N-terminal sequence of purified GP25 was identical to that of CbpC, a 21 kDa cellulose-binding protein (CBP) of R. albus 8. The nucleotide sequence of the gp25 gene was determined by PCR and genomic walking procedures. The gp25 gene encoded a protein of 165 aa with a calculated molecular mass of 16940 Da that showed 72.9% identity with CbpC and presented homologies with type IV pilins of Gram-negative pathogenic bacteria. Negative-staining electron microscopy revealed fine and flexible pili surrounding R. albus 20 cells while mutant cells were not piliated. In addition, immunoelectron microscopy showed that the anti-Adh serum probing mainly GP25, completely decorated the pili surrounding R. albus 20, thereby showing that GP25 was a major pilus subunit. This study shows for the first time the presence of pili at the surface of R. albus and identifies GP25 as their major protein subunit. Though GP25 was not identified as a CBP, isolated pili were shown to bind cellulose. In conclusion, these pili, which belong to the family of type IV pili, mediate adhesion of R. albus 20 to cellulose.
Article
Endobronchial infection is associated with pulmonary tuberculosis in the majority of cases. We have investigated the adherence of Mycobacterium tuberculosis to the human respiratory mucosa. Organ cultures constructed with human tissue were infected with M. tuberculosis in the presence or absence of mycobacterial fibronectin attachment cell surface proteins and examined by scanning electron microscopy. M. tuberculosis adhered mainly to extracellular matrix (ECM) in areas of mucosal damage, but not to ciliated mucosa, intact extruded cells, basement membrane or collagen fibres. Bacteria also adhered to fibrous but not globular mucus and occasionally to healthy unciliated mucosa, open tight junctions and to extruded cells that had degenerated, exposing their contents. There was a significant reduction (p<0.05) in the number of bacteria adhering to ECM after pre-incubation of bacteria with fibronectin and after pre-incubation of the tissue with M. avium fibronectin attachment protein (FAP) and M. bovis antigen 85B protein, in a concentration dependent manner. The combined effect of FAP and antigen 85B protein was significantly greater than either protein alone. Bacterial adherence to fibrous mucus was not influenced by fibronectin. We conclude that M. tuberculosis adheres to ECM in areas of mucosal damage at least in part via FAP and antigen 85B protein.
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The mechanisms by which DNA is taken up into the bacterial cell during natural genetic transformation are poorly understood. Although related components essential to the uptake of DNA during transformation have been defined in Gram-negative species, it remains unclear whether DNA binding and uptake are dissociable events. Therefore, DNA uptake has been the earliest definable step in any Gram-negative transformation pathway. In the human pathogen Neisseria gonorrhoeae, sequence-specific DNA uptake requires an intact type IV pili (Tfp) biogenesis machinery along with three molecules that are dispensable for Tfp expression: ComP (a pilin subunit-like molecule), PilT (a cytoplasmic protein involved in pilus retraction) and ComE (a periplasmic protein with intrinsic DNA-binding activity). By conditionally altering the levels of ComP and PilT expression, we show here that DNA binding and uptake are resolvable events. Consequently, we are able to demonstrate that PilT is largely dispensable for functional DNA binding and, therefore, contributes specifically to uptake. Furthermore, sequence specificity in this system is imposed at the level of DNA binding, a process that is influenced by both ComP and PilE. However, sequence-specific DNA binding is not attributable to an intrinsic property of the Tfp subunit protein. Finally, we demonstrate the existence of a robust, non-specific DNA-binding activity associated with the expression of both Tfp and PilT, which is unrelated to transformation but obscures the observation of specific binding events.
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Colonization of the human small intestine by Vibrio cholerae requires the type 4 toxin co-regulated pilus (TCP). Genes encoding the structure and biogenesis functions of TCP are organized within an operon located on the Vibrio Pathogenicity Island (VPI). In an effort to elucidate the functions of proteins involved in TCP biogenesis, in frame deletions of all of the genes within the tcp operon coding for putative pilus biogenesis proteins have been constructed and the resulting mutants characterized with respect to the assembly and function of TCP. As a result of this analysis, we have identified the product of one of these genes, tcpF, as a novel secreted colonization factor. Chromosomal deletion of tcpF yields a mutant that retains in vitro phenotypes associated with the assembly of functional TCP yet is severely attenuated for colonization of the infant mouse intestine. Furthermore, we have determined that the mechanism by which TcpF is translocated across the bacterial outer membrane requires the TCP biogenesis machinery and is independent of the type II extracellular protein secretion (EPS) system. These results suggest a dual role for the TCP biogenesis apparatus in V. cholerae pathogenesis and a novel mechanism of intestinal colonization mediated by a soluble factor.
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An intact Haemophilus ducreyi flp operon is essential for microcolony formation in vitro. tadA is the 9th of 15 genes in the operon and has homology to NTPases of type IV secretion systems. Fifteen human volunteers were experimentally infected with both H. ducreyi 35000HP and the tadA mutant, 35000HP.400. Papules developed at similar rates at sites inoculated with the mutant and parent, while pustules formed at 36.4% of parent sites and at 0% of mutant sites (P = 0.001). Compared to 35000HP, 35000HP.400 had only a modest but significant reduction in lesion scores in the temperature-dependent rabbit model of chancroid. These data suggest that proteins secreted by the flp locus are required for full expression of virulence by H. ducreyi in humans but have less of a role in virulence in an animal model of infection.
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Bacterial adherence and biofilm formation in implant-associated infection may vary depending on species of microorganisms and characteristics of implant surfaces. We evaluated the differences in adherence and biofilm formation between Staphylococcus epidermidis and Mycobacterium tuberculosis on various spinal implant surfaces. In implant-associated infections, bacteria in biofilm are resistant to antibiotics or host defense mechanism. The removal of implants is usually necessary to eradicate infection. On the contrary, in tuberculous infection, complete debridement and antituberculous chemotherapy without removal of implant have been regarded as a safe procedure. S. epidermidis and M. tuberculosis were cultured with 4 types of metal segments, smooth or rough-surfaced stainless steel, and titanium alloy, respectively. After isolation of colonized microorganisms and biofilm with trypsin treatment and culture on plate media, colony-forming units were counted. The features of adherence and biofilm formation were observed under scanning electron microscopy. Biofilm-forming S. epidermidis showed heavy adhesion and multiplication on the surface of all 4 metal segments, 16.5 times more colony-forming units than nonbiofilm-forming ones. On scanning electron microscopy, there were many aggregated microcolonies with thick biofilm in biofilm-forming S. epidermidis but much less in nonbiofilm-forming S. epidermidis. M. tuberculosis were rarely adhered to metal surfaces and showed scanty biofilm formation. On the contrary to S. epidermidis, adherence and biofilm formation of M. tuberculosis on implant surface are less likely, and it can provide the basis of successful instrumentation in spine tuberculosis.
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Immunohistochemical study was done by harvesting articular cartilage of the facet joints during the decompressive surgery for spinal stenosis. To observe the expression of estrogen receptor on the articular cartilage of the facet joints in degenerative spondylolisthesis (DS) SUMMARY OF BACKGROUND DATA: Few attempts have been made to evaluate the effect of sex-hormone, although DS is more common in females than in males. After harvesting the articular cartilage of the facet joints in 17 DS and in 15 spinal stenosis (SS) patients, the expression of estrogen receptor and the severity of facet arthritis were observed by H-E and immunohistochemical staining, respectively. Measurements of both staining were made by using a semiquantitative analysis. The significantly increased expression of estrogen receptor correlated with the severity of facet arthritis (r = 0.78, P < 0.05). There was a significantly increased expression of estrogen receptor of the facet joint in DS compared with SS (P < 0.01). The histologic-histochemical grading of cartilage lesion in DS was 12.4 (SEM, 0.6), which was significantly higher than in SS (P < 0.05). These findings suggest that the higher expression of estrogen receptor might aggravate degenerative change of the facet articular cartilage and might also be considered one of the causative factors for DS in postmenopausal women.