Listeria monocytogenesσ^BRegulates Stress Response and Virulence Functions

Abstract

While the stress-responsive alternative sigma factor B has been identified in different species of Bacillus, Listeria, and Staphylococcus, the B regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify B -dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate B -dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted B -dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and ⌬sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant B -dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-foldhigher expression in the wild type). Of the 55 genes exhibiting B -dependent expression, 54 were preceded by a sequence resembling the B promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the B -dependent nature of a subset of eight selected promoter regions. Notably, the B -dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, B also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that B contributes to L. monocytogenes gene expression during infection.

σ B contributes to Listeria monocytogenes invasion by controlling expression of inlA and inlB

“…Recognition of the invasion-defective phenotype of the DsigB strain in non-phagocytic cells (Kim et al, 2004), of P4 inlA as a s B -dependent promoter, and of P2 inlB as a putative s B -dependent promoter (Kazmierczak et al, 2003) identified s B as an important factor contributing to regulation of inlA and inlB. The inlA and inlB genes are transcribed both individually and in an operon (Lingnau et al, 1995).…”

“…The inlA and inlB genes are transcribed both individually and in an operon (Lingnau et al, 1995). To date, six promoters have been identified in the inlAB locus, which include a confirmed PrfA-regulated promoter (P3 inlA ) and a confirmed s B -dependent promoter (P4 inlA ) upstream of inlA, and a putative s B -dependent promoter (P2 inlB ) upstream of inlB (Kazmierczak et al, 2003;Lingnau et al, 1995). The PrfA-regulated promoter, P3 inlA , is the only promoter reported to generate a bicistronic transcript (Lingnau et al, 1995); therefore, we reasoned that any s B -mediated effects on inlB expression would probably occur through P2 inlB .…”

“…upstream of inlB (P2 inlB ) (Kazmierczak et al, 2003) suggests that contributions of s B to L. monocytogenes invasion may not be solely limited to modulation of inlA expression.…”

The SOS response of Listeria monocytogenes is involved in stress resistance and mutagenesis

“…Several classes of stress-response genes have been described in L. monocytogenes, which are activated during exposure to specific or general stresses. In particular, the class I and class III heat-shock and the SigB response have been investigated thoroughly (Derre et al, 1999;Hanawa et al, 1999;Kazmierczak et al, 2003). Recently, it has been shown that the SOS response of L. monocytogenes is activated upon mild heat exposure (van der Veen et al, 2007).…”

lmo1273, a novel gene involved in Listeria monocytogenes virulence

“…Another four genes encode proteins related to stress. Finally, 30 of the 71 genes are preceded by a sigma B promoter as identified by Kazmierczak et al (2003), suggesting that they might also be regulated in response to stress conditions. Interestingly, more than 50 % of the genes identified by this transcriptomic analysis (49/88 identified) were also found to be regulated during infection of macrophages or Caco-2 cells (Joseph et al, 2006).…”