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Two novel members of the LhrC family of small RNAs in Listeria monocytogenes with overlapping regulatory functions but distinctive expression profiles.

ABSTRACT: Multicopy small RNAs (sRNAs) have gained recognition as an important feature of bacterial gene regulation. In the human pathogen Listeria monocytogenes, 5 homologous sRNAs, called LhrC1-5, control gene expression by base pairing to target mRNAs though 3 conserved UCCC motifs common to all 5 LhrCs. We show here that the sRNAs Rli22 and Rli33-1 are structurally and functionally related to LhrC1-5, expanding the LhrC family to 7 members, which makes it the largest multicopy sRNA family reported so far. Rli22 and Rli33-1 both contain 2 UCCC motifs important for post-transcriptional repression of 3 LhrC target genes. One such target, oppA, encodes a virulence-associated oligo-peptide binding protein. Like LhrC1-5, Rli22 and Rli33-1 employ their UCCC motifs to recognize the Shine-Dalgarno region of oppA mRNA and prevent formation of the ribosomal complex, demonstrating that the 7 sRNAs act in a functionally redundant manner. However, differential expression profiles of the sRNAs under infection-relevant conditions suggest that they might also possess non-overlapping functions. Collectively, this makes the LhrC family a unique case for studying the purpose of sRNA multiplicity in the context of bacterial virulence.

SUBMITTER: Mollerup MS

PROVIDER: S-EPMC5013991 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The LhrC family of small regulatory RNAs (sRNAs) is known to be induced when the foodborne pathogen Listeria monocytogenes is exposed to infection-relevant conditions, such as human blood. Here we demonstrate that excess heme, the core component of hemoglobin in blood, leads to a strong induction of the LhrC family members LhrC1-5. The heme-dependent activation of lhrC1-5 relies on the response regulator LisR, which is known to play a role in virulence and stress tolerance. Importantly, our studies revealed that LhrC1-5 and LisR contribute to the adaptation of L. monocytogenes to excess heme. Regarding the regulatory function of the sRNAs, we demonstrate that LhrC1-5 act to down-regulate the expression of known LhrC target genes under heme-rich conditions: oppA, tcsA, and lapB, encoding surface exposed proteins with virulence functions. These genes were originally identified as targets for LhrC-mediated control under cell envelope stress conditions, suggesting a link between the response to heme toxicity and cell envelope stress in L. monocytogenes. We also investigated the role of LhrC1-5 in controlling the expression of genes involved in heme uptake and utilization: lmo2186 and lmo2185, encoding the heme-binding proteins Hbp1 and Hbp2, respectively, and lmo0484, encoding a heme oxygenase-like protein. Using in vitro binding assays, we demonstrated that the LhrC family member LhrC4 interacts with mRNAs encoded from lmo2186, lmo2185, and lmo0484. For lmo0484, we furthermore show that LhrC4 uses a CU-rich loop for basepairing to the AG-rich Shine-Dalgarno region of the mRNA. The presence of a link between the response to heme toxicity and cell envelope stress was further underlined by the observation that LhrC1-5 down-regulate the expression of lmo0484 in response to the cell wall-acting antibiotic cefuroxime. Collectively, this study suggests a role for the LisR-regulated sRNAs LhrC1-5 in a coordinated response to excess heme and cell envelope stress in L. monocytogenes.

Project description:While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate sigma(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 sigma(B)-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Delta 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 sigma(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-fold-higher expression in the wild type). Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions. Notably, the sigma(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, sigma(B) also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that sigma(B) contributes to L. monocytogenes gene expression during infection.

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Two novel members of the LhrC family of small RNAs in Listeria monocytogenes with overlapping regulatory functions but distinctive expression profiles.

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