The Listeria monocytogenes Bile Stimulon under Acidic Conditions Is Characterized by Strain-Specific Patterns and the Upregulation of Motility, Cell Wall Modification Functions, and the PrfA Regulon

Abstract

Listeria monocytogenes uses a variety of transcriptional regulation strategies to adapt to the extra-host environment, the gastrointestinal tract, and the intracellular host environment. While the alternative sigma factor SigB has been proposed to be a key transcriptional regulator that facilitates L. monocytogenes adaptation to the gastrointestinal environment, the L. monocytogenes' transcriptional response to bile exposure is not well-understood. RNA-seq characterization of the bile stimulon was performed in two L. monocytogenes strains representing lineages I and II. Exposure to bile at pH 5.5 elicited a large transcriptomic response with ~16 and 23% of genes showing differential transcription in 10403S and H7858, respectively. The bile stimulon includes genes involved in motility and cell wall modification mechanisms, as well as genes in the PrfA regulon, which likely facilitate survival during the gastrointestinal stages of infection that follow bile exposure. The fact that bile exposure induced the PrfA regulon, but did not induce further upregulation of the SigB regulon (beyond that expected by exposure to pH 5.5), suggests a model where at the earlier stages of gastrointestinal infection (e.g., acid exposure in the stomach), SigB-dependent gene expression plays an important role. Subsequent exposure to bile induces the PrfA regulon, potentially priming L. monocytogenes for subsequent intracellular infection stages. Some members of the bile stimulon showed lineage- or strain-specific distribution when 27 Listeria genomes were analyzed. Even though sigB null mutants showed increased sensitivity to bile, the SigB regulon was not found to be upregulated in response to bile beyond levels expected by exposure to pH 5.5. Comparison of wildtype and corresponding ΔsigB strains newly identified 26 SigB-dependent genes, all with upstream putative SigB-dependent promoters.

Keywords: Listeria monocytogenes; PrfA; RNA-seq; lineages; sigB.

Figure 1

The Bile Stimulon. (A) Pie charts showing the number of L. monocytogenes total transcripts found to be affected by exposure to bile at pH 5.5 in strains H7858 and 10403S, with number of ncRNAs in parenthesis. H7858 and 10403S genomes include, respectively, a total of 3029 and 2956 annotated genes and ncRNAs. (B) Proportional Venn diagram showing the overlap of transcripts with significantly higher (Upregulated) or lower (Downregulated) levels after exposure to bile in H7858 (left) and 10403S (right), with number of non-coding RNAs in parenthesis.

Figure 2

Analysis of differential expression on the RNA-seq data of L. monocytogenes exposed to pH 5.5 with and without bile. Scatterplot representing the average expression level of genes exposed to pH 5.5 with bile (Y axis) vs. without bile (X axis) in L. monocytogenes 10403S (Top) and H7858 (Bottom). The known regulons of SigB and PrfA are labeled, as well as the significantly upregulated genes belonging to transport, cell wall, and motility related functions. The x = y line is shown in solid black, indicating a ratio of one (non-differentially expressed).

Figure 3

Association between genes classified here into the bile stimulon and SigB or PrfA dependent genes. For strains 10403S and H7858, two-sided Fisher's exact test was used to assess whether genes up- or down-regulated in bile were associated with (A) the SigB regulon or (B) the PrfA regulon. Odds ratio values are plotted with error bars representing the 95% confidence intervals. The y-axis is shown in log scale. FDR correction for multiple testing was applied and adjusted p < 0.05 were considered significant. Empty circles represent odds ratio values that were exactly zero and were therefore plotted on the x-axis. N.S. indicates odds ratio values that are not significantly different from 1; all odds ratio values that are not marked with NS were found to be significantly different from 1.