The Nanomechanical Properties of Lactococcus lactis Pili Are Conditioned by the Polymerized Backbone Pilin

Pili produced by Lactococcus lactis subsp. lactis are putative linear structures consisting of repetitive subunits of the major pilin PilB that forms the backbone, pilin PilA situated at the distal end of the pilus, and an anchoring pilin PilC that tethers the pilus to the peptidoglycan. We determined the nanomechanical properties of pili using optical-tweezers force spectroscopy. Single pili were exposed to optical forces that yielded force-versus-extension spectra fitted using the Worm-Like Chain model. Native pili subjected to a force of 0–200 pN exhibit an inextensible, but highly flexible ultrastructure, reflected by their short persistence length. We tested a panel of derived strains to understand the functional role of the different pilins. First, we found that both the major pilin PilB and sortase C organize the backbone into a full-length organelle and dictate the nanomechanical properties of the pili. Second, we found that both PilA tip pilin and PilC anchoring pilin were not essential for the nanomechanical properties of pili. However, PilC maintains the pilus on the bacterial surface and may play a crucial role in the adhesion- and biofilm-forming properties of L. lactis.     

Pilus biogenesis of Gram‐positive bacteria: Roles of sortases and implications for assembly

Successful adherence, colonization, and survival of Gram‐positive bacteria require surface proteins, and multiprotein assemblies called pili. These surface appendages are attractive pharmacotherapeutic targets and understanding their assembly mechanisms is essential for identifying a new class of ‘anti‐infectives’ that do not elicit microbial resistance. Molecular details of the Gram‐negative pilus assembly are available indepth, but the Gram‐positive pilus biogenesis is still an emerging field and investigations continue to reveal novel insights into this process. Pilus biogenesis in Gram‐positive bacteria is a biphasic process that requires enzymes called pilus‐sortases for assembly and a housekeeping sortase for covalent attachment of the assembled pilus to the peptidoglycan cell wall. Emerging structural and functional data indicate that there are at least two groups of Gram‐positive pili, which require either the Class C sortase or Class B sortase in conjunction with LepA/SipA protein for major pilin polymerization. This observation suggests two distinct modes of sortase‐mediated pilus biogenesis in Gram‐positive bacteria. Here we review the structural and functional biology of the pilus‐sortases from select streptococcal pilus systems and their role in Gram‐positive pilus assembly.     

Pilin and Sortase Residues Critical for Endocarditis- and Biofilm-Associated Pilus Biogenesis in Enterococcus faecalis

Enterococci commonly cause hospital-acquired infections, such as infective endocarditis and catheter-associated urinary tract infections. In animal models of these infections, a long hairlike extracellular protein fiber known as the endocarditis- and biofilm-associated (Ebp) pilus is an important virulence factor for Enterococcus faecalis. For Ebp and other sortase-assembled pili, the pilus-associated sortases are essential for fiber formation as they create covalent isopeptide bonds between the sortase recognition motif and the pilin-like motif of the pilus subunits. However, the molecular requirements governing the incorporation of the three pilus subunits (EbpA, EbpB, and EbpC) have not been investigated in E. faecalis. Here, we show that a Lys residue within the pilin-like motif of the EbpC subunit was necessary for EbpC polymerization. However, incorporation of EbpA into the pilus fiber only required its sortase recognition motif (LPXTG), while incorporation of EbpB only required its pilin-like motif. Only the sortase recognition motif would be required for incorporation of the pilus tip subunit, while incorporation of the base subunit would only require the pilin recognition motif. Thus, these data support a model with EbpA at the tip and EbpB at the base of an EbpC polymer. In addition, the housekeeping sortase, SrtA, was found to process EbpB and its predicted catalytic Cys residue was required for efficient cell wall anchoring of mature Ebp pili. Thus, we have defined molecular interactions involved in fiber polymerization, minor subunit organization, and pilus subcellular compartmentalization in the E. faecalis Ebp pilus system. These studies advance our understanding of unique molecular mechanisms of sortase-assembled pilus biogenesis.     

Assembly of pili on the surface of Corynebacterium diphtheriae

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.     

Housekeeping sortase facilitates the cell wall anchoring of pilus polymers in Corynebacterium diphtheriae

Many surface proteins in Gram-positive bacteria are covalently linked to the cell wall through a transpeptidation reaction catalysed by the enzyme sortase. Corynebacterium diphtheriae encodes six sortases, five of which are devoted to the assembly of three distinct types of pilus fibres--SrtA for the SpaA-type pilus, SrtB/SrtC for the SpaD-type pilus, and SrtD/SrtE for the SpaH-type pilus. We demonstrate here the function of SrtF, the so-called housekeeping sortase, in the cell wall anchoring of pili. We show that a multiple deletion mutant strain expressing only SrtA secretes a large portion of SpaA polymers into the culture medium, with concomitant decrease in the cell wall-linked pili. The same phenotype is observed with the mutant that is missing SrtF alone. By contrast, a strain that expresses only SrtF displays surface-linked pilins but no polymers. Therefore, SrtF can catalyse the cell wall anchoring of pilin monomers as well as pili, but it does not polymerize pilins. We show that SrtA and SrtF together generate wild-type levels of the SpaA-type pilus on the bacterial surface. Furthermore, by regulating the expression of SpaA in the cell, we demonstrate that the SrtF function becomes critical when the SpaA level is sufficiently high. Together, these findings provide key evidence for a two-stage model of pilus assembly: pilins are first polymerized by a pilus-specific sortase, and the resulting fibre is then attached to the cell wall by either the cognate sortase or the housekeeping sortase.     

Sortases and pilin elements involved in pilus assembly of Corynebacterium diphtheriae

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.     

Acyl Enzyme Intermediates in Sortase-Catalyzed Pilus Morphogenesis in Gram-Positive Bacteria

In gram-positive bacteria, covalently linked pilus polymers are assembled by a specific transpeptidase enzyme called pilus-specific sortase. This sortase is postulated to cleave the LPXTG motif of a pilin precursor between threonine and glycine and to form an acyl enzyme intermediate with the substrate. Pilus polymerization is believed to occur through the resolution of this intermediate upon specific nucleophilic attack by the conserved lysine located within the pilin motif of another pilin monomer, which joins two pilins with an isopeptide bond formed between threonine and lysine. Here, we present evidence for sortase reaction intermediates in Corynebacterium diphtheriae. We show that truncated SrtA mutants that are loosely bound to the cytoplasmic membrane form high-molecular-weight complexes with SpaA polymers secreted into the extracellular milieu. These complexes are not formed with SpaA pilin mutants that have alanine substitutions in place of threonine in the LPXTG motif or lysine in the pilin motif. The same phenotype is observed with alanine substitutions of either the conserved cysteine or histidine residue of SrtA known to be required for catalysis. Remarkably, the assembly of SpaA pili, or the formation of intermediates, is abolished with a SrtA mutant missing the membrane-anchoring domain. We infer that pilus polymerization involves the formation of covalent pilin-sortase intermediates, which occurs within a molecular platform on the exoplasmic face of the cytoplasmic membrane that brings together both sortase and its cognate substrates in close proximity to each other, likely surrounding a secretion apparatus. We present electron microscopic data in support of this picture.Adherence to specific host tissue is a key step in bacterial colonization and the establishment of a successful infection by bacterial pathogens. Bacteria express a variety of adhesive cell surface molecules to bind host cells or other substrates in their natural habitat. The proteinaceous filaments known as pili or fimbriae are a clinically important class of these molecules. Both gram-negative and gram-positive bacteria express pili (6, 8). The gram-positive bacterial pili are unique in three respects (12, 25, 31). First, they represent heterodimeric or heterotrimeric protein polymers in which individual pilin subunits are covalently joined to each other (2, 9, 32). Second, the polymer itself is covalently attached to the cell wall (3, 31). Third, unlike pilus assembly in gram-negative bacteria, many of which require chaperones (26), the polymerization of the gram-positive pili and their cell wall attachment require specific transpeptidase enzymes called sortases (31).Mazmanian and colleagues discovered the sortase SrtA as an enzyme that linked the surface protein A of Staphylococcus aureus to its cell wall (15). Genome sequences revealed that sortases are ubiquitously expressed in gram-positive bacteria, including significant pathogens, such as Actinomyces naeslundii, Bacillus cereus, Corynebacterium diphtheriae, Enterococcus faecalis, Streptococcus agalactiae, and Streptococcus pneumoniae (4, 5, 28). Sortases are classified according to their functions and phylogenic relationships (4, 5). The class that closely matches SrtA of S. aureus in structure and function is now called a housekeeping sortase. Its function is to attach numerous surface proteins to the cell wall (16). Common to each of these cell surface proteins is a cell wall sorting signal with an LPXTG motif that is absolutely necessary for cell wall anchoring (18). Elegant genetic, biochemical, and structural work by the Schneewind laboratory illuminated the universal reaction mechanism of protein sorting in the gram-positive cell wall (14). Cell wall anchoring of surface proteins is catalyzed in two steps. In the first step, SrtA cleaves the TG peptide bond of the LPXTG motif of protein A and forms an acyl enzyme intermediate involving the threonine of protein A and the catalytic cysteine of sortase (22, 27, 29). In the second step, the cleaved protein A is transferred to the cell wall when a nucleophile amine from the lipid II precursor attacks and resolves the acyl enzyme intermediate (20, 21, 30). This seminal work formed the basis of our current model of pilus assembly catalyzed by pilus-specific sortases (12).We have used C. diphtheriae as a model for studies of the mechanism of pilus biogenesis. The corynebacterial genome encodes six different sortases (32). We now know that while five of these sortases (SrtA to -E) are devoted to pilus assembly, even the housekeeping sortase, SrtF, is required for efficient attachment of pili to the cell wall (23). Corynebacteria produce three distinct types of heterotrimeric pili, which are encoded by three pilus islands, each encoding three pilins (namely, SpaABC, SpaDEF, and SpaGHI) plus one or two cognate sortases essential for the assembly of the respective pilus (7, 24, 32). In each case, the prototype pilus represents a shaft structure made of a specific major pilin (namely, SpaA, SpaD, and SpaH) (12). Each type of pilus also contains a minor pilin at the tip (SpaC, SpaF, and SpaG) and another minor pilin dispersed along the shaft, as well as at the base of the pilus (SpaB, SpaE, and SpaI) (12). How are these polymers assembled, and how are they attached to the cell wall? All pilin proteins are predicted to contain in their amino termini a hydrophobic signal sequence necessary for export to the exoplasm by the Sec machinery. In addition, like the cell wall-anchored protein A of S. aureus, a cell wall sorting signal including the LPXTG motif is also present at the carboxy terminus of each of the Spa proteins of corynebacteria and other pilus proteins found in different gram-positive organisms (17). It is thus logical to imagine that the pilus-specific sortase utilizes the LPXTG motif for pilus polymerization, its cell wall anchoring, or both. Substantial genetic, biochemical, and ultrastructural analyses have proved this prediction. Consequently, Ton-That and Schneewind proposed a model of pilus assembly which posited that the basic mechanism of catalysis is conserved between cell wall sorting of surface proteins and the assembly of the pilus (31).According to our current working model (Fig. ​(Fig.1A),1A), the prototype SpaA pilus is assembled as follows. SrtA, which is essential and also specific for SpaA pilus formation, captures and cleaves cognate pilins at the LPXTG motif and forms an acyl enzyme intermediate. To form a dimer of SpaA and SpaC, the proposed tip entity, a conserved lysine in the SpaA pilin motif attacks the Cys-Thr bond of the SpaC-SrtA acyl enzyme intermediate. Shaft formation ensues by the cyclic addition of SpaA to the SpaC-SpaA dimer and the SpaC-SpaA_n oligomer formed in the preceding reaction. When a SpaB is attached to the growing pilus terminus by a similar mechanism involving a critical lysine of SpaB, it acts as a switch, terminating pilus polymerization in favor of cell wall anchoring (11). This happens by the classic resolution reaction mentioned above, which involves the lipid II precursor (28), followed by its linkage to the cell wall (11). Alternatively, the SpaB-containing pilus can elongate further by adding a SpaA subunit to SpaB (11). This model explains all the available genetic and biochemical data we have obtained so far in the corynebacterial system, as well as other systems reported by various investigators.Open in a separate windowFIG. 1.(A) Working model of pilus assembly in C. diphtheriae. Spa pilins are synthesized in the cytoplasm and transported across the cytoplasmic membrane by the Sec machinery. The membrane-bound pilus-specific sortase SrtA cleaves the Spa pilins at the LPXTG motif and forms an acyl enzyme intermediate with the substrates. Pilus polymerization occurs when this intermediate is resolved by a nucleophilic attack by the lysine residue within the pilin motif of an adjacent intermediate. Cell wall anchoring terminates pilus polymerization when SpaB is incorporated into the pilus base by the housekeeping sortase, SrtF (see the text for details). (B) Membrane localization of the pilus-specific sortase SrtA. Corynebacteria grown to mid-log phase were separated from the culture medium (M) by centrifugation. The cell wall (W) was removed from its protoplast by muramidase treatment of the cells. The protoplasts were lysed, and membrane (P*) and cytoplasmic (C) compartments were obtained by ultracentrifugation. Protein samples were separated on 4 to 12% Tris-glycine gradient gels and detected by immunoblotting them with the specific antisera α-SrtA, α-SecA, and α-SpaA. The positions of molecular mass markers (kDa) are indicated. WT, wild type.Significantly, there has been no report demonstrating the proposed intermediates of pilus assembly, to our knowledge. The present study was initiated to explore this key element of our model of pilus assembly, as well as the localization of the sortase in the membrane and its organization in the exoplasmic membrane in relation to the cognate pilins and the general secretion machinery.     

Contribution of Individual Ebp Pilus Subunits of Enterococcus faecalis OG1RF to Pilus Biogenesis,Biofilm Formation and Urinary Tract Infection

The endocarditis and biofilm-associated pilus (Ebp) operon is a component of the core genome of Enterococcus faecalis that has been shown to be important for biofilm formation, adherence to host fibrinogen, collagen and platelets, and in experimental endocarditis and urinary tract infection models. Here, we created single and double deletion mutants of the pilus subunits and sortases; next, by combining western blotting, immunoelectron microscopy, and using ebpR in trans to increase pilus production, we identified EbpA as the tip pilin and EbpB as anchor at the pilus base, the latter attached to cell wall by the housekeeping sortase, SrtA. We also confirmed EbpC and Bps as the major pilin and pilin-specific sortase, respectively, both required for pilus polymerization. Interestingly, pilus length was increased and the number of pili decreased by deleting ebpA, while control overexpression of ebpA in trans restored wild-type levels, suggesting a dual role for EbpA in both initiation and termination of pilus polymerization. We next investigated the contribution of each pilin subunit to biofilm formation and UTI. Significant reduction in biofilm formation was observed with deletion of ebpA or ebpC (P<0.001) while ebpB was found to be dispensable; a similar result was seen in kidney CFUs in experimental UTI (ΔebpA, ΔebpC, P≤0.0093; ΔebpB, non-significant, each vs. OG1RF). Hence, our data provide important structural and functional information about these ubiquitous E. faecalis pili and, based on their demonstrated importance in biofilm and infection, suggest EbpA and EbpC as potential targets for antibody-based therapeutic approaches.     

Cell surface display of minor pilin adhesins in the form of a simple heterodimeric assembly in Corynebacterium diphtheriae

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.     

Insights into Streptococcus agalactiae PI-2b pilus biosynthesis and role in adherence to host cells

The core PI-2b pilus present in “hypervirulent” ST-17 Streptococcus agalactiae strains consists of three pilin subunits (Spb1, Ap1 and Ap2) assembled by sortase SrtC1 and cell-wall anchored by Srt2. Spb1 was shown to be the major pilin and Ap2 the anchor pilin. Ap1 is a putative adhesin. Two additional genes, orf and lep, are part of this operon. The contribution of Lep and Ap1 to the biogenesis of the PI-2b pilus was investigated. Concerning the role of PI-2b, we found that higher PI-2b expression resulted in higher adherence to human brain endothelial cells and higher phagocytosis by human THP1 macrophages.     

Functional Identification of Conserved Residues Involved in Lactobacillus rhamnosus Strain GG Sortase Specificity and Pilus Biogenesis

In Gram-positive bacteria, sortase-dependent pili mediate the adhesion of bacteria to host epithelial cells and play a pivotal role in colonization, host signaling, and biofilm formation. Lactobacillus rhamnosus strain GG, a well known probiotic bacterium, also displays on its cell surface mucus-binding pilus structures, along with other LPXTG surface proteins, which are processed by sortases upon specific recognition of a highly conserved LPXTG motif. Bioinformatic analysis of all predicted LPXTG proteins encoded by the L. rhamnosus GG genome revealed a remarkable conservation of glycine residues juxtaposed to the canonical LPXTG motif. Here, we investigated and defined the role of this so-called triple glycine (TG) motif in determining sortase specificity during the pilus assembly and anchoring. Mutagenesis of the TG motif resulted in a lack or an alteration of the L. rhamnosus GG pilus structures, indicating that the TG motif is critical in pilus assembly and that they govern the pilin-specific and housekeeping sortase specificity. This allowed us to propose a regulatory model of the L. rhamnosus GG pilus biogenesis. Remarkably, the TG motif was identified in multiple pilus gene clusters of other Gram-positive bacteria, suggesting that similar signaling mechanisms occur in other, mainly pathogenic, species.     

Assembly of pili on the surface of Bacillus cereus vegetative cells

Vegetative forms of Bacillus cereus are reported to form pili, thin protein filaments that protrude up to 1 mum from the bacterial surface. Pili are assembled from two precursor proteins, BcpA and BcpB, in a manner requiring a pilus-associated sortase enzyme (SrtD). Pili are also formed on the surface of Bacillus anthracis expressing bcpA-srtD-bcpB. BcpA is distributed throughout the entire pilus, whereas BcpB appears positioned at its tip. In agreement with the hypothesis for pilus assembly in Gram-positive bacteria, BcpA encompasses the YPK pilin motif and the LPXTG sorting signal, each of which is absolutely required for the incorporation of BcpA and BcpB into pili. In contrast to BcpB, which relies on the presence of BcpA for incorporation into pili, BcpA fibre assembly occurs even in the absence of BcpB. B. anthracis sortase A (srtA), but not sortase B (srtB) or C (srtC), is required for proper anchoring of pili to the bacterial envelope, suggesting that BcpA/BcpB pili are linked to peptidoglycan cross-bridges.     

Molecular principles of antigenic variation in Neisseria gonorrhoeae

The genome of Neisseria gonorrhoeae harbours many gene loci for the production of variant pili. Strain MS11 has two expression genes (pilE) with promoter and complete coding sequences. The remaining genes are silent (pilS) lacking the promoter and the conservative amino terminals coding sequences of pilin. The pilus genes consist of six variable minicassettes (mc's), that are flancked by strictly conserved sequences. Upon phase (P⁺ to P⁺) and antigenic (P⁺ to P^–, or vice versa) transitions minicassettes from silent loci are transferred from silent pilus gene copies to the expression gene by gene conversion. P^– variants resulting from such rearrangements still produce pilin mRNA as well as pilin, but only a few are found on the surface of those gonococci.     

Structural differences between the Streptococcus agalactiae housekeeping and pilus-specific sortases: SrtA and SrtC1

The assembly of pili on the cell wall of Gram-positive bacteria requires transpeptidase enzymes called sortases. In Streptococcus agalactiae, the PI-1 pilus island of strain 2603V/R encodes two pilus-specific sortases (SrtC1 and SrtC2) and three pilins (GBS80, GBS52 and GBS104). Although either pilus-specific sortase is sufficient for the polymerization of the major pilin, GBS80, incorporation of the minor pilins GBS52 and GBS104 into the pilus structure requires SrtC1 and SrtC2, respectively. The S. agalactiae housekeeping sortase, SrtA, whose gene is present at a different location and does not catalyze pilus polymerization, was shown to be involved in cell wall anchoring of pilus polymers. To understand the structural basis of sortases involved in such diverse functions, we determined the crystal structures of S. agalactiae SrtC1 and SrtA. Both enzymes are made of an eight-stranded beta-barrel core with variations in their active site architecture. SrtA exhibits a catalytic triad arrangement similar to that in Streptococcus pyogenes SrtA but different from that in Staphylococcus aureus SrtA. In contrast, the SrtC1 enzyme contains an N-terminal helical domain and a 'lid' in its putative active site, which is similar to that seen in Streptococcus pneumoniae pilus-specific sortases, although with subtle differences in positioning and composition. To understand the effect of such differences on substrate recognition, we have also determined the crystal structure of a SrtC1 mutant, in which the conserved DP(W/F/Y) motif was replaced with the sorting signal motif of GBS80, IPNTG. By comparing the structures of WT wild type SrtA and SrtC1 and the 'lid' mutant of SrtC1, we propose that structural elements within the active site and the lid may be important for defining the role of specific sortase in pili biogenesis.     

Molecular Analyses of the Natural Transformation Machinery and Identification of Pilus Structures in the Extremely Thermophilic Bacterium Thermus thermophilus Strain HB27

Thermus thermophilus HB27, an extremely thermophilic bacterium, exhibits high competence for natural transformation. To identify genes of the natural transformation machinery of T. thermophilus HB27, we performed homology searches in the partially completed T. thermophilus genomic sequence for conserved competence genes. These analyses resulted in the detection of 28 open reading frames (ORFs) exhibiting significant similarities to known competence proteins of gram-negative and gram-positive bacteria. Disruption of 15 selected potential competence genes led to the identification of 8 noncompetent mutants and one transformation-deficient mutant with a 100-fold reduced transformation frequency. One competence protein is similar to DprA of Haemophilus influenzae, seven are similar to type IV pilus proteins of Pseudomonas aeruginosa or Neisseria gonorrhoeae (PilM, PilN, PilO, PilQ, PilF, PilC, PilD), and another deduced protein (PilW) is similar to a protein of unknown function in Deinococcus radiodurans R1. Analysis of the piliation phenotype of T. thermophilus HB27 revealed the presence of single pilus structures on the surface of the wild-type cells, whereas the noncompetent pil mutants of Thermus, with the exception of the pilF mutant, were devoid of pilus structures. These results suggest that pili and natural transformation in T. thermophilus HB27 are functionally linked.     

Insights into type IV pilus biogenesis and dynamics from genetic analysis of a C-terminally tagged pilin: a role for O-linked glycosylation

Type IV pili are surface organelles essential for pathogenicity of many Gram-negative bacteria. In Neisseria gonorrhoeae, the major subunit of type IV pili, PilE, is a target of its general O-linked glycosylation system. This system modifies a diverse set of periplasmic and extracellular gonococcal proteins with a variable set of glycans. Here we show that expression of a particular hexa-histidine-tagged PilE was associated with growth arrest. By studying intra- and extragenic suppressors, we found that this phenotype was dependent on pilus assembly and retraction. Based on these results, we developed a sensitive tool to identify factors with subtle effects on pilus dynamics. Using this approach, we found that glycan chain length has differential effects on the growth arrest that appears to be mediated at the level of pilin subunit-subunit interactions and bidirectional remodelling of pilin between its membrane-associated and assembled states. Gonococcal pilin glycosylation thus plays both an intracellular role in pilus dynamics and potential extracellular roles mediated through type IV pili. In addition to demonstrating the effect of glycosylation on pilus dynamics, the study provides a new way of identifying factors with less dramatic effects on processes involved in type IV pilus biogenesis.     

The Group A Streptococcus serotype M2 pilus plays a role in host cell adhesion and immune evasion

Group A Streptococcus (GAS), or Streptococcus pyogenes, is a human pathogen that causes diseases ranging from skin and soft tissue infections to severe invasive diseases, such as toxic shock syndrome. Each GAS strain carries a particular pilus type encoded in the variable f ibronectin‐binding, c ollagen‐binding, T antigen (FCT) genomic region. Here, we describe the functional analysis of the serotype M2 pilus encoded in the FCT‐6 region. We found that, in contrast to other investigated GAS pili, the ancillary pilin 1 lacks adhesive properties. Instead, the backbone pilin is important for host cell adhesion and binds several host factors, including fibronectin and fibrinogen. Using a panel of recombinant pilus proteins, GAS gene deletion mutants and Lactococcus lactis gain‐of‐function mutants we show that, unlike other GAS pili, the FCT‐6 pilus also contributes to immune evasion. This was demonstrated by a delay in blood clotting, increased intracellular survival of the bacteria in macrophages, higher bacterial survival rates in human whole blood and greater virulence in a Galleria mellonella infection model in the presence of fully assembled FCT‐6 pili.     

Assembly mechanism of FCT region type 1 pili in serotype M6 Streptococcus pyogenes

The human pathogen Streptococcus pyogenes produces diverse pili depending on the serotype. We investigated the assembly mechanism of FCT type 1 pili in a serotype M6 strain. The pili were found to be assembled from two precursor proteins, the backbone protein T6 and ancillary protein FctX, and anchored to the cell wall in a manner that requires both a housekeeping sortase enzyme (SrtA) and pilus-associated sortase enzyme (SrtB). SrtB is primarily required for efficient formation of the T6 and FctX complex and subsequent polymerization of T6, whereas proper anchoring of the pili to the cell wall is mainly mediated by SrtA. Because motifs essential for polymerization of pilus backbone proteins in other Gram-positive bacteria are not present in T6, we sought to identify the functional residues involved in this process. Our results showed that T6 encompasses the novel VAKS pilin motif conserved in streptococcal T6 homologues and that the lysine residue (Lys-175) within the motif and cell wall sorting signal of T6 are prerequisites for isopeptide linkage of T6 molecules. Because Lys-175 and the cell wall sorting signal of FctX are indispensable for substantial incorporation of FctX into the T6 pilus shaft, FctX is suggested to be located at the pilus tip, which was also implied by immunogold electron microscopy findings. Thus, the elaborate assembly of FCT type 1 pili is potentially organized by sortase-mediated cross-linking between sorting signals and the amino group of Lys-175 positioned in the VAKS motif of T6, thereby displaying T6 and FctX in a temporospatial manner.