Transcriptomic analysis of Clostridium perfringens and its infected murine host.
Creators: name: Low, Lee-Yean
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: Clostridium perfringens is a Gram positive anaerobic bacterium that causes a variety of toxin-mediated diseases in humans, such as clostridial myonecrosis (gas gangrene). The production of the important virulence factors of clostridial myonecrosis, α-toxin and perfringolysin O, is regulated by the VirSR two-component regulatory system. The RevR orphan response regulator is also involved in the virulence of C. perfringens as a revR mutant was attenuated for virulence in the mouse myonecrosis model, when compared to the wild-type strain. To further characterize the VirSR and RevR regulatory networks, comparative transcriptomic analysis on the virR and revR regulatory mutants, and the wild type using strand-specific RNA-seq was carried out. The results revealed that VirR and RevR act as global negative and positive regulators, respectively. A total of 18 genes, including the sporulation-specific spoIVA, sigG and sigF genes, were regulated reciprocally by RevR and VirR. RNA-seq reads visualized as a coverage plot showed that there were 97 previously unannotated transcripts in the intergenic regions (SR1-97). These transcripts may potentially encode novel proteins or small regulatory RNA molecules. One of these potential transcripts, SR76, which is located in the intergenic region between the ribU and tex genes, was further characterized. The Northern blot results showed that SR76 was co-transcribed with ribU at the early logarithmic phase and probably is the 5’UTR of the ribU. In silico analysis of SR76 revealed that it is a putative FMN riboswitch, which regulates its target genes in the presence or absence of its putative ligand, FMN. An SR76 null mutant was constructed by allelic exchange. QRT-PCR analysis showed that in the SR76 mutant, ribU was expressed in a higher level compared to the wild type, suggesting that SR76 may regulate gene expression through the formation of a terminator, thus leading to the inhibition of ribU expression. To understand host-pathogen interactions in clostridial myonecrosis, comparative RNA-seq analysis was carried out to measure the expression of both bacterial and murine genes in a murine C. perfringens infection model. The in vivo bacterial transcriptome obtained was compared with the transcriptome of its equivalent in vitro culture. A new subset of putative virulence genes that may be implicated in survival, adaptation and virulence at the early stage of infection were determined from the up-regulated genes during C. perfringens in vivo growth. The pscX gene (cpe0489) was the most highly up-regulated (&gt; log2 7.70) gene in vivo. It encodes a putative flippase, which is postulated to be involved in the synthesis or assembly of the polysaccharides in Gram positive bacteria. This study also provides evidence that the host NLRP3 inflammasome and NFκB pathways were activated as a result of defending the invasion of C. perfringens. This thesis presents the first successful study to simultaneously examine host and pathogen transcriptomes in an infected muscle lesion and thus provides invaluable insights into the infectious process in clostridial myonecrosis.