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Actinomyces oris plays an important role in oral biofilm development. Like many gram-positive bacteria, A. oris produces a sizable number of surface proteins that are anchored to bacterial peptidoglycan by a conserved transpeptidase named the housekeeping sortase SrtA; however, the biological role of many A. oris surface proteins in biofilm formation is largely unknown. Here, we report that the glycoprotein GspA-a genetic suppressor of srtA deletion lethality-not only promotes biofilm formation but also maintains cell membrane integrity under cation stress. In comparison to wild-type cells, under elevated concentrations of mono- and divalent cations the formation of mono- and multi-species biofilms by mutant cells devoid of gspA was significantly diminished, although planktonic growth of both cell types in the presence of cations was indistinguishable. Because gspA overexpression is lethal to cells lacking gspA and srtA, we performed a genetic screen to identify GspA determinants involving cell viability. DNA sequencing and biochemical characterizations of viable clones revealed that mutations of two critical cysteine residues and a serine residue severely affected GspA glycosylation and biofilm formation. Furthermore, mutant cells lacking gspA were markedly sensitive to sodium dodecyl sulfate, a detergent that solubilizes the cytoplasmic membranes, suggesting the cell envelope of the gspA mutant was altered. Consistent with this observation, the gspA mutant exhibited increased membrane permeability, independent of GspA glycosylation, compared to the wild-type strain. Altogether, the results support the notion that the cell wall-anchored glycoprotein GspA provides a defense mechanism against cation stress in biofilm development promoted by A. oris.

A gram-negative colonizer of the oral cavity, Fusobacterium nucleatum not only interacts with many pathogens in the oral microbiome but also has the ability to spread to extraoral sites including placenta and amniotic fluid, promoting preterm birth. To date, however, the molecular mechanism of interspecies interactions-termed coaggregation-by F. nucleatum and how coaggregation affects bacterial virulence remain poorly defined. Here, we employed genome-wide transposon mutagenesis to uncover fusobacterial coaggregation factors, revealing the intertwined function of a two-component signal transduction system (TCS), named CarRS, and a lysine metabolic pathway in regulating the critical coaggregation factor RadD. Transcriptome analysis shows that CarR modulates a large regulon including radD and lysine metabolic genes, such as kamA and kamD, the expression of which are highly up-regulated in the DeltacarR mutant. Significantly, the native culture medium of DeltakamA or DeltakamD mutants builds up abundant amounts of free lysine, which blocks fusobacterial coaggregation with streptococci. Our demonstration that lysine-conjugated beads trap RadD from the membrane lysates suggests that lysine utilizes RadD as its receptor to act as a metabolic inhibitor of coaggregation. Lastly, using a mouse model of preterm birth, we show that fusobacterial virulence is significantly attenuated with the DeltakamA and DeltacarR mutants, in contrast to the enhanced virulence phenotype observed upon diminishing RadD (DeltaradD or DeltacarS mutant). Evidently, F. nucleatum employs the TCS CarRS and environmental lysine to modulate RadD-mediated interspecies interaction, virulence, and nutrient acquisition to thrive in the adverse environment of oral biofilms and extraoral sites.

Controlled RNA degradation is a crucial process in bacterial cell biology for maintaining proper transcriptome homeostasis and adaptation to changing environments. mRNA turnover in many Gram-positive bacteria involves a specialized ribonuclease called RNase J (RnJ). To date, however, nothing is known about this process in the diphtheria-causative pathogen Corynebacterium diphtheriae, nor is known the identity of this ribonuclease in this organism. Here, we report that C. diphtheriae DIP1463 encodes a predicted RnJ homolog, comprised of a conserved N-terminal β-lactamase domain, followed by β-CASP and C-terminal domains. A recombinant protein encompassing the β-lactamase domain alone displays 5′-exoribonuclease activity, which is abolished by alanine-substitution of the conserved catalytic residues His186 and His188. Intriguingly, deletion of DIP1463/rnj in C. diphtheriae reduces bacterial growth and generates cell shape abnormality with markedly augmented cell width. Comparative RNA-seq analysis revealed that RnJ controls a large regulon encoding many factors predicted to be involved in biosynthesis, regulation, transport, and iron acquisition. One upregulated gene in the ∆rnj mutant is ftsH, coding for a membrane protease (FtsH) involved in cell division, whose overexpression in the wild-type strain also caused cell-width augmentation. Critically, the ∆rnj mutant is severely attenuated in virulence in a Caenorhabditis elegans model of infection, while the FtsH-overexpressing and toxin-less strains exhibit full virulence as the wild-type strain. Evidently, RNase J is a key ribonuclease in C. diphtheriae that post-transcriptionally influences the expression of numerous factors vital to corynebacterial cell physiology and virulence. Our findings have significant implications for basic biological processes and mechanisms of corynebacterial pathogenesis.

In Gram-positive bacteria, the peptidoglycan serves as a placeholder for surface display of a unique class of monomeric and polymeric proteins, or pili - the precursors of which harbor a cell wall sorting signal with LPXTG motif that is recognized by a conserved transpeptidase enzyme called sortase. Since this original discovery over two decades ago, extensive genetic, biochemical and structural studies have illuminated the basic mechanisms of sortase-mediated cell wall anchoring of surface proteins and pili. We now know how LPXTG-containing surface proteins are folded post-translocationally, how sortase enzymes recognize substrates, and how a remnant of the cell wall sorting signal modulates intramembrane signaling. In this review, we will highlight new findings from a few model experimental paradigms and present future prospects for the field.

This article describes several established approaches for genetic manipulation of Corynebacterium diphtheriae, the causative agent of diphtheria that is known to have provided key evidence for Koch's postulates on the germ theory. First, it includes a detailed gene deletion method that generates nonpolar, in-frame, markerless deletion mutants, utilizing the levansucrase SacB as a counter-selectable marker. Second, it provides a thorough protocol for rescuing deletion mutants using Escherichia coli-Corynebacterium shuttle vectors. Finally, a Tn5 transposon mutagenesis procedure is described. In principle, these protocols can be used for other Corynebacterium species, including Corynebacterium glutamicum and Corynebacterium matruchotii. © 2020 Wiley Periodicals LLC Basic Protocol 1: Gene deletion in Corynebacterium diphtheriae Basic Protocol 2: Complementation of a mutant strain Basic Protocol 3: Tn5 transposon mutagenesis of Corynebacterium diphtheriae.

Corynebacterium diphtheriae is the leading cause of pharyngeal diphtheria, a respiratory disease characterized by formation of a pseudomembrane at the site of infection. Although outbreaks of C. diphtheriae infections are rare nowadays, the emergence of multidrug-resistant C. diphtheriae strains is one of the most significant public health concerns worldwide. Although C. diphtheriae has been studied for more than a century and diphtheria toxin and pili have been identified as major virulence factors, little is known about factors involved in bacterial colonization and development of disease. Here, we describe the utilization of Caenorhabditis elegans as a cost-effective, versatile model of infection to evaluate C. diphtheriae virulence. We provide detailed protocols for nematode synchronization and for evaluation of nematode survival and formation of a deformed anal region induced by C. diphtheriae infection. These protocols will permit future high-throughput screenings of virulence factors in C. diphtheriae and advance our knowledge of C. diphtheriae pathogenesis. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synchronization of nematodes Basic Protocol 2: Assay for nematode survival following C. diphtheriae infection Basic Protocol 3: Assays for bacterial colonization and formation of deformed anal region.

Considered a commensal, the Gram-negative anaerobe Fusobacterium nucleatum is a key member of the oral microbiome due to its wide range of interactions with many oral microbes. While the periodontal pathogenic properties of this organism have widely been examined, its connotation with extra-oral infections, including preterm birth and colorectal cancer, has now become apparent. Nonetheless, little is known about the mechanisms of pathogenicity and the associated virulence factors of F. nucleatum, most likely due to limited genetic tools and facile methodology. Here, we describe molecular techniques for the genetic manipulation of F. nucleatum, including markerless, nonpolar gene deletion, complementation, and Tn5 transposon mutagenesis. Further, we provide methodology to assess virulence potential of F. nucleatum using a mouse model of preterm birth. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of a galK mutant strain Basic Protocol 2: Complementation of a mutant strain Basic Protocol 3: Tn5 transposon mutagenesis of F. nucleatum Basic Protocol 4: Mouse model of preterm birth.

Adhesive pili in Gram-positive bacteria represent a variety of extracellular multiprotein polymers that mediate bacterial colonization of specific host tissues and associated pathogenesis. Pili are assembled in two distinct but coupled steps, an orderly crosslinking of pilin monomers and subsequent anchoring of the polymer to peptidoglycan, catalyzed by two transpeptidase enzymes - the pilus-specific sortase and the housekeeping sortase. Here, we review this biphasic assembly mechanism based on studies of two prototypical models, the heterotrimeric pili in Corynebacterium diphtheriae and the heterodimeric pili in Actinomyces oris, highlighting some newly emerged basic paradigms. The disparate mechanisms of protein ligation mediated by the pilus-specific sortase and the spatial positioning of adhesive pili on the cell surface modulated by the housekeeping sortase are among the notable highlights.

Significance Vancomycin-resistant enterococci are hospital-associated pathogens that evolved resistance to most antibiotics used in clinical practice. Daptomycin, a lipopeptide antibiotic used as frontline therapy for severe multidrug-resistant enterococcal infections, targets the bacterial cell membrane (CM). The LiaFSR stress response system orchestrates daptomycin and antimicrobial peptide (AMP) resistance by modulating CM phospholipid content or localization. Here, we identify a single protein (LiaX) that senses antimicrobial molecules and regulates changes in CM phospholipid architecture. We show that LiaX-mediated modulation of antibiotic and AMP resistance affects virulence during infection caused by a recalcitrant hospital pathogen. Targeting this response in multidrug-resistant organisms may be a therapeutic intervention to restore susceptibility to cell envelope-targeting antibiotics and increase the ability of the immune system to clear pathogens., Bacteria have developed several evolutionary strategies to protect their cell membranes (CMs) from the attack of antibiotics and antimicrobial peptides (AMPs) produced by the innate immune system, including remodeling of phospholipid content and localization. Multidrug-resistant Enterococcus faecalis, an opportunistic human pathogen, evolves resistance to the lipopeptide daptomycin and AMPs by diverting the antibiotic away from critical septal targets using CM anionic phospholipid redistribution. The LiaFSR stress response system regulates this CM remodeling via the LiaR response regulator by a previously unknown mechanism. Here, we characterize a LiaR-regulated protein, LiaX, that senses daptomycin or AMPs and triggers protective CM remodeling. LiaX is surface exposed, and in daptomycin-resistant clinical strains, both LiaX and the N-terminal domain alone are released into the extracellular milieu. The N-terminal domain of LiaX binds daptomycin and AMPs (such as human LL-37) and functions as an extracellular sentinel that activates the cell envelope stress response. The C-terminal domain of LiaX plays a role in inhibiting the LiaFSR system, and when this domain is absent, it leads to activation of anionic phospholipid redistribution. Strains that exhibit LiaX-mediated CM remodeling and AMP resistance show enhanced virulence in the Caenorhabditis elegans model, an effect that is abolished in animals lacking an innate immune pathway crucial for producing AMPs. In conclusion, we report a mechanism of antibiotic and AMP resistance that couples bacterial stress sensing to major changes in CM architecture, ultimately also affecting host–pathogen interactions.

The Gram-positive cell envelope serves as a molecular platform for surface display of capsular polysaccharides, wall teichoic acids (WTAs), lipoteichoic acids (LTAs), lipoproteins, surface proteins and pili. WTAs, LTAs, and sortase-assembled pili are a few features that make the Gram-positive cell envelope distinct from the Gram-negative counterpart. Interestingly, a set of LytR-CpsA-Psr family proteins, found in all Gram-positives but limited to a minority of Gram-negative organisms, plays divergent functions, while decorating the cell envelope with glycans. Furthermore, a phylum of Gram-positive bacteria, the actinobacteria, appear to employ oxidative protein folding as the major folding mechanism, typically occurring in an oxidizing environment of the Gram-negative periplasm. These distinctive features will be highlighted, along with recent findings in the cell envelope biogenesis.

In Gram-positive bacteria, the conserved LCP family enzymes studied to date are known to attach glycopolymers, including wall teichoic acid, to the cell envelope. It is unknown if these enzymes catalyze glycosylation of surface proteins. We show here in the actinobacterium Actinomyces oris by X-ray crystallography and biochemical analyses that A. oris LcpA is an LCP homolog, possessing pyrophosphatase and phosphotransferase activities known to belong to LCP enzymes that require conserved catalytic Arg residues, while harboring a unique disulfide bond critical for protein stability. Importantly, LcpA mediates glycosylation of the surface protein GspA via phosphotransferase activity. Our studies provide the first experimental evidence of an archetypal LCP enzyme that promotes glycosylation of a cell wall-anchored protein in Gram-positive bacteria., The widely conserved LytR-CpsA-Psr (LCP) family of enzymes in Gram-positive bacteria is known to attach glycopolymers, including wall teichoic acid, to the cell envelope. However, it is undetermined if these enzymes are capable of catalyzing glycan attachment to surface proteins. In the actinobacterium Actinomyces oris, an LCP homolog here named LcpA is genetically linked to GspA, a glycoprotein that is covalently attached to the bacterial peptidoglycan by the housekeeping sortase SrtA. Here we show by X-ray crystallography that LcpA adopts an α-β-α structural fold, akin to the conserved LCP domain, which harbors characteristic catalytic arginine residues. Consistently, alanine substitution for these residues, R149 and R266, abrogates GspA glycosylation, leading to accumulation of an intermediate form termed GspALMM, which is also observed in the lcpA mutant. Unlike other LCP proteins characterized to date, LcpA contains a stabilizing disulfide bond, mutations of which severely affect LcpA stability. In line with the established role of disulfide bond formation in oxidative protein folding in A. oris, deletion of vkor, coding for the thiol-disulfide oxidoreductase VKOR, also significantly reduces LcpA stability. Biochemical studies demonstrated that the recombinant LcpA enzyme possesses pyrophosphatase activity, enabling hydrolysis of diphosphate bonds. Furthermore, this recombinant enzyme, which weakly interacts with GspA in solution, catalyzes phosphotransfer to GspALMM. Altogether, the findings support that A. oris LcpA is an archetypal LCP enzyme that glycosylates a cell wall-anchored protein, a process that may be conserved in Actinobacteria, given the conservation of LcpA and GspA in these high-GC-content organisms.

Enterococcus faecium is an important cause of hospital-associated infections, including urinary tract infections (UTIs), bacteremia, and infective endocarditis. Pili have been shown to play a role in the pathogenesis of Gram-positive bacteria, including E. faecium . We previously demonstrated that a nonpiliated Δ empABC :: cat derivative of E. faecium TX82 was attenuated in biofilm formation and in a UTI model. Here, we studied the contributions of the individual pilus subunits EmpA, EmpB, and EmpC to pilus architecture, biofilm formation, adherence to extracellular matrix (ECM) proteins, and infection. We identified EmpA as the tip of the pili and found that deletion of empA reduced biofilm formation to the same level as deletion of the empABC operon, a phenotype that was restored by reconstituting in situ the empA gene. Deletion of empB also caused a reduction in biofilm, while EmpC was found to be dispensable. Significant reductions in adherence to fibrinogen and collagen type I were observed with deletion of empA and empB , while deletion of empC had no adherence defect. Furthermore, we showed that each deletion mutant was significantly attenuated in comparison to the isogenic parental strain, TX82, in a mixed-inoculum UTI model ( P < 0.001 to 0.048), that reconstitution of empA restored virulence in the UTI model, and that deletion of empA also resulted in attenuation in an infective endocarditis model ( P = 0.0088). Our results indicate that EmpA and EmpB, but not EmpC, contribute to biofilm and adherence to ECM proteins; however, all the Emp pilins are important for E. faecium to cause infection in the urinary tract.

Disulfide bonds are important for the stability and function of many secreted proteins. In Gram-negative bacteria, these linkages are catalyzed by thiol-disulfide oxidoreductases (Dsb) in the periplasm. Protein oxidation has been well studied in these organisms, but it has not fully been explored in Gram-positive bacteria, which lack traditional periplasmic compartments. Recent bioinformatics analyses have suggested that the high-GC-content bacteria (i.e., actinobacteria) rely on disulfide-bond-forming pathways. In support of this, Dsb-like proteins have been identified in Mycobacterium tuberculosis , but their functions are not known. Actinomyces oris and Corynebacterium diphtheriae have recently emerged as models to study disulfide bond formation in actinobacteria. In both organisms, disulfide bonds are catalyzed by the membrane-bound oxidoreductase MdbA. Remarkably, unlike known Dsb proteins, MdbA is important for pathogenesis and growth, which makes it a potential target for new antibacterial drugs. This review will discuss disulfide-bond-forming pathways in bacteria, with a special focus on Gram-positive bacteria.

The actinobacterium Corynebacterium matruchotii has been implicated in nucleation of oral microbial consortia leading to biofilm formation. Due to the lack of genetic tools, little is known about basic cellular processes, including protein secretion and folding, in this organism. We report here a survey of the C. matruchotii genome, which encodes a large number of exported proteins containing paired cysteine residues, and identified an oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA (MdbA Cd ). Crystallization studies uncovered that the 1.2-Å resolution structure of C. matruchotii MdbA (MdbA Cm ) possesses two conserved features found in actinobacterial MdbA enzymes, a thioredoxin-like fold and an extended α-helical domain. By reconstituting the disulfide bond-forming machine in vitro , we demonstrated that MdbA Cm catalyzes disulfide bond formation within the actinobacterial pilin FimA. A new gene deletion method supported that mdbA is essential in C. matruchotii . Remarkably, heterologous expression of MdbA Cm in the C. diphtheriae Δ mdbA mutant rescued its known defects in cell growth and morphology, toxin production, and pilus assembly, and this thiol-disulfide oxidoreductase activity required the catalytic motif CXXC. Altogether, the results suggest that MdbA Cm is a major thiol-disulfide oxidoreductase, which likely mediates posttranslocational protein folding in C. matruchotii by a mechanism that is conserved in Actinobacteria . IMPORTANCE The actinobacterium Corynebacterium matruchotii has been implicated in the development of oral biofilms or dental plaque; however, little is known about the basic cellular processes in this organism. We report here a high-resolution structure of a C. matruchotii oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA. By biochemical analysis, we demonstrated that C. matruchotii MdbA catalyzes disulfide bond formation in vitro . Furthermore, a new gene deletion method revealed that deletion of mdbA is lethal in C. matruchotii . Remarkably, C. matruchotii MdbA can replace C. diphtheriae MdbA to maintain normal cell growth and morphology, toxin production, and pilus assembly. Overall, our studies support the hypothesis that C. matruchotii utilizes MdbA as a major oxidoreductase to catalyze oxidative protein folding.

The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae.

Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the Δvkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria.

The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6 Enterococcus faecium proteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence in E. faecium and possibly other Gram-positive bacterial species.

Posttranslocational protein folding in the Gram-positive biofilm-forming actinobacterium Actinomyces oris is mediated by a membrane-bound thiol-disulfide oxidoreductase named MdbA, which catalyzes oxidative folding of nascent polypeptides transported by the Sec translocon. Reoxidation of MdbA involves a bacterial v itamin K ep o xide r eductase (VKOR)-like protein that contains four cysteine residues, C93/C101 and C175/C178, with the latter forming a canonical CXXC thioredoxin-like motif; however, the mechanism of VKOR-mediated reoxidation of MdbA is not known. We present here a topological view of the A. oris membrane-spanning protein VKOR with these four exoplasmic cysteine residues that participate in MdbA reoxidation. Like deletion of the VKOR gene, alanine replacement of individual cysteine residues abrogated polymicrobial interactions and biofilm formation, concomitant with the failure to form adhesive pili on the bacterial surface. Intriguingly, the mutation of the cysteine at position 101 to alanine (C101A mutation) resulted in a high-molecular-weight complex that was positive for MdbA and VKOR by immunoblotting and was absent in other alanine substitution mutants and the C93A C101A double mutation and after treatment with the reducing agent β-mercaptoethanol. Consistent with this observation, affinity purification followed by immunoblotting confirmed this MdbA-VKOR complex in the C101A mutant. Furthermore, ectopic expression of the Mycobacterium tuberculosis VKOR analog in the A. oris VKOR deletion (ΔVKOR) mutant rescued its defects, in contrast to the expression of M. tuberculosis VKOR variants known to be nonfunctional in the disulfide relay that mediates reoxidation of the disulfide bond-forming catalyst DsbA in Escherichia coli . Altogether, the results support a model of a disulfide relay, from its start with the pair C93/C101 to the C175-X-X-C178 motif, that is required for MdbA reoxidation and appears to be conserved in members of the class Actinobacteria . IMPORTANCE It has recently been shown in the high-GC Gram-positive bacteria (or Actinobacteria ) Actinomyces oris and Corynebacterium diphtheriae that oxidative folding of nascent polypeptides transported by the Sec machinery is catalyzed by a membrane-anchored oxidoreductase named MdbA. In A. oris , reoxidation of MdbA requires a bacterial VKOR-like protein, and yet, how VKOR mediates MdbA reoxidation is unknown. We show here that the A. oris membrane-spanning protein VKOR employs two pairs of exoplasmic cysteine residues, including the canonical CXXC thioredoxinlike motif, to oxidize MdbA via a disulfide relay mechanism. This mechanism of disulfide relay is essential for pilus assembly, polymicrobial interactions, and biofilm formation and appears to be conserved in members of the class Actinobacteria , including Mycobacterium tuberculosis .

The Gram-positive bacterium Actinomyces oris , a key colonizer in the development of oral biofilms, contains 18 LPXTG motif-containing proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria, A. oris expresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal “glyco-stress,” caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortase srtA is genetically disrupted. Consistent with this finding, we show here that a mutant lacking lepB2 and srtA was unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neither lepB1 nor lepB2 abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, the lepB2 deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, the lepB2 mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely, lepB1 was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity. IMPORTANCE Sec-mediated translocation of bacterial protein precursors across the cytoplasmic membrane involves cleavage of their signal peptide by a signal peptidase (SPase). Like many Gram-positive bacteria, A. oris expresses two SPases, LepB1 and LepB2. The latter is a genetic suppressor of lethal “glyco-stress” caused by membrane accumulation of glycosylated GspA when the housekeeping sortase srtA is genetically disrupted. We show here that LepB1 and LepB2 are capable of processing GspA, whereas only LepB2 is required for cleavage of fimbrial signal peptides. This is the first example of a type I SPase dedicated to LPXTG motif-containing fimbrial proteins. Thus, A. oris provides an experimental model with which to investigate the specificity mechanism of type I SPases.

Pathogenic bacteria adhere despite severe mechanical perturbations induced by the host, such as coughing. In Gram-positive bacteria, extracellular protein appendages termed pili are necessary for adherence under mechanical stress. However, little is known about the behavior of Gram-positive pili under force. Here, we demonstrate a mechanism by which Gram-positive pili are able to dissipate mechanical energy through mechanical unfolding and refolding of isopeptide bond-delimited polypeptide loops present in Ig-type CnaA domains. Using single-molecule force spectroscopy, we find that these loops of the pilus subunit SpaA of the SpaA-type pilus from Corynebacterium diphtheriae and FimA of the type 2 pilus from Actinomyces oris unfold and extend at forces that are the highest yet reported for globular proteins. Loop refolding is limited by the hydrophobic collapse of the polypeptide and occurs in milliseconds. Remarkably, both SpaA and FimA initially refold to mechanically weaker intermediates that recover strength with time or ligand binding. Based on the high force extensibility, CnaA-containing pili can dissipate ∼28-fold as much energy compared with their inextensible counterparts before reaching forces sufficient to cleave covalent bonds. We propose that efficient mechanical energy dissipation is key for sustained bacterial attachment against mechanical perturbations.

As a pioneer colonizer of the oral cavity, Actinomyces oris expresses proteinaceous pili (also called fimbriae) to mediate the following two key events in biofilm formation: adherence to saliva deposits on enamel and interbacterial associations. Assembly of type 2 fimbriae that directly facilitate coaggregation with oral streptococci and Actinomyces biofilm development requires the class C sortase SrtC2. Although the general sortase-associated mechanisms have been elucidated, several structural attributes unique to the class C sortases require functional investigation. Mutational studies reported here suggest that the N-terminal transmembrane (TM) region of SrtC2, predicted to contain a signal peptide sequence, is cleaved off the mature protein and that this processing is critical for the proper integration of the enzyme at the cytoplasmic membrane, which is mediated by the extended hydrophobic C terminus containing a TM domain and a cytoplasmic tail. Deletion of this putative TM or the entire cytoplasmic domain abolished the enzyme localization and functionality. Alanine substitution of the conserved catalytic Cys-His dyad abrogated the SrtC2 enzymatic activity. In contrast, mutations designed to alter a “lid” domain that covers the catalytic pocket of a class C sortase showed no effect on enzyme activity. Finally, each of the deleterious mutations that affected SrtC2 activity or membrane localization also eliminated Actinomyces species biofilm development and bacterial coaggregation with streptococci. We conclude that the N terminus of SrtC2, which contains the signal sequence, is required for proper protein translocation and maturation, while the extended C-terminal hydrophobic region serves as a stable membrane anchor for proper enzyme functionality.

Among Actinomyces spp., Actinomyces oris is the most abundant species in the human oral cavity. This chapter provides an account of the current knowledge of a key adhesive principle, the fimbriae of A. oris, which are considered to be the main players for the cell-cell and cell-substratum interactions involving the early colonizers of dental plaque. The genome sequence of A. oris has led to the identification and characterization of various fimbrial components and the specific enzymes, called sortases, that are responsible for the ordered assembly of fimbrial subunits into covalently cross-linked polymers and subsequent incorporation into the cell wall. The genome sequence also revealed the presence of an unusually large number of putative cell wall-anchored proteins, some of which must serve as additional adhesive principles facilitating the adherence of bridge organisms and late colonizers. Importantly, not only is the S. aureus srtA mutant defective in cell wall anchoring of LPXTG-containing surface proteins but also it is attenuated in animal models of infection. The Actinomyces fimbrial system is a versatile adhesive principle for promoting bacterial coaggregation and host tissue adherence that leads to the development of one of the most complex biofilms, the dental plaque.

By combining X-ray crystallography and modelling, we describe here the atomic structure of distinct adhesive moieties of FimA, the shaft fimbrillin of Actinomyces type 2 fimbriae, which uniquely mediates the receptor-dependent intercellular interactions between Actinomyces and oral streptococci as well as host cells during the development of oral biofilms. The FimA adhesin is built with three IgG-like domains, each of which harbours an intramolecular isopeptide bond, previously described in several Gram-positive pilins. Genetic and biochemical studies demonstrate that although these isopeptide bonds are dispensable for fimbrial assembly, cell-cell interactions and biofilm formation, they contribute significantly to the proteolytic stability of FimA. Remarkably, FimA harbours two autonomous adhesive modules, which structurally resemble the Staphylococcus aureus Cna B domain. Each isolated module can bind the plasma glycoprotein asialofetuin as well as the polysaccharide receptors present on the surface of oral streptococci and epithelial cells. Thus, FimA should serve as an excellent paradigm for the development of therapeutic strategies and elucidating the precise molecular mechanisms underlying the interactions between cellular receptors and Gram-positive fimbriae.

Interaction of Actinomyces oris with salivary proline-rich proteins (PRPs), which serve as fimbrial receptors, involves type 1 fimbriae. Encoded by the gene locus fimQ-fimP-srtC1 , the type 1 fimbria is comprised of the fimbrial shaft FimP and the tip fimbrillin FimQ. Fimbrial polymerization requires the fimbria-specific sortase SrtC1, which catalyzes covalent linkage of fimbrial subunits. Using genetics, biochemical methods, and electron microscopy, we provide evidence that the tip fimbrillin, FimQ, is involved in fimbrial assembly and interaction with PRPs. Specifically, while deletion of fimP completely abolished the type 1 fimbrial structures, surface display of monomeric FimQ was not affected by this mutation. Surprisingly, deletion of fimQ significantly reduced surface assembly of the type 1 fimbriae. This defect was rescued by recombinant FimQ ectopically expressed from a plasmid. In agreement with the role of type 1 fimbriae in binding to PRPs, aggregation of A. oris with PRP-coated beads was abrogated in cells lacking srtC1 or fimP . This aggregation defect of the Δ fimP mutant was mainly due to significant reduction of FimQ on the bacterial surface, as the aggregation was not observed in a strain lacking fimQ . Increasing expression of FimQ in the Δ fimP mutant enhanced aggregation, while overexpression of FimP in the Δ fimQ mutant did not. Furthermore, recombinant FimQ, not FimP, bound surface-associated PRPs in a dose-dependent manner. Thus, not only does FimQ function as the major adhesin of the type 1 fimbriae, it also plays an important role in fimbrial assembly.

Pilus assembly in Gram-positive bacteria occurs by a two-step mechanism, whereby pilins are polymerized and then covalently anchored to the cell wall. In Corynebacterium diphtheriae, the pilin-specific sortase SrtA catalyses polymerization of the SpaA-type pilus, consisting of the shaft pilin SpaA, tip pilin SpaC and minor pilin SpaB. Cell wall anchoring of the SpaA polymers is triggered when SrtA incorporates SpaB into the pilus base via lysine-mediated transpeptidation; anchoring to the cell wall peptidoglycan is subsequently catalysed by the housekeeping sortase SrtF. Here we show that SpaB and SpaC formed a heterodimer independent of SpaA polymerization. SrtA was absolutely required for the formation of the SpaBC heterodimer, while SrtF facilitated the optimal cell wall anchoring of this heterodimer. Alanine substitution of the SpaB lysine residue K139 or truncation of the SpaB cell wall-sorting signal (CWSS) abolished assembly of the SpaBC heterodimer, hence underscoring SpaB function in transpeptidation and cell wall linkage. Importantly, sortase specificity for the cell wall-anchoring step was found to be dependent on the LAFTG motif within the SpaB CWSS. Thus, C. diphtheriae employs a common sortase-catalysed mechanism involving lysine-mediated transpeptidation to generate both adhesive pilus and simple heterodimeric structures on the bacterial the cell wall.

The Gram-positive pathogen Streptococcus agalactiae or group B streptococcus (GBS) is the leading cause of bacterial septicemia, pneumonia and meningitis among neonates around the world. The pathogen assembles two types of pili on its surface, named PI-1 and PI-2, that mediate bacterial adherence to host cells. The GBS PI-1 pilus is formed by the major pilin GBS80, which forms the pilus shaft, and two minor pilins GBS104 and GBS52, which are incorporated into the pilus structure. While considerable structural information exists on Gram-negative pili, the structural study of Gram-positive pili is an emerging area of research. Here, the purification, crystallization and initial phasing of the 35 kDa major fragment of the backbone pilin GBS80 are reported. Crystals were obtained in two different space groups: P2(1) and C2. SAD data collected from an iodide-derivative crystal at the home source were used to obtain initial phases and interpretable electron-density maps.

Summary Streptococcus agalactiae is the leading cause of neonatal pneumonia, sepsis, and meningitis. The pathogen assembles heterotrimeric pilus structures on its surface; however, their function in pathogenesis is poorly understood. We report here the crystal structure of the pilin GBS52, which reveals two IgG-like fold domains, N1 and N2. Each domain is comprised of seven antiparallel β strands, an arrangement similar to the fold observed in the Staphylococcus aureus adhesin Cna. Consistent with its role as an adhesin, deletion of gbs52 gene significantly reduces bacterial adherence to pulmonary epithelial cells. Moreover, latex beads linked to the GBS52 protein adhere to pulmonary but not to many other epithelial cells; binding to the former is specifically inhibited by antibodies against GBS52. Nonetheless, substantial binding is only observed with N2 domain-conjugated beads. This study presents the structure of a Gram-positive pilin that utilizes a distinct IgG fold variant to mediate pathogen adherence to a specific tissue.

Adherence to host tissues mediated by pili is pivotal in the establishment of infection by many bacterial pathogens. Corynebacterium diphtheriae assembles on its surface three distinct pilus structures. The function and the mechanism of how various pili mediate adherence, however, have remained poorly understood. Here we show that the SpaA-type pilus is sufficient for the specific adherence of corynebacteria to human pharyngeal epithelial cells. The deletion of the spaA gene, which encodes the major pilin forming the pilus shaft, abolishes pilus assembly but not adherence to pharyngeal cells. In contrast, adherence is greatly diminished when either minor pilin SpaB or SpaC is absent. Antibodies directed against either SpaB or SpaC block bacterial adherence. Consistent with a direct role of the minor pilins, latex beads coated with SpaB or SpaC protein bind specifically to pharyngeal cells. Therefore, tissue tropism of corynebacteria for pharyngeal cells is governed by specific minor pilins. Importantly, immunoelectron microscopy and immunofluorescence studies reveal clusters of minor pilins that are anchored to cell surface in the absence of a pilus shaft. Thus, the minor pilins may also be cell wall anchored in addition to their incorporation into pilus structures that could facilitate tight binding to host cells during bacterial infection.

Multiple pilus gene clusters have been identified in several gram-positive bacterial genomes sequenced to date, including the Actinomycetales , clostridia, streptococci, and corynebacteria. The genome of Corynebacterium diphtheriae contains three pilus gene clusters, two of which have been previously characterized. Here, we report the characterization of the third pilus encoded by the spaHIG cluster. By using electron microscopy and biochemical analysis, we demonstrate that SpaH forms the pilus shaft, while SpaI decorates the structure and SpaG is largely located at the pilus tip. The assembly of the SpaHIG pilus requires a specific sortase located within the spaHIG pilus gene cluster. Deletion of genes specific for the synthesis and polymerization of the other two pilus types does not affect the SpaHIG pilus. Moreover, SpaH but not SpaI or SpaG is essential for the formation of the filament. When expressed under the control of an inducible promoter, the amount of the SpaH pilin regulates pilus length; no pili are assembled from an SpaH precursor that has an alanine in place of the conserved lysine of the SpaH pilin motif. Thus, the spaHIG pilus gene cluster encodes a pilus structure that is independently assembled and antigenically distinct from other pili of C. diphtheriae . We incorporate these findings in a model of sortase-mediated pilus assembly that may be applicable to many gram-positive pathogens.

The type III secretion signal of Yersinia enterocolitica YopN was mapped using a gene fusion approach. yopN codons 1 to 12 were identified as critical for signal function. Several synonymous mutations that abolish secretion of hybrid proteins without altering the codon specificity of yopN mRNA were identified.

Summary Corynebacterium diphtheriae SpaA pili are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals, and SpaC seems to be positioned at the pilus tip. Pilus assembly in C. diphtheriae requires the pilin motif and the C-terminal sorting signal of SpaA, and is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side-chain amino groups of pilin motif sequences to generate covalent linkages between pilin subunits. We show here that two elements of SpaA pilin precursor, the pilin motif and the sorting signal, are together sufficient to promote the polymerization of an otherwise secreted protein by a process requiring the function of the sortase A gene (srtA). Five other sortase genes are dispensable for SpaA pilus assembly. Further, the incorporation of SpaB into SpaA pili requires a glutamic acid residue within the E box motif of SpaA, a feature that is found to be conserved in other Gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs. When the main fimbrial subunit of Actinomyces naeslundii type I fimbriae, FimA, is expressed in corynebacteria, C. diphtheriae strain NCTC13129 polymerized FimA to form short fibres. Although C. diphtheriae does not depend on other actinomycetal genes for FimA polymerization, this process involves the pilin motif and the sorting signal of FimA as well as corynebacterial sortase D (SrtD). Thus, pilus assembly in Gram-positive bacteria seems to occur by a universal mechanism of ordered cross-linking of precursor proteins, the multiple conserved features of which are recognized by designated sortase enzymes.

The formation of adhesive pili on the surface of Gram-negative bacteria has been studied in detail, whereas the pilus assembly pathways in Gram-positive bacteria remain to be characterized. Gram-positive microbes use the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins; a strategy that involves sorting signals of surface protein precursors and sortase, a transpeptidase that cleaves sorting signals and links the C-terminus of surface proteins via an amide bond to the peptidoglycan cross-bridge. Recent studies with Actinomyces naeslundii, Corynebacterium diphtheriae and Streptococcus parasanguis suggest that some sortase enzymes catalyze protein polymerization leading to the formation of pili on the surface of Gram-positive bacteria. Arthobacter photogonimos and Ruminococcus albus appear to use different strategies for pilus assembly, consistent with the notion that Gram-positive organisms, similar to Gram-negative bacteria, have evolved multiple molecular strategies for the formation of pili on microbial surfaces.

Pili of Gram-negative pathogens are formed from pilin precursor molecules by non-covalent association within the outer membrane envelope. Gram-positive microbes employ the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins, however, an assembly pathway for pili has not yet been revealed. We show here that pili of Corynebacterium diphtheriae are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals and SpaC seems positioned at the pilus tip. Assembled pili are released from the bacterial surface by treatment with murein hydrolase, suggesting that the pilus fibres may be anchored to the cell wall envelope. All three pilin subunit proteins are synthesized as precursors carrying N-terminal signal peptides and C-terminal sorting signals. Some, but not all, of the six sortase genes encoded in the genome of C. diphtheriae are required for precursor processing, pilus assembly or cell wall envelope attachment. Pilus assembly is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits. This covalent tethering of adjacent pilin subunits appears to have evolved in many Gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs.

Sortase (SrtA), an enzyme that anchors surface proteins to the cell wall of Gram-positive bacteria, cleaves sorting signals at the LPXTG motif. We have identified a second sortase (SrtB) in the Gram-positive pathogen Staphylococcus aureus that is required for anchoring of a surface protein with a NPQTN motif. Purified SrtB cleaves NPQTN-bearing peptides in vitro , and a srtB mutant is defective in the persistence of animal infections. srtB is part of an iron-regulated locus called i ron-responsive s urface d eterminants ( isd ), which also contains a ferrichrome transporter and surface proteins with NPQTN and LPXTG motifs. Cell wall-anchored surface proteins and the isd locus seem involved in a novel mechanism of iron acquisition that is important for bacterial pathogenesis.

Sortase, a cysteine-transpeptidase conserved in Gram-positive bacteria, anchors on the cell wall many surface proteins that facilitate bacterial pathogenesis and fitness. Genetic disruption of the housekeeping sortase in several Gram-positive pathogens reported thus far attenuates virulence, but not bacterial growth. Paradoxically, we discovered that depletion of the housekeeping sortase SrtA was lethal for Actinomyces oris; yet, all of its predicted cell wall-anchored protein substrates (AcaA-N) were individually dispensable for cell viability. Using Tn5-transposon mutagenesis to identify factors that upend lethality of srtA deletion, we uncovered a set of genetic suppressors harbouring transposon insertions within genes of a locus encoding AcaC and a LytR-CpsA-Psr (LCP)-like protein. AcaC was shown to be highly glycosylated and dependent on LCP for its glycosylation. Upon SrtA depletion, the glycosylated form of AcaC, hereby renamed GspA, was accumulated in the membrane. Overexpression of GspA in a mutant lacking gspA and srtA was lethal; conversely, cells overexpressing a GspA mutant missing a membrane-localization domain were viable. The results reveal a unique glycosylation pathway in A. oris that is coupled to cell wall anchoring catalysed by sortase SrtA. Significantly, this novel phenomenon of glyco-stress provides convenient cell-based assays for developing a new class of inhibitors against Gram-positive pathogens.

The formation of dental plaque, a highly complex biofilm that causes gingivitis and periodontitis, requires specific adherence among many oral microbes, including the coaggregation of Actinomyces oris with Streptococcus oralis that helps to seed biofilm development. Here, we report the discovery of a key coaggregation factor for this process. This protein, which we named coaggregation factor A (CafA), is one of 14 cell surface proteins with the LPXTG motif predicted in A. oris MG1, whose function was hitherto unknown. By systematic mutagenesis of each of these genes and phenotypic characterization, we found that the Actinomyces/Streptococcus coaggregation is only abolished by deletion of cafA. Subsequent biochemical and cytological experiments revealed that CafA constitutes the tip of a unique form of the type 2 fimbria long known for its role in coaggregation. The direct and predominant role of CafA in adherence is evident from the fact that CafA or an antibody against CafA inhibits coaggregation, whereas the shaft protein FimA or a polyclonal antibody against FimA has no effect. Remarkably, FimA polymerization was blocked by deletion of genes for both CafA and FimB, the previously described tip protein of the type 2 fimbria. Together, these results indicate that some surface proteins not linked to a pilus gene cluster in Gram-positive bacteria may hijack the pilus. These unique tip proteins displayed on a common pilus shaft may serve distinct physiological functions. Furthermore, the pilus shaft assembly in Gram-positive bacteria may require a tip, as is true for certain Gram-negative bacterial pili.

Bacteriophage muralytic enzymes degrade the cell wall envelope of staphylococci to release phage particles from the bacterial cytoplasm. Murein hydrolases of staphylococcal phages phi11, 80alpha, 187, Twort, and phiPVL harbor a central domain that displays sequence homology to known N-acetylmuramyl-L-alanyl amidases; however, their precise cleavage sites on the staphylococcal peptidoglycan have thus far not been determined. Here we examined the properties of the phi11 enzyme to hydrolyze either the staphylococcal cell wall or purified cell wall anchor structures attached to surface protein. Our results show that the phi11 enzyme has D-alanyl-glycyl endopeptidase as well as N-acetylmuramyl-L-alanyl amidase activity. Analysis of a deletion mutant lacking the amidase-homologous sequence, phi11(Delta181-381), revealed that the D-alanyl-glycyl endopeptidase activity is contained within the N-terminal 180 amino acid residues of the polypeptide chain. Sequences similar to this N-terminal domain are found in the murein hydrolases of staphylococcal phages but not in those of phages that infect other Gram-positive bacteria such as Listeria or Bacillus.

Pili or fimbriae are known virulence factors that facilitate bacterial colonization of specific host tissues and pathogenesis. Corynebacterium diphtheriae, the causative agent of pharyngeal diphtheria, harbors three pilus gene clusters encoding the heterotrimeric SpaA-, SpaD-, and SpaH-type pili. The current model for Gram-positive pilus assembly is based on the studies of the SpaA-type pili, which may serve as a major virulence determinant for C. diphtheriae because they are specifically required for adherence to pharyngeal epithelial cells. The SpaA-type pilus is comprised of the shaft pilin SpaA and minor proteins SpaB and SpaC, which make up the base and tip of the surface structure, respectively. Pilus biogenesis requires tandem sortase enzymes that covalently link SpaABC monomers and subsequently anchor the resulting heterotrimeric polymer to the bacterial peptidoglycan. Here, a decade of research aimed to reveal the assembly, structure, and role of C. diphtheriae pili in pathogenesis is discussed.

Toxigenic Corynebacterium diphtheriae strains cause diphtheria in humans. The toxigenic C. diphtheriae isolate NCTC13129 produces three distinct heterotrimeric pili that contain SpaA, SpaD, and SpaH, making up the shaft structure. The SpaA pili are known to mediate bacterial adherence to pharyngeal epithelial cells. However, to date little is known about the expression of different pili in various clinical isolates and their importance in bacterial pathogenesis. Here, we characterized a large collection of C. diphtheriae clinical isolates for their pilin gene pool by PCR and for the expression of the respective pilins by immunoblotting with antibodies against Spa pilins. Consistent with the role of a virulence factor, the SpaA-type pili were found to be prevalent among the isolates, and most significantly, corynebacterial adherence to pharyngeal epithelial cells was strictly correlated with isolates that were positive for the SpaA pili. By comparison, the isolates were heterogeneous for the presence of SpaD- and SpaH-type pili. Importantly, using Caenorhabditis elegans as a model host for infection, we show here that strain NCTC13129 rapidly killed the nematodes, the phenotype similar to isolates that were positive for toxin and all pilus types. In contrast, isogenic mutants of NCTC13129 lacking SpaA-type pili or devoid of toxin and SpaA pili exhibited delayed killing of nematodes with similar kinetics. Consistently, nontoxigenic or toxigenic isolates that lack one, two, or all three pilus types were also attenuated in virulence. This work signifies the important role of pili in corynebacterial pathogenesis and provides a simple host model to identify additional virulence factors.

Pili or fimbriae are recognized as essential virulence determinants assembled on the bacterial surface. Gram-positive bacteria produce covalently linked pilus structures that are distinct from gram-negative counterparts. In this chapter, we describe three commonly used techniques to extract, detect, and visualize pili from gram-positive bacteria: (1) Western blot analysis, (2) Immuno-Electron Microscopy, and (3) Atomic Force Microscopy.

Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae . Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ω tox + phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox + corynephage. DNA binding sites of the tox -controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.

The Gram-positive pathogen Streptococcus agalactiae, known as group B Streptococcus (GBS), is the leading cause of bacterial septicemia, pneumonia, and meningitis among neonates. GBS assembles two types of pili—pilus islands (PIs) 1 and 2—on its surface to adhere to host cells and to initiate colonization for pathogenesis. The GBS PI-1 pilus is made of one major pilin, GBS80, which forms the pilus shaft, and two secondary pilins, GBS104 and GBS52, which are incorporated into the pilus at various places. We report here the crystal structure of the 35-kDa C-terminal fragment from GBS80, which is composed of two IgG-like domains (N2–N3). The structure was solved by single-wavelength anomalous dispersion using sodium-iodide-soaked crystals and diffraction data collected at the home source. The N2 domain exhibits a cnaA/DEv-IgG fold with two calcium-binding sites, while the N3 domain displays a cnaB/IgG-rev fold. We have built a model for full-length GBS80 (N1, N2, and N3) with the help of available homologous major pilin structures, and we propose a model for the GBS PI-1 pilus shaft. The N2 and N3 domains are arranged in tandem along the pilus shaft, whereas the respective N1 domain is tilted by approximately 20° away from the pilus axis. We have also identified a pilin-like motif in the minor pilin GBS52, which might aid its incorporation at the pilus base.