Differential Regulation ofListeria monocytogenesInternalin and Internalin-Like Genes by σ^Band PrfA as Revealed by Subgenomic Microarray Analyses

Abstract

The Listeria monocytogenes genome contains more than 20 genes that encode cell surface–associated internalins. To determine the contributions of the alternative sigma factor σB and the virulence gene regulator PrfA to internalin gene expression, a subgenomic microarray was designed to contain two probes for each of 24 internalin-like genes identified in the L. monocytogenes 10403S genome. Competitive microarray hybridization was performed on RNA extracted from (i) the 10403S parent strain and an isogenic ΔsigB strain; (ii) 10403S and an isogenic ΔprfA strain; (iii) a (G155S) 10403S derivative that expresses the constitutively active PrfA (PrfA*) and the ΔprfA strain; and (iv) 10403S and an isogenic ΔsigBΔprfA strain. σB- and PrfA-dependent transcription of selected genes was further confirmed by quantitative reverse-transcriptase polymerase chain reaction. For the 24 internalin-like genes examined, (i) both σB and PrfA contributed to transcription of inlA and inlB, (ii) only σB contributed to transcription of inlC2, inlD, lmo0331, and lmo0610; (iii) only PrfA contributed to transcription of inlC and lmo2445; and (iv) neither σB nor PrfA contributed to transcription of the remaining 16 internalin-like genes under the conditions tested.

Transcriptomic and Phenotypic Analyses Identify Coregulated, Overlapping Regulons among PrfA, CtsR, HrcA, and the Alternative Sigma Factors σ B , σ C , σ H , and σ L in Listeria monocytogenes

“…In conjunction with previous findings (20) (5,29,30,50,56,69). In terms of contribution to biological functions, genes coregulated by B and PrfA (e.g., inlA [34,38,49]) have previously been shown to be critical for virulence (18,42), even though contributions of B and PrfA to inlA transcription may be apparent only under specific growth conditions (38,48). Our data here also indicate a Full data sets are available in Fig.…”

Prevalence, Characterization, and Antimicrobial Resistance of Listeria monocytogenes Isolates from Bovine Hides and Carcasses

“…Many putative virulence markers in L. monocytogenes have been identified, and the surface-associated internalins are claimed to play a role in the pathogenesis of human listeriosis (14). In the present study, three main internalin genes (inlA, inlC, and inlJ) as well as the lmo2672 marker were detected in all 54 L. monocytogenes isolates, which suggests that these strains may be potentially virulent for consumers.…”

Listeria monocytogenesσBModulates PrfA-Mediated Virulence Factor Expression

“…A PrfA* strain was selected for these studies to ensure constitutively high PrfA activity and, consequently, to increase the likelihood of identifying regulatory interactions between highly active PrfA (i.e., PrfA*) and B . L. monocytogenes strains with the PrfA*(G155S) allele have PrfA activity levels similar to those found in intravacuolar bacteria, as determined by hly and actA transcript levels, even when the PrfA* strain is grown under in vitro conditions (36,46). While PrfA* strains are important tools for studying PrfA-dependent transcription outside the mammalian host (45,49,62), one cannot exclude the possibility that intracellular L. monocytogenes organisms differ from extracellular PrfA* strains in some aspects of PrfA-dependent gene regulation (e.g., levels and activity status of PrfA).…”

“…To illustrate, because entry into stationary phase induces Bdependent transcription (10,59), early-stationary-phase cells were used to ensure high B activity. An L. monocytogenes strain with a prfA*(G155S) allele was used to constitutively express high levels of PrfA-regulated virulence genes (46,52,70). L. monocytogenes stock cultures of the strains used in this study (Table 1) were stored at Ϫ80°C in brain heart infusion broth (BHI broth; Difco, Detroit, MI) plus 15% glycerol.…”

“…Compared to wild-type PrfA, PrfA* has a greater affinity for the PrfA box (19), which appears to result in increased relative expression of PrfAregulated target genes. As a consequence, PrfA* strains are useful tools for in vitro simulation of the high PrfA activity levels typical of intracellular L. monocytogenes cells, i.e., transcript levels of the PrfA-dependent gene plcA are similar in a PrfA* strain grown in a liquid medium (46) and in intracellular L. monocytogenes.…”