Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens

Abstract

The anaerobic bacterium Clostridium perfringens mediates clostridial myonecrosis, or gas gangrene, by producing a number of extracellular toxins and enzymes. Transposon mutagenesis with Tn916 was used to isolate a pleiotropic mutant of C. perfringens that produced reduced levels of phospholipase C, protease and sialidase, and did not produce any detectable perfringolysin O activity. Southern hybridization revealed that a single copy of Tn916 had inserted into a 2.7 kb HindIII fragment in the C. perfringens chromosome. A 4.3kb PstI fragment, which spanned the Tn916 insertion site, was cloned from the wild-type strain. When subcloned into a shuttle vector and introduced into C. perfringens this fragment was able to complement the Tn916-derived mutation. Transformation of the mutant with plasmids containing the 2.7 kb HindIII fragment, or the 4.3 kb PstI fragment, resulted in toxin and enzyme levels greater than or equal to those of the wild-type strain. The PstI fragment was sequenced and found to potentially encode seven open reading frames, two of which appeared to be arranged in an operon and shared sequence similarity with members of two-component signal transduction systems. The putative virR gene encoded a protein with a deduced molecular weight of 30,140, and with sequence similarity to activators in the response regulator family of proteins. The next gene, virS, into which Tn916 had inserted, was predicted to encode a membrane-spanning protein with a deduced molecular weight of 51,274. The putative VirS protein had sequence similarity to sensor proteins and also contained a histidine residue highly conserved in the histidine protein kinase family of sensor proteins. Virulence studies carried out using a mouse model implicated the virS gene in the pathogenesis of histotoxic C. perfringens infections. It was concluded that a two-component sensor regulator system that activated the expression of a number of extracellular toxins and enzymes involved in virulence had been cloned and sequenced. A model that described the regulation of extracellular toxin production in C. perfringens was constructed.

The Spatial Organization of the VirR Boxes Is Critical for VirR-Mediated Expression of the Perfringolysin O Gene, pfoA , from Clostridium perfringens

“…C. perfringens strains were grown at 37°C in Trypticase-peptone-glucose broth (34), brain heart infusion broth (Oxoid), fluid thioglycolate medium (Difco), or nutrient agar (31) supplemented with erythromycin (50 g ml Ϫ1 ). For the screening of perfringolysin O production, C. perfringens transformants were grown on horse blood agar (19). All agar cultures of C. perfringens were incubated in an atmosphere of 10% (vol/vol) H 2 and 10% (vol/vol) CO 2 in N 2 .…”

“…Phosphorylated VirS then acts as a phosphate donor for the phosphorylation of the conserved Asp-57 residue of VirR. Once activated, VirR then modulates the transcription of its target genes either directly or by altering the transcription of other regulatory genes, in particular by the action of VR-RNA (6,7,18,41).…”

“…It is characterized by its ability to produce many extracellular toxins and enzymes (33), including alpha-toxin (phospholipase C) and theta-toxin (perfringolysin O), which have been shown to act synergistically in gas gangrene (1,2,12,44). The production of these toxins, as well as collagenase (kappa-toxin), sialidase (19), and alpha-clostripain (40), is regulated by a two-component signal transduction system that comprises the VirS sensor histidine kinase and the VirR response regulator. The mutation or inactivation of either the virS or virR gene alters the organism's ability to produce the various toxins and enzymes, with perfringolysin O and alphaclostripain production being totally dependent on a functional VirS-VirR system (19,37,40).…”

“…The production of these toxins, as well as collagenase (kappa-toxin), sialidase (19), and alpha-clostripain (40), is regulated by a two-component signal transduction system that comprises the VirS sensor histidine kinase and the VirR response regulator. The mutation or inactivation of either the virS or virR gene alters the organism's ability to produce the various toxins and enzymes, with perfringolysin O and alphaclostripain production being totally dependent on a functional VirS-VirR system (19,37,40). In addition to its role in extracellular toxin and enzyme production, the VirS-VirR regulatory network is also involved in the regulation of several housekeeping genes and the hyp7 gene, which encodes a regulatory RNA molecule, VR-RNA (6,41).…”

Gene Regulation, Two Component Regulatory Systems, and Adaptive Responses in Treponema Denticola

“…Although LytTR domains are widespread among human and plant bacterial pathogens, they are present in only a small subset of RRs (typically 1 or 2 per bacterial genome) (Galperin 2008). LytTR RRs contribute to the regulation of diverse processes and activities, including biofilm formation (Lizewski et al 2004), toxin production (Ba-Thein et al 1996;Lyristis et al 1994), type IV pili synthesis (Belete et al 2008), antimicrobial peptide production (Risoen et al 1998), flagellar assembly, flagellar function (Martin et al 2013), natural competence (Streptococcus pneumoniae) (de Saizieu et al 2000), extracellular polysaccharide biosynthesis (Pseudomonas aeruginosa) (Lizewski et al 2004;Morici et al 2007), overall fitness and virulence gene expression (Abdelnour et al 1993;Martin et al 2013). It is noteworthy that among the Spirochaetaceae, only T. denticola, T. bryantii (identifier WP_022932649) and Sphaerochaeta globosa (WP_013608153) encode LytTR domain-containing proteins (Frederick et al 2008.…”