Proteome and Transcriptome Analysis of the Virulence Genes Regulated by the VirR/VirS System in Clostridium perfringens

Abstract

The proteins under the control of the two-component system VirR/VirS in Clostridium perfringens were analyzed by using two-dimensional gel electrophoresis of the culture supernatant from the wild type and the virR mutant. Based on matrix-assisted laser desorption ionization-time of flight/mass spectrometry, seven positively regulated proteins and eight negatively regulated proteins were identified. Transcriptome analysis confirmed that 7 of the 15 proteins were regulated by the VirR/VirS system at the transcriptional level, but the remaining proteins were modified with a VirR/VirS-directed protease at the posttranslation and secretion levels. We purified and characterized the VirR/VirS-directed protease from the culture supernatant and identified it as a kind of clostripain. Because this proteolytic activity was strongly inhibited by leupeptin and antipain, it was concluded that this protease was a member of the family of cysteine proteases of C. perfringens.Clostridium perfringens is the primary causative agent of clostridial myonecrosis, also known as gas gangrene (25). This anaerobic organism invades traumatized or ischemic tissue, and although the infection is relatively localized, the bacteria produce numerous extracellular toxins that are responsible for the extensive tissue destruction and necrosis seen in classical cases of gas gangrene (1, 9, 30).Two-component regulatory systems, consisting of a membrane sensor (histidine kinase) and a cytoplasmic response regulator, enable bacteria to sense and respond to environmental conditions. In response to an appropriate signal, autophosphorylation occurs at a conserved histidine residue in the cytoplasmic domain of the sensor. The phosphate group is then transferred to an aspartate residue on the response regulator, which in turn stimulates or represses target genes at the transcriptional level. The importance of these two-component systems, in control of both metabolism and virulence factor regulation, has been demonstrated in a wide range of bacterial species (8).Previous works have indicated that one of the two-component systems, VirR/VirS, of C. perfringens globally controls the production of the virulence factors alpha-toxin (phospholipase C), theta-toxin (perfringolysin O), kappa-toxin (collagenase), sialidase, protease, and hemagglutinin (18,27). Studies on virR mutants have also disclosed that the VirR/VirS system regulates the expression of the genes plc (alpha-toxin gene), pfoA (theta-toxin gene), and colA (kappa-toxin gene) at the transcriptional level (5). Banu et al. (3) identified the other genes that were regulated either positively or negatively by the VirR/ VirS system by means of a differential display method using the wild type and the virR mutant of C. perfringens.Proteome analysis is an excellent tool for analyzing the final products of these regulated genes. The profiles of proteome and transcriptome should be different, based on differences in the posttranscriptional regulation that control the translation rate (12) and half-lives of prot...

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

“…The genome sequence of C. perfringens strain 13 was recently published, and VirR boxes were identified upstream of four additional open reading frames. Two of these open reading frames, CPE0845 and CPE0920, were found to encode hypothetical proteins, while CPE0846 and hyp7 encode the protease alpha-clostripain (40) and the regulatory RNA molecule, VR-RNA (6, 41), respectively. The expression of both CPE0846 and hyp7 has been demonstrated to be VirR dependent, and it is assumed that the expression of CPE0845 and CPE0920 is regulated by VirR in a similar manner (6,40,41).…”

“…By performing in vitro binding studies, we showed that VirR binds to each of these repeats independently, i.e., binding to the VirR boxes is not cooperative (11). The identification of the VirR boxes (11), in conjunction with the sequencing of the C. perfringens strain 13 genome (38), has led to the identification of putative VirRbinding sites upstream of other genes (38), two of which have been demonstrated to be regulated by VirR (40,41).…”

“…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).…”

Caracterização molecular dos principais fatores de virulência e genótipos de Clostridium perfringens isolados de frangos com enterite necrótica.

“…Sistema de dois componentes VirS/VirR, composto por uma proteína sensora de membrana e outra que funciona como regulador de transcrição (BANU et al, 2000;BA-THEIN et al, 1996;CHEUNG et al, 2009;SHIMIZU et al, 2002b); 2) Sistema de a uma camada celular de enterócitos Caco-2, provoca forte estímulo para a produção das toxinas α, β, β2, e perfringolisina. Aparentemente, este processo é mediado pelo Sistema VirS/VirR, mostrando-se a importância dos sistemas regulatórios na patogênese das doenças causadas por C. perfringens (McCLANE, 2010).…”