RNA-seq analysis of virR and revR mutants of Clostridium perfringens.

BACKGROUND: Clostridium perfringens causes toxin-mediated diseases, including gas gangrene (clostridial myonecrosis) and food poisoning in humans. The production of the toxins implicated in gas gangrene, α-toxin and perfringolysin O, is regulated by the VirSR two-component regulatory system. In addition, RevR, an orphan response regulator, has been shown to affect virulence in the mouse myonecrosis model. RevR positively regulates the expression of genes that encode hydrolytic enzymes, including hyaluronidases and sialidases. RESULTS: To further characterize the VirSR and RevR regulatory networks, comparative transcriptomic analysis was carried out with strand-specific RNA-seq on C. perfringens strain JIR325 and its isogenic virR and revR regulatory mutants. Using the edgeR analysis package, 206 genes in the virR mutant and 67 genes in the revR mutant were found to be differentially expressed. Comparative analysis revealed that VirR acts as a global negative regulator, whilst RevR acts as a global positive regulator. Therefore, about 95 % of the differentially expressed genes were up-regulated in the virR mutant, whereas 81 % of the differentially expressed genes were down-regulated in the revR mutant. Importantly, we identified 23 genes that were regulated by both VirR and RevR, 18 of these genes, which included the sporulation-specific spoIVA, sigG and sigF genes, were regulated positively and negatively by RevR and VirR, respectively. Furthermore, analysis of the mapped RNA-seq reads visualized as depth of coverage plots showed that there were 93 previously unannotated transcripts in intergenic regions. These transcripts potentially encode small RNA molecules. CONCLUSION: In conclusion, using strand-specific RNA-seq analysis, this study has identified differentially expressed chromosomal and pCP13 native plasmid-encoded genes, antisense transcripts, and transcripts within intergenic regions that are controlled by the VirSR or RevR regulatory systems.

Concurrent Host-Pathogen Transcriptional Responses in a Clostridium perfringens Murine Myonecrosis Infection.

To obtain an insight into host-pathogen interactions in clostridial myonecrosis, we carried out comparative transcriptome analysis of both the bacterium and the host in a murine Clostridium perfringens infection model, which is the first time that such an investigation has been conducted. Analysis of the host transcriptome from infected muscle tissues indicated that many genes were upregulated compared to the results seen with mock-infected mice. These genes were enriched for host defense pathways, including Toll-like receptor (TLR) and Nod-like receptor (NLR) signaling components. Real-time PCR confirmed that host TLR2 and NLRP3 inflammasome genes were induced in response to C. perfringens infection. Comparison of the transcriptome of C. perfringens cells from the infected tissues with that from broth cultures showed that host selective pressure induced a global change in C. perfringens gene expression. A total of 33% (923) of C. perfringens genes were differentially regulated, including 10 potential virulence genes that were upregulated relative to their expression in vitro These genes encoded putative proteins that may be involved in the synthesis of cell wall-associated macromolecules, in adhesion to host cells, or in protection from host cationic antimicrobial peptides. This report presents the first successful expression profiling of coregulated transcriptomes of bacterial and host genes during a clostridial myonecrosis infection and provides new insights into disease pathogenesis and host-pathogen interactions.IMPORTANCEClostridium perfringens is the causative agent of traumatic clostridial myonecrosis, or gas gangrene. In this study, we carried out transcriptional analysis of both the host and the bacterial pathogen in a mouse myonecrosis infection. The results showed that in comparison to mock-infected control tissues, muscle tissues from C. perfringens-infected mice had a significantly altered gene expression profile. In particular, the expression of many genes involved in the innate immune system was upregulated. Comparison of the expression profiles of C. perfringens cells isolated from the infected tissues with those from equivalent broth cultures identified many potential virulence genes that were significantly upregulated in vivo These studies have provided a new understanding of the range of factors involved in host-pathogen interactions in a myonecrosis infection.

Recurrent candidaemia in a neonate with Hirschsprung's disease: fluconazole resistance and genetic relatedness of eight Candida tropicalis isolates.

The incidence of candidaemia among immunocompromised patients in Malaysia is increasing at an alarming rate. Isolation of clinical strains that are resistant to fluconazole has also risen markedly. We report here the repeated isolation of Candida tropicalis from the blood of a neonatal patient with Hirschsprung's disease. In vitro fluconazole susceptibility tests of the eight isolates obtained at different time points showed that seven of the isolates were resistant and one isolate was scored as susceptible dose-dependent. Random amplification of polymorphic DNA fingerprinting of the isolates using three primers and subsequent phylogenetic analysis revealed that these isolates were highly similar strains having minor genetic divergence, with a mean pairwise similarity coefficient of 0.893+/-0.041. The source of the infectious agent was thought to be the central venous catheter, as culture of its tip produced fluconazole-resistant C. tropicalis. This study demonstrates the utility of applying molecular epidemiology techniques to complement traditional mycological culture and drug susceptibility tests for accurate and appropriate management of recurrent candidaemia and highlights the need for newer antifungals that can combat the emergence of fluconazole-resistant C. tropicalis strains.