Phenotypic and Transcriptomic Analyses Demonstrate Interactions between the Transcriptional Regulators CtsR and Sigma B inListeria monocytogenes

Abstract

Listeria monocytogenesB positively regulates the transcription of class II stress response genes; CtsR negatively regulates class III stress response genes. To identify interactions between these two stress response systems, we constructed L. monocytogenes ⌬ctsR and ⌬ctsR ⌬sigB strains, as well as a ⌬ctsR strain expressing ctsR in trans under the control of an IPTG (isopropyl-␤-D-thiogalactopyranoside)-inducible promoter. These strains, along with a parent and a ⌬sigB strain, were assayed for motility, heat resistance, and invasion of human intestinal epithelial cells, as well as by whole-genome transcriptomic and quantitative real-time PCR analyses. Both ⌬ctsR and ⌬ctsR ⌬sigB strains had significantly higher thermotolerances than the parent strain; however, full heat sensitivity was restored to the ⌬ctsR strain when ctsR was expressed in trans. Although log-phase ⌬ctsR was not reduced in its ability to infect human intestinal cells, the ⌬ctsR ⌬sigB strain showed significantly lower invasion efficiency than either the parent strain or the ⌬sigB strain, indicating that interactions between CtsR and B contribute to invasiveness. Statistical analyses also confirmed interactions between the ctsR and the sigB null mutations in both heat resistance and invasion phenotypes. Microarray transcriptomic analyses and promoter searches identified (i) 42 CtsR-repressed genes, (ii) 22 genes with lower transcript levels in the ⌬ctsR strain, and (iii) at least 40 genes coregulated by both CtsR and B , including genes encoding proteins with confirmed or plausible roles in virulence and stress response. Our data demonstrate that interactions between CtsR and B play an important role in L. monocytogenes stress resistance and virulence.

Listeria monocytogenesσBModulates PrfA-Mediated Virulence Factor Expression

“…To illustrate, an L. monocytogenes ⌬sigB strain had significantly higher hly transcript levels under growth conditions similar to those used in this study (59). In another study (33), the same ⌬sigB strain also showed slightly but significantly higher plcA transcript levels than those in the parent strain when bacteria were exposed to B -inducing conditions (salt stress). The ⌬sigB strain has also been shown to have higher hemolytic activity than 10403S, supporting increased virulence gene expression in the absence of B (10,53).…”

“…The P2 prfA promoter region consists of two overlapping promoters, one recognized by A and one recognized by B (53,60,67), indicating a positive regulatory role for B in prfA expression. This positive regulatory role for B initially appeared to be contradicted by observations of increased virulence gene expression and hemolytic activity in a ⌬sigB strain (10,33,53,59). However, in the gram-positive pathogen Staphylococcus aureus, B appears to have both positive and negative roles in regulating expression of virulence factors, positively influencing expression of a number of adhesins and negatively influencing expression of numerous exoenzymes and toxins (3).…”

Population Diversity of Listeria monocytogenes LO28: Phenotypic and Genotypic Characterization of Variants Resistant to High Hydrostatic Pressure

“…The first cluster contains five immotile variants showing extreme HHP and heat resistance. Resistance to various stresses, as seen for most variants, might be explained by simultaneous activation through regulatory networks of the different stress mechanisms, as previously described for HrcA, CtsR, and B in L. monocytogenes (10,11). The second cluster contains two stressresistant variants with anaerobic growth and aerobic biofilm formation similar to the wild type (Fig.…”

Identification of a σ B -Dependent Small Noncoding RNA in Listeria monocytogenes

“…In addition, a large number of genes were found to be negatively influenced by B , in particular genes encoding flagellar and ribosomal proteins. Intriguingly, many of the B -dependent effects observed in these studies appear to be indirect, i.e., through secondary gene regulatory systems, such as the stress-related transcriptional regulators HrcA and CtsR, the key virulence regulator PrfA, and the RNA chaperone Hfq (15,16).…”