The PrfA regulon of Listeria monocytogenes is induced by growth in low-oxygen microaerophilic conditions

Abstract

Listeria monocytogenes is a food-borne pathogen that must adapt to several environments both inside and outside the host. One such environment is the microaerophilic conditions encountered in the host intestine proximal to the mucosal surface. The aim of this study was to investigate the expression of the PrfA regulon in response to microaerophilic growth conditions in the presence of either glucose or glycerol as a carbon source using four transcriptional (Phly, PactA, P/prfA and P/plcA) gene fusions. Further, RNAseq analysis was used to identify global changes in gene expression during growth in microaerophilic conditions. Following microaerophilic growth, there was a PrfA-dependent increase in transcription from the Phly, PactA and P/plcA promoters, indicating that microaerophilic growth induces the PrfA regulon regardless of the carbon source with increased expression of the PrfA, LLO and ActA proteins. A sigB mutation had no effect on the induction of the PrfA regulon under microaerophilic conditions when glucose was used as a carbon source. In contrast, when glycerol was the carbon source, a sigB mutation increased expression from the Phly and PactA promoters regardless of the level of oxygen. The RNAseq analysis showed that 273 genes were specifically regulated by microaerophilic conditions either up or down including the PrfA regulon virulence factors. Overall, these data indicated that L. monocytogenes PrfA regulon is highly responsive to the low-oxygen conditions likely to be encountered in the small intestine and that SigB has an input into the regulation of the PrfA regulon when glycerol is the sole carbon source.

Keywords: Listeria monocytogenes; PrfA regulon; RNAseq; microaerophilic.

Fig. 1.. The level of Gfp expression under aerobic and microaerophilic conditions using glucose or glycerol as a carbon source in MD10 media. (a) The transcription from phly promoter. (b) The transcription from pactA promoter. The fluorescence unit divided by OD₆₀₀ shows the transcription level of the strains. Black colour represents aerobic conditions, while brown colour represents the micraerophilic condition. The results show that microaerophilic conditions increase the Gfp expression when glucose or glycerol was used as a carbon source and able to relieve the catabolite repression of glucose. The data are the mean of three independent experiments. The error bar is the mean and sd. Significant differences (****P<0.00001) were calculated using two-way ANOVA.

Fig. 2.. The effect of a prfA mutation on the level of expression in either glucose or glycerol as a carbon source in MD10 media. (a) The transcription in microaerophilic conditions. (b) The transcription in aerobic conditions. The fluorescence unit divided by OD₆₀₀ shows the transcription level of the strains. Four strains were used in experiments: two as a control in black colour (Lm InlA phly::Gfp and Lm InlA pactA::Gfp) and the other two with prfA mutation in brown colour (Lm InlA ∆prfA phly::Gfp and Lm InlA ∆prfA pactA::Gfp). The results revealed that prfA mutants abolished detectable transcription from phly and pactA when glucose or glycerol was used as a carbon source under aerobic or microaerophilic conditions. The data are the mean of three independent experiments. The error bar is the mean and sd. Significant differences (***P<0.0001; ****P<0.00001) were calculated using one-way ANOVA.

Fig. 3.. Representative expression of PrfA-regulated proteins under different growth conditions. The protein was extracted from L. monocytogenes EGDe׃׃InlA^m grown in MD10 medium and analysed by Western blotting with anti-PrfA, anti-LLO, anti-ActA and anti-P60 antibodies. A-glu and A-gly mean (under aerobic condition using glucose or glycerol as a carbon source, respectively), M-glu and M-gly mean (under microaerophilic condition using glucose or glycerol as a carbon source, respectively) and Nd (no detected band). ∆prfA was used as a negative control, and P60 levels were used as a loading control.

Fig. 4.. The transcription from PrfA promoters under aerobic and microaerophilic conditions using glucose or glycerol as a carbon source in MD10 media. (a) The operon structure and promoter organization of the plcA-prfA locus involve the expression of PrfA. (b) The transcription from p/prfA and p/prfA promoters. The fluorescence unit divided by OD₆₀₀ shows the transcription level of the strains. The black colour shows the transcription under aerobic conditions, while brown colour shows the transcription under microaerophilic conditions. The error bar is the mean and sd. Significant differences (ns, non-significant; **P<0.05) were calculated using two-way ANOVA.

Fig. 5.. The effect of a sigB mutation on the level of expression in either glucose or glycerol as a carbon source in MD10 media. (a) The transcription in microaerophilic conditions. (b) The transcription in aerobic conditions. The fluorescence unit divided by OD₆₀₀ shows the transcription level of the strains. Four strains were used in experiments: two as a control in black colour (Lm InlA phly::Gfp and Lm InlA pactA::Gfp) and the other two with sigB mutation in brown colour (Lm InlA ∆sigB phly::Gfp and Lm InlA ∆sigB pactA::Gfp). The results revealed that sigB mutants increased the transcription from phly and pactA when glycerol was used as a carbon source regardless of the oxygen conditions. The data are the mean of at least three independent experiments. The error bar is the mean and sd. Significant differences (**P<0.001; ***P<0.0001; ****P<0.00001) were calculated using one-way ANOVA.

Fig. 6.. Transcriptomic analysis of L. monocytogenes under microaerophilic or aerobic conditions using glucose or glycerol as a carbon source in MD10 media. (a) PCA of gene expression for L. monocytogenes inlA in microaerophilic and aerobic conditions using glycerol or glucose as a carbon source. All four conditions were replicated twice. Each data point represents one replicate. Aerobic conditions are represented in red, and microaerophilic conditions are represented in green. When glycerol was used as a carbon source, this is represented by triangles, and when glucose was used as a carbon source, this is shown by circles. Variance explained by PC1 and PC2 is shown on the x-axis and y-axis, respectively. (b) The Venn diagram showing overlapping gene sets between the analysed conditions. The Venn diagram on the left shows genes differentially upregulated, while the Venn diagram on the right shows genes differentially downregulated. The blue group represents genes differentially expressed in aerobic conditions using glycerol as a carbon source compared to aerobic conditions using glucose as a carbon source. Green represents genes differentially expressed in microaerophilic conditions using glucose as a carbon source compared to aerobic conditions using glucose as a carbon source. Purple represents genes differentially expressed in microaerophilic conditions using glycerol as a carbon source compared to microaerophilic conditions using glucose as a carbon source. Red represents genes differentially expressed in microaerophilic conditions using glycerol as a carbon source compared to aerobic conditions using glycerol as a carbon source. The numbers indicate the number of genes, and the overlaps highlight the number of gene sets that are shared between conditions.

Fig. 7.. RNAseq analysis of L. monocytogenes under microaerophilic versus aerobic conditions using glucose or glycerol as a carbon source in MD10 media. (a, b) Genes that have significant transcriptomic changes under microaerophilic conditions specific to glucose as a carbon source. (c, d) Genes with significant transcriptomic changes under microaerophilic conditions specific to glycerol as a carbon source. (a, c) The volcano plot showing the differential gene expression in two biological replicates under microaerophilic versus aerobic conditions. An individual gene is represented by circles, while filtering differentially expressed genes is represented by dotted lines. Circles in red show annotated genes of known function that are significantly down/upregulated, while circles in blue show PrfA regulon genes that are all significantly upregulated. (b, d) A heat map showing log2 fold change of annotated genes of known function with significant padj <0.05 for two replicates. Gene values are ordered from highest to lowest fold change, from top to bottom. The key indicates the log2 fold change values for each colour.