Various cell-surface multisubunit protein polymers, known as pili or fimbriae, have a pivotal role in the colonization of specific host tissues by many pathogenic bacteria. In contrast to Gram-negative bacteria, Gram-positive bacteria assemble pili by a distinct mechanism involving a transpeptidase called sortase. Sortase crosslinks individual pilin monomers and ultimately joins the resulting covalent polymer to the cell-wall peptidoglycan. Here we review current knowledge of this mechanism and the roles of Gram-positive pili in the colonization of specific host tissues, modulation of host immune responses and the development of bacterial biofilms.
SummaryAdherence 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.
SummaryMany 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 surfacelinked 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.
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.Over 35 years ago, using electron microscopy of corynebacterial species, Yanagawa and colleagues observed proteinaceous filaments, namely, pili or fimbriae, on the surface of many corynebacterial species (30, 31). Since then, pili have been identified in several gram-positive bacteria (2, 4, 10-13, 19, 21, 29). However, the mechanisms of pilus assembly have remained obscure. It has been proposed that gram-positive bacterial pili are covalently linked to the cell wall peptidoglycan and require sortase (14,20,25,32), a transpeptidase that is found in all gram-positive organisms (5, 17, 23). The available genome sequences have revealed multiple pilus gene clusters with a putative sortase(s) in each of several gram-positive bacterial species, including the Actinomycetales, clostridia, corynebacteria, and streptococci (9, 26). In Corynebacterium diphtheriae, with genes encoding pilin subunits and putative sortases organized into three clusters, it has been shown that the organism assembles two different pilus structures encoded by spaABC and spaDEF gene clusters, designated SpaA-type and SpaD-type pili, respectively (9, 26). It is likely that the third cluster, spaHIG, also encodes a distinct pilus organelle.Previous work has shown that the SpaA-type pilus of corynebacteria is composed of three pilin subunits with SpaA polymers forming the pilus shaft, SpaB observed at regular intervals, and SpaC positioned at the tip (26). Similarly, the SpaD-type pilus is composed of SpaD, SpaE, and SpaF, which are counterparts of SpaA, SpaB, and SpaC, respectively (9). Although C. diphtheriae has six sortase genes, srtA, srtB, srt...
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.