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.     

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.     

Pilus Biogenesis in Lactococcus lactis: Molecular Characterization and Role in Aggregation and Biofilm Formation

The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used.     

The GpsB files: the truth is out there

Peptidoglycan (PG), an essential stress‐bearing component of the bacterial cell wall, is synthesised by penicillin binding proteins (PBPs). PG synthesis at the cell division septum is necessary for constructing new poles of progeny cells, and cells cannot elongate without inserting new PG in the side‐wall. The cell division regulator GpsB appears to co‐ordinate PG synthesis at the septum during division and at the side‐wall during elongation in rod‐shaped and ovococcoid Gram‐positive bacteria. How the control over PG synthesis is exerted is unknown. In this issue of Molecular Microbiology, Rued et al. show that in pneumococci GpsB forms complexes with PBP2a and PBP2b, and that deletion or depletion of GpsB prevents closure of the septal ring that in itself is PBP2x‐dependent. Loss of GpsB can be suppressed by spontaneous mutations, including within the gene encoding the only PP2C Ser/Thr phosphatase in Streptococcus pneumoniae, indicating that GpsB plays a key – but unknown – role in protein phosphorylation in pneumococci. Rued et al. combine phenotypic and genotypic analyses of mutant strains that suggest discrepancies in the literature concerning GpsB might have arisen from accumulation of unidentified suppressors, highlighting the importance and power of strain validation and whole genome sequencing in this context.     

Polyamine‐independent growth and biofilm formation,and functional spermidine/spermine N‐acetyltransferases in Staphylococcus aureus and Enterococcus faecalis

Polyamines such as spermidine and spermine are primordial polycations that are ubiquitously present in the three domains of life. We have found that Gram‐positive bacteria Staphylococcus aureus and Enterococcus faecalis have lost either all or most polyamine biosynthetic genes, respectively, and are devoid of any polyamine when grown in polyamine‐free media. In contrast to bacteria such as Pseudomonas aeruginosa, Campylobacter jejuni and Agrobacterium tumefaciens, which absolutely require polyamines for growth, S. aureus and E. faecalis grow normally over multiple subcultures in the absence of polyamines. Furthermore, S. aureus and E. faecalis form biofilms normally without polyamines, and exogenous polyamines do not stimulate growth or biofilm formation. High levels of external polyamines, including norspermidine, eventually inhibit biofilm formation through inhibition of planktonic growth. We show that spermidine/spermine N‐acetyltransferase (SSAT) homologues encoded by S. aureus USA300 and E. faecalis acetylate spermidine, spermine and norspermidine, that spermine is the more preferred substrate, and that E. faecalis SSAT is almost as efficient as human SSAT with spermine as substrate. The polyamine auxotrophy, polyamine‐independent growth and biofilm formation, and presence of functional polyamine N‐acetyltransferases in S. aureus and E. faecalis represent a new paradigm for bacterial polyamine biology.     

Photodynamic and peptide-based strategy to inhibit Gram-positive bacterial biofilm formation

Abstract

The self-produced extracellular polymeric matrix of biofilms renders them difficult to eliminate once they are established. This makes the inhibition of biofilm formation key to successful treatment of biofilm infection. Antimicrobial photodynamic therapy (aPDT) and antimicrobial peptides offer a new approach as antibiofilm strategies. In this study sub-lethal doses of aPDT (with chlorin-e6 (Ce6-PDT) or methylene blue (MB-PDT)) and the peptides AU (aurein 1.2 monomer) or (AU)₂K (aurein 1.2?C-terminal dimer) were combined to evaluate their ability to prevent biofilm development by Enterococcus faecalis. Biofilm formation was assessed by resazurin reduction, confocal microscopy, and infrared spectroscopy. All treatments successfully prevented biofilm development. The (AU)₂K dimer had a stronger effect, both alone and combined with aPDT, while the monomer AU had significant activity when combined with Ce6-PDT. Additionally, it is shown that the peptides bind to the lipoteichoic acid of the E. faecalis cell wall, pointing to a possible key mechanism of biofilm inhibition.     

Nanographene oxides carrying antisense walR RNA regulates the Enterococcus faecalis biofilm formation and its susceptibility to chlorhexidine

Enterococcus faecalis is the dominant pathogen for persistent periapical periodontitis. The chlorhexidine (CHX) is used as conversional irrigation agents during endodontic root canal therapy. It was reported that the antisense walR RNA (ASwalR) suppressed the biofilm organization. The aim of this study was to investigate the antimicrobial effects of novel graphene oxide (GO)-polyethylenimine (PEI)-based antisense walR (ASwalR) on the inhibition of E. faecalis biofilm and its susceptibility to chlorhexidine. The recombinant ASwalR plasmids were modified with a gene encoding enhanced green fluorescent protein (ASwalR-eGFP) as a reporter gene so that the transformation efficiency could be evaluated by the fluorescence intensity. The GO-PEI-based ASwalR vector transformation strategy was developed to be transformed into E. faecalis and to over-produce ASwalR in biofilms. Colony forming units (CFU) and confocal laser scanning microscopy were used to investigate whether the antibacterial properties of antisense walR interference strategy sensitize E. faecalis biofilm to the CHX. The results indicated that overexpression of ASwalR by GO-PEI-based transformation strategy could inhibit biofilm formation, decrease the EPS synthesis and increase the susceptibility of E. faecalis biofilms to CHX. Our reports demonstrated that antisense walR RNA will be a supplementary strategy in treating E. faecalis with irrigation agents.     

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.     

Mechanism for Sortase Localization and the Role of Sortase Localization in Efficient Pilus Assembly in Enterococcus faecalis

Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and secretion of virulence factors out of the cell. Since many secreted virulence factors in gram-positive organisms are subsequently attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion and cell wall attachment in Enterococcus faecalis. We discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the enterococcus. The SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted, implying a single site of secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that this positively charged domain can act as a localization retention signal for the focal compartmentalization of membrane proteins.Understanding the transport and processing of proteins in their journey from the cytosol to the extracellular milieu has driven significant advances in elucidating the molecular interactions between an organism and its environment. These interactions are particularly important at the host-pathogen interface, where bacterial adhesins, toxins, and other virulence factors interact with host tissues (31). In gram-negative organisms, transit from the cytosol to the extracellular environment occurs by several mechanisms that either bypass the periplasm or use it as an organelle to process and fold proteins destined for secretion (46). Gram-positive organisms lack a membrane-bound periplasm but nevertheless secrete many virulence factors that require posttranslational modification (21). It has been proposed that the space between the cell membrane and cell wall provides a protected environment for folding and processing of secreted proteins in gram-positive bacteria (23, 24, 36, 52). Once translocated across the membrane, many virulence factors, such as the Streptococcus pyogenes SpeB protease, are secreted into the extracellular milieu (4), while adhesins are retained at the bacterial surface, where they mediate attachment to host tissues. A large subset of adhesins characterized as virulence factors in gram-positive organisms, such as S. pyogenes M protein and Staphylococcus aureus protein A, are covalently linked to the cell wall by the presence of a cell wall sorting (CWS) signal (1, 8, 41). The CWS signal is comprised of a C-terminal LPXTG motif, a transmembrane domain, and a positively charged tail (41). Proteins containing this CWS signal are recognized by a sortase enzyme, which cleaves the CWS motif between the threonine-glycine bond. Subsequent transpeptidation links the protein to a lipid II intermediate prior to its incorporation into the cell wall (26, 47). The protein-lipid II complex is processed by penicillin binding proteins, which results in the incorporation of the CWS protein into the mature cell wall (48). Despite the great deal known about the biochemical and mechanistic aspects of cell wall synthesis and sorting, details of the spatio-temporal coordination of cell wall synthesis, sorting, and secretion are unclear. Nevertheless, a close association linking these separate processes appears to be critical, because CWS proteins become properly exposed on the surface of the bacteria only after their sortase-mediated incorporation into the cell wall (25).Enterococcus faecalis commonly causes urinary tract infections, endocarditis, intra-abdominal infections, and bacteremia, and it relies on CWS proteins, including Esp, aggregation substance (AS), and pili, to cause disease (18, 27, 39, 42). While these studies demonstrate the importance of cell wall proteins in E. faecalis pathogenesis, the basic mechanisms by which these proteins are localized to the cell surface or secreted remains unclear. We show here that secretion, protein trafficking, and cell wall processing are colocalized at single foci in E. faecalis through the presence of a positively charged retention domain within the localized protein itself, indicating that these processes are compartmentalized into an organelle.     

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.     

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.     

Virtual screening of LPXTG competitive SrtA inhibitors targeting signal transduction mechanism in Bacillus anthracis: a combined experimental and theoretical study

Abstract

Members of the sortase enzyme super family decorate the surfaces of Bacillus anthracis cell wall with proteins that play key roles in microbial pathogenesis and its biofilm formation. Bacillus anthracis Sortase-A (Ba-SrtA) is a potential target for new therapeutics as it is required for B. anthracis survival and replication within macrophages. An understanding of the binding site pocket and substrate recognition mechanism by SrtA enzymes may serve to be beneficial in the rational development of sortase inhibitors. Here, the LPXTG signal peptide-based competitive inhibitors are screened against the Ba-SrtA and compounds with reasonable inhibition, specificity, and mechanisms of inactivation of SrtA have been covered. The screened compounds are experimentally validated against the phylogenetically similar Gram-positive pathogen B. cereus. In situ microscopic visualizations suggest that these screened compounds showed the microbial and biofilm inhibitory activity against B. cereus. It facilitates the further development of these molecules into useful anti-infective agents to treat infections caused by B. anthracis and other Gram-positive pathogens. These results provide insight into basic design principles for generating new clinically relevant lead molecules. It also provides an alternative strategy where a screened ligand molecule can be used in combination to battle increasingly against the Gram-positive pathogens.     

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.     

Impact of peptidoglycan O-acetylation on autolytic activities of the Enterococcus faecalis N-acetylglucosaminidase AtlA and N-acetylmuramidase AtlB

Autolysins are potentially lethal enzymes that partially hydrolyze peptidoglycan for incorporation of new precursors and septum cleavage after cell division. Here, we explored the impact of peptidoglycan O-acetylation on the enzymatic activities of Enterococcus faecalis major autolysins, the N-acetylglucosaminidase AtlA and the N-acetylmuramidase AtlB. We constructed isogenic strains with various O-acetylation levels and used them as substrates to assay E. faecalis autolysin activities. Peptidoglycan O-acetylation had a marginal inhibitory impact on the activities of these enzymes. In contrast, removal of cell wall glycopolymers increased the AtlB activity (37-fold), suggesting that these polymers negatively control the activity of this enzyme.     

The Enterococcal Surface Protein, Esp, Is Involved in Enterococcus faecalis Biofilm Formation

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.     

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.     

A sweet new role for LCP enzymes in protein glycosylation

The peptidoglycan that surrounds Gram‐positive bacteria is affixed with a range of macromolecules that enable the microbe to effectively interact with its environment. Distinct enzymes decorate the cell wall with proteins and glycopolymers. Sortase enzymes covalently attach proteins to the peptidoglycan, while LytR‐CpsA‐Psr (LCP) proteins are thought to attach teichoic acid polymers and capsular polysaccharides. Ton‐That and colleagues have discovered a new glycosylation pathway in the oral bacterium Actinomyces oris in which sortase and LCP enzymes operate on the same protein substrate. The A. oris LCP protein has a novel function, acting on the cell surface to transfer glycan macromolecules to a protein, which is then attached to the cell wall by a sortase. The reactions are tightly coupled, as elimination of the sortase causes the lethal accumulation of glycosylated protein in the membrane. Since sortase enzymes are attractive drug targets, this novel finding may provide a convenient cell‐based tool to discover inhibitors of this important enzyme family.     

Lytic transglycosylases RlpA and MltC assist in Vibrio cholerae daughter cell separation

The cell wall is a crucial structural feature in the vast majority of bacteria and comprises a covalently closed network of peptidoglycan (PG) strands. While PG synthesis is important for survival under many conditions, the cell wall is also a dynamic structure, undergoing degradation and remodeling by ‘autolysins’, enzymes that break down PG. Cell division, for example, requires extensive PG remodeling, especially during separation of daughter cells, which depends heavily upon the activity of amidases. However, in Vibrio cholerae, we demonstrate that amidase activity alone is insufficient for daughter cell separation and that lytic transglycosylases RlpA and MltC both contribute to this process. MltC and RlpA both localize to the septum and are functionally redundant under normal laboratory conditions; however, only RlpA can support normal cell separation in low‐salt media. The division‐specific activity of lytic transglycosylases has implications for the local structure of septal PG, suggesting that there may be glycan bridges between daughter cells that cannot be resolved by amidases. We propose that lytic transglycosylases at the septum cleave PG strands that are crosslinked beyond the reach of the highly regulated activity of the amidase and clear PG debris that may block the completion of outer membrane invagination.     

Role of interspecies interactions in dual-species biofilms developed in vitro by uropathogens isolated from polymicrobial urinary catheter-associated bacteriuria

Most catheter-associated urinary tract infections are polymicrobial. Here, uropathogen interactions in dual-species biofilms were studied. The dual-species associations selected based on their prevalence in clinical settings were Klebsiella pneumoniae–Escherichia coli, E. coli–Enterococcus faecalis, K. pneumoniae–E. faecalis, and K. pneumoniae–Proteus mirabilis. All species developed single-species biofilms in artificial urine. The ability of K. pneumoniae to form biofilms was not affected by E. coli or E. faecalis co-inoculation, but was impaired by P. mirabilis. Conversely, P. mirabilis established a biofilm when co-inoculated with K. pneumoniae. Additionally, E. coli persistence in biofilms was hampered by K. pneumoniae but not by E. faecalis. Interestingly, E. coli, but not K. pneumoniae, partially inhibited E. faecalis attachment to the surface and retarded biofilm development. The findings reveal bacterial interactions between uropathogens in dual-species biofilms ranged from affecting initial adhesion to outcompeting one bacterial species, depending on the identity of the partners involved.