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

Abstract

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.

Keywords: C. perfringens; Gene transcription; RNA-seq; Reciprocal gene expression.

Fig. 1

Relative gene expression of the virR and revR mutants compared to the wild type. Strand-specific RNA-seq libraries of the wild-type strain JIR325 and its virR and revR mutants were generated from RNA extracted from each strain. Comparative transcriptomic analysis was carried out and differentially expressed genes in the a virR and b revR mutants were identified using the “DESeq” R package. In the MA (log ratios versus mean average) plots that are shown, each point represents a gene. Red dots represent genes with a significant change in expression with a cutoff of FDR > 0.01 and an absolute log₂ fold change > 1. Black dots denote genes with a fold change that is not within the range of the set cutoff. The “Y” axis represents the log₂ fold change compared to the wild type and the “X” axis is the average log₂ normalized counts in two independent biological replicates

Fig. 2

QRT-PCR validation of selected genes in the virR and revR mutants. RNA was extracted from cells grown in TPG broth for 5 h, which corresponded to the late logarithmic growth phase. RNA was converted to cDNA using reverse transcriptase, and was subsequently used as the template in QRT-PCR, which was performed to validate the relative expression of the pfoA, ccp, plc, and cspB genes in the virR mutant (a) and the expression of the pfoA, plc, nirC and cspB genes in the revR mutant (b). Expression levels are shown relative to the expression of the housekeeping gene, rpoA, and are the average of at least three independent biological replicates (n ≥ 3, mean ± SEM). The asterisk (*) denotes a statistically significant difference (p ≤ 0.05) as calculated with the student’s unpaired t-test

Fig. 3

Gene Ontology (GO) and KEGG enrichment analysis. Bar charts denote the log₁₀ FDR (False Discovery Rate) of over-represented GO (grey) and KEGG (black) categories in the a virR and b revR mutants as identified by the “Goseq” Bioconductor package. Abbreviations: RNA BTFA: RNA polymerase binding transcription factor activity; PTTA: phosphate transmembrane transporter activity

Fig. 4

Genes that were regulated by both VirR and RevR. The Venn diagram generated with Vennt software represents the number of genes that were regulated by VirR (183; blue), RevR (44; pink) or both (23; purple). The 18 genes that were controlled by both regulators are indicated. The five genes that were regulated in a similar manner by VirR and RevR are not listed here

Fig. 5

Relative expression of pCP13 genes in the virR and revR mutants compared to the wild type. a The expression levels of all plasmid-encoded genes in the virR (blue) and revR (red) mutants. Note that no FDR and fold-change cutoff criteria were applied to the data represented here. b Real-time PCR was performed to validate differentially expressed pCP13 plasmid genes in the virR and revR mutants. Expression levels were relative to the expression of the housekeeping gene, rpoA, and were the average of at least three independent biological replicates (n ≥ 3, mean ± SEM). The asterisk (*) denotes a statistically significant difference (p ≤ 0.05) as calculated with the student’s unpaired t-test

Fig. 6

Expression levels and depth of coverage plots of VR-RNA, VirT and VirU. Depth of coverage plots of a VR-RNA (cpe0957), b VirT (cpe0845) and c VirU (cpe0920) in the wild-type strain, and the virR and revR mutants are shown as visualized in Artemis. The genetic organization of each sRNA is shown on the top of each figure. Mapped RNA-seq data are displayed as a plot showing sequence depth for the forward (red) and reverse (green) strand. In the depth of coverage plots, the Y axes represent the coverage depth in reads per million mapped reads (RPM). Expressed sRNAs are clearly seen as peaks in the depth of coverage plots

Fig. 7

Validation of selected sRNA transcripts. Depth of coverage plots of a SR14, b SR16 c SR42, d SR50 and e SR73 together with their respective genetic organization and RT-PCR results are shown. Depth of coverage plots from the RNA-seq data of the wild-type JIR325 are depicted, where red and green graphs represent transcription on the sense- and antisense-strand, respectively. The Y axes show the coverage depth in reads per million mapped reads (RPM) and the X axes show the corresponding genetic organization. The approximate transcript lengths are indicated in brackets. Whole genes are represented by filled arrows, while open arrows indicate partial genes. RT-PCR was carried out to validate these unannotated transcripts. L, PCR marker ladder; 1, DNA control; 2, wild-type RNA with RT reaction; 3, wild-type RNA with no RT; 4, virR mutant RNA with RT; 5, virR mutant RNA with no RT; 6, revR mutant RNA with RT; 7, revR mutant RNA with no RT. The estimated sizes of the RT-PCR products (in kb) are shown on the left of the agarose gel images

Fig. 8

Time-course Northern blotting analysis of SR73. RNA was isolated from the wild-type JIR325 cultured in TPG broth at the indicated times (1, 2, 3, 4 and 5 h). A total of 10 μg of RNA extracted from the wild-type strain was used in Northern blotting analysis to examine the expression of SR73. The loading control shows the total RNA samples that were separated by agarose gel electrophoresis and later transferred to a Nylon membrane for Northern blot analysis. The genes flanking SR73 are indicated as green arrows, while the yellow bar shows the position of the SR73 probe. The probe was generated using forward (→) and reverse (←) primers as designated in the gene map. The hybridization of the probe to the target RNA was detected by CDP-Star chemiluminescence. The black solid arrow marks the transcript that hybridized to the probe and the estimated size of the transcripts (in kb) is shown on the right of the Northern blot image