Exploring the physiological role and mode of action of the small RNA LhrC of Listeria monocytogenes and a recently discovered sigmaB-regulated sRNA of Staphylococcus aureus to check for their possible involvement in pathogenicity
Final Report Abstract
Throughout the last decade it has been recognized that non-coding RNAs in bacteria are not rare and singular cases, but rather play an important and extensive regulatory role in the adaption to changing environmental conditions or even for the virulence of pathogenic bacteria. The multicopy sRNA LhrC was one of the first sRNAs identified in the food-borne pathogen L. monocytogenes. It has previously been shown to be induced in blood and inside macrophages and can thus be proposed to be involved in virulence. Within the scope of this project several conditions leading to cell surface stress (β-lactam-antibiotics, bile salts, high osmolarity, low pH, ethanol or SDS) could be pinpointed as specific inducers of LhrC expression. In this process, the two-component system LisRK was shown to be indispensable for expression of all five copies of LhrC. Successive mutation in the lhrC promoter region appointed a few specific bases that are required for LhrC induction. These bases could be part of a motif that is recognized by LisR directly (there is no LisR consensus binding sequence identified yet), or by another still unknown factor that connects LisR to its target genes. Construction of a mutant lacking all five copies of LhrC (∆lhrC-1-5) turned out to be a challenging task. Eventually on hand, this mutant has been analyzed in stress tolerance assays and exhibited a hampered growth in the presence of cefuroxime, bile salts and ethanol. A bioinformatics search for possible mRNA targets of the sRNA disclosed lapB amongst others to be directly bound by LhrC. This mRNA encodes a cell wall associated protein that was recently identified as an important virulence factor. Quantitative RT-PCR on lapB showed increased amounts of the mRNA in ∆lhrC-1-5 compared to wild type after cefuroxime stress. Thus, one could suggest that lapB is bound by LhrC, and the RNA-RNA complex is prone to degradation eventually resulting in down regulation of LapB. Reporter gene fusions and Western Blot analyses on LapB protein corroborated that LhrC impedes lapB translation by blocking the ribosome binding site of the mRNA. Although LhrC binds to Hfq, it does not require the RNA chaperone in terms of stability or for lapB interaction. Structural probing and gel mobility shift assays unveiled the mechanism of LhrC-lapB binding to be not quite simple, but rather concerning three different sites of LhrC (two of them CU-rich loops), and entailing a structural rearrangement in the sRNA. Because LhrC is primarily induced after cell surface stress, a comprehensive comparison particularly of the membrane/surface proteome of wild type and ∆lhrC-1-5 should reveal possible targets of the sRNA. Thus, a protocol for the enrichment of the membrane proteome was established in L. monocytogenes allowing for a coverage of about 57% of the theoretical membrane proteome. A preferential protein quantification via metabolic protein labeling could not be implemented. Therefore, proteins have been quantified by a label free approach. The most significant result of this global analysis was the detection of 14 proteins that are dysregulated in all three biological replicates when wild type and ∆lhrC-1-5 are compared after cefuroxime stress (7 up- and 7 down regulated proteins). LhrC's involvement in virulence and the implication of several CU-rich loops in target binding is redolent of RNAIII in S. aureus. Speculating LhrC could play as a central role for virulence as RNAIII, this sRNA of L. monocytogenes surely deserves further profound studies.
Publications
- "LhrC - a quintuplets sRNA of Listeria monocytogenes and its role during infection and antimicrobial conditions". Joined meeting "Molecular mechanisms of inflammation and infection" of the three universities UHH (University Medical Center Hamburg-Eppendorf), AU (Aarhus University) and SDU (University of Southern Denmark) in Sandbjerg (Denmark) on the 31st of October and 1st of November 2011
- "An Hfq-binding sRNA in Listeria monocytogenes regulates a virulence adhesion in an Hfqindependent manner". FEBS international workshop "New Developments in RNA Biology" in Tavira (Algarve, Portugal) from the 1st to the 4th of September 2012
- "LhrC - a quintuplets sRNA of Listeria monocytogenes and its role during infection and antimicrobial conditions". Conference "2nd Mol Micro Meeting Würzburg (M3W2)" in Würzburg (Germany) from the 25th to the 27th of April 2012
