Regulation of Virulence by the RevR Response Regulator in Clostridium perfringens

Abstract

Clostridium perfringens causes clostridial myonecrosis or gas gangrene and produces several extracellular hydrolytic enzymes and toxins, many of which are regulated by the VirSR signal transduction system. The revR gene encodes a putative orphan response regulator that has similarity to the YycF (WalR), VicR, PhoB, and PhoP proteins from other Gram-positive bacteria. RevR appears to be a classical response regulator, with an N-terminal receiver domain and a C-terminal domain with a putative winged helix-turn-helix DNA binding region. To determine its functional role, a revR mutant was constructed by allelic exchange and compared to the wild type by microarray analysis. The results showed that more than 100 genes were differentially expressed in the mutant, including several genes involved in cell wall metabolism. The revR mutant had an altered cellular morphology; unlike the short rods observed with the wild type, the mutant cells formed long filaments. These changes were reversed upon complementation with a plasmid that carried the wild-type revR gene. Several genes encoding extracellular hydrolytic enzymes (sialidase, hyaluronidase, and ␣-clostripain) were differentially expressed in the revR mutant. Quantitative enzyme assays confirmed that these changes led to altered enzyme activity and that complementation restored the wild-type phenotype. Most importantly, the revR mutant was attenuated for virulence in the mouse myonecrosis model compared to the wild type and the complemented strains. These results provide evidence that RevR regulates virulence in C. perfringens; it is the first response regulator other than VirR to be shown to regulate virulence in this important pathogen.

A Novel Toxin Regulator, the CPE1446-CPE1447 Protein Heteromeric Complex, Controls Toxin Genes in Clostridium perfringens

“…To date, only a few transcriptional regulators have been identified in C. perfringens (4,13,25). The present study discovered that the CPE1447-CPE1446 complex conserved across many clostridial species is a novel toxin gene regulator, and we also identified a possible new regulatory cascade.…”

Transcriptional Profile during Deoxycholate-Induced Sporulation in a Clostridium perfringens Isolate Causing Foodborne Illness

“…4; also see Table S7 in the supplemental material). Hiscox et al (58,59) revealed that nagH was positively regulated by the response regulator RevR in strain 13. They also showed that genes involved in the formation of the spore coat, septum, and cortex were downregulated in the revR null mutant (58).…”

“…The orthologs of glgC, AC7_A0087 (encoding glycogen synthase) and glgB, are known as the E regulon in B. subtilis (57). In the group of lipid transport and metabolism (group I), nagH, encoding hyaluronidase (-toxin), was shown to be regulated by the DNA-responding regulator RevR in C. perfringens strain 13 (58,59). In the group of translation (group J), argS, cysS, and ileS, encoding arginyl-, cysteinyl-, and isoleucyl-tRNA synthetase, respectively, were specifically upregulated for DCA.…”

Utility of the Clostridial Site-Specific Recombinase TnpX To Clone Toxic-Product-Encoding Genes and Selectively Remove Genomic DNA Fragments

“…cDNA was amplified from 4 g of RNA by using random hexamers, as described previously (32,33,35). Quantitative PCR using cDNA was performed as described previously (32,33,35). The oligonucleotides used for reverse transcription-quantitative PCR (qRT-PCR) are listed in Table 2.…”

“…RNA was treated for DNA contamination by using Turbo DNase (Ambion) and was determined to be DNA free by the failure to amplify the tet(A)P gene present on pCW3 (data not shown). cDNA was amplified from 4 g of RNA by using random hexamers, as described previously (32,33,35). Quantitative PCR using cDNA was performed as described previously (32,33,35).…”