The multicopy sRNA LhrC of the intracellular pathogen Listeria monocytogenes has been shown to be induced under infection-relevant conditions, but its physiological role and mechanism of action is not understood. In an attempt to pinpoint the exact terms of LhrC expression, cell envelope stress could be defined as a specific inducer of LhrC. In this process, the two-component system LisRK was shown to be indispensable for expression of all five copies of LhrC. lapB mRNA, encoding a cell wall associated protein that was recently identified as an important virulence factor, was disclosed to be directly bound by LhrC leading to an impediment of its translation. Although LhrC binds to Hfq, it does not require the RNA chaperone for stability or lapB mRNA interaction. The mechanism of LhrC-lapB mRNA binding was shown to involve three redundant CU-rich sites and a structural rearrangement in the sRNA. This study represents an extensive depiction of a so far uncharacterized multicopy sRNA and reveals interesting new aspects concerning its regulation, virulence association and mechanism of target binding.

Listeria monocytogenes is the causative agent of the foodborne disease listeriosis. During infection, L. monocytogenes produces an array of non-coding RNAs, including the multicopy sRNA LhrC. These five, nearly identical sRNAs are highly induced in response to cell envelope stress and target the virulence adhesin lapB at the post-transcriptional level. Here, we demonstrate that LhrC controls expression of additional genes encoding cell envelope-associated proteins with virulence function. Using transcriptomics and proteomics, we identified a set of genes affected by LhrC in response to cell envelope stress. Three targets were significantly down-regulated by LhrC at both the RNA and protein level: lmo2349, tcsA and oppA. All three genes encode membrane-associated proteins: A putative substrate binding protein of an amino acid ABC transporter (Lmo2349); the CD4+ T cell-stimulating antigen TcsA, and the oligopeptide binding protein OppA, of which the latter 2 are required for full virulence of L. monocytogenes. For OppA, we show that LhrC acts by direct base paring to the ribosome binding site of the oppA mRNA, leading to an impediment of its translation and a decreased mRNA level. The sRNA-mRNA interaction depends on 2 of 3 CU-rich regions in LhrC allowing binding of 2 oppA mRNAs to a single LhrC molecule. Finally, we found that LhrC contributes to infection in macrophage-like cells. These findings demonstrate a central role for LhrC in controlling the level of OppA and other virulence-associated cell envelope proteins in response to cell envelope stress.

Methods used to isolate Listeria spp. from raw milk were compared during a 13-month period (April 1989–April 1990). Raw milk was obtained bimonthly from 12 dairy farms during this time period. Three enrichments were compared: Listeria enrichment broth (LEB); University of Vermont medium (UVM), followed by a secondary enrichment with Fraser broth; and LPALCAMY. Four selective plating media, including Listeria selective agar-Oxford formulation; Modified McBride Listeria agar (MMLA); Lithium chloride-phenylethanol-moxalactam (LPM) agar; and L-PALCAM agar, were compared. L-PALCAMY enrichment broth enhanced isolation of L. monocytogenes and other Listeria spp. The greatest numbers of positive Listeria samples were obtained on Oxford and PALCAM agars, while the poorest isolation was obtained using the combination of LEB with MMLA. L. monocytogenes was found to be present in 3.0% (9/300) of the samples analyzed. The incidence of Listeria spp. was 28.0%; Listeria innocua, 26.7%; and Listeria welshimeri, 1.7%. Listeria ivanovii was not isolated. The incidence of Listeria was highest during the warmer months of April–July, while the lowest incidences occurred in December and February. Somatic cell counts (SCC) and standard plate counts (SPC) were determined by the cooperative that supplied the raw milk. In general, a producer's milk with high SPC corresponded to a high incidence of Listeria spp., while low SPC corresponded to a low incidence. A particular producer's milk with the highest incidence of Listeria spp. also had the highest SCC.