Assembly of Distinct Pilus Structures on the Surface of Corynebacterium diphtheriae

Abstract

Different surface organelles contribute to specific interactions of a pathogen with host tissues or infectious partners. Multiple pilus gene clusters potentially encoding different surface structures have been identified in several gram-positive bacterial genomes sequenced to date, including actinomycetales, clostridia, corynebacteria, and streptococci. Corynebacterium diphtheriae has been shown to assemble a pilus structure, with sortase SrtA essential for the assembly of a major subunit SpaA and two minor proteins, SpaB and SpaC. We report here the characterization of a second pilus consisting of SpaD, SpaE, and SpaF, of which SpaD and SpaE form the pilus shaft and SpaF may be located at the pilus tip. The structure of the SpaDEF pilus contains no SpaABC pilins as detected by immunoelectron microscopy. Neither deletion of spaA nor sortase srtA abolishes SpaDEF pilus formation. The assembly of the SpaDEF pilus requires specific sortases located within the SpaDEF pilus gene cluster. Although either sortase SrtB or SrtC is sufficient to polymerize SpaDF, the incorporation of SpaE into the SpaD pili requires sortase SrtB. In addition, an alanine in place of the lysine of the SpaD pilin motif abrogates pilus polymerization. Thus, SpaD, SpaE, and SpaF constitute a different pilus structure that is independently assembled and morphologically distinct from the SpaABC pili and possibly other pili of C. diphtheriae.Protruding out of the bacterial surface are proteinaceous filaments, named pili or fimbriae, which mediate bacterial attachment and colonization of host tissues (24,31,32,42). Pili are also known to interact with other bacteria in a microbiofilm (45), are involved in the translocation of DNA across biological membranes (4, 16), and can serve as phage receptors (6). The structure and function of pili in gram-negative bacteria have been studied in great detail (30,34). In contrast, the mechanisms of pilus assembly in gram-positive bacteria are less well understood, although pili or fimbriae have been identified in several gram-positive organisms, including Actinomyces spp. (8), Arthrobacter photogonimos (11), Corynebacterium diphtheriae (43), Ruminococcus albus (28), Streptococcus parasanguis (42), and, more recently, Streptococcus agalactiae (15). It has been proposed that gram-positive bacterial pili are covalently linked to peptidoglycan and require sortase (18,25,37,45), a transpeptidase which is found in all gram-positive organisms (21,26,35). Studies by Cisar et al. and Yeung et al. in Actinomyces revealed two fimbrial types, designated 1 and 2 (5, 46, 47). FimP and FimA precursor proteins, which harbor the C-terminal cell wall sorting signal, assemble type 1 and 2 fimbriae, respectively, in a manner that requires the expression of sortase-like genes (44,48,49). The assembly of S. parasanguis FW213 fimbriae involves the major structural subunit Fap1, which contains a C-terminal sorting signal (29, 41, 42), suggesting the role of sortase in fimbrial biogenesis.In C. diphtheriae (12, 20), we found that cory...

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

“…FimA pili are easily observed by EM in WT A. oris, and can exceed 2 μm in length (13). Although other Gram-positive bacteria have shorter pili, the expression of genes responsible for pilus biogenesis may be up-regulated to produce longer pili (31,32). One consequence of this observation is the hypothesis that pilus biogenesis is force-dependent, up-regulated only when longer pili are needed to dissipate large mechanical perturbations of the surrounding environment.…”

The Corynebacterium diphtheriae shaft pilin SpaA is built of tandem Ig-like modules with stabilizing isopeptide and disulfide bonds

“…The general principles of assembly were first established through studies on the SpaA pili expressed by Corynebacterium diphtheriae. These pili, encoded by the gene cluster spaA-spaB-srtA-spaC, comprise a polymeric shaft formed by SpaA, SpaC located at the tip, and SpaB found at the base and occasionally along the shaft (6,(8)(9)(10). In our current model, successive major pilin SpaA subunits are joined by the action of the pilus-specific sortase SrtA.…”

Is it feasible to control pathogen infection by competitive binding of probiotics to the host?

“…Corynebacterium, 16,17 Clostridia, 16,17 Enterococci, 18 and Streptococcus, 19 have been recently found also in probiotic Lactobacillus rhamnosus GG and Bifidobacterium bifidum PRL2010, demonstrating that they were involved not only with adherence but also with favorable immune response of the host. 20,21 Nonpilus adhesins of several bacteria also play pivotal roles in adherence to the host and its immune response.…”