Regulatory roles of two small RNAs in the human pathogen Listeria monocytogenes and the evaluation of an alternative infection model

Abstract: Listeriosis is a potentially lethal disease caused by the Gram-positive facultative intracellular pathogen Listeria monocytogenes (L.m.). L.m. is found ubiquitously in the environment and infects humans via ingestion of contaminated food. Contaminated products are usually derived from ruminants and involve dairy products and different kinds of processed meat. Listeriosis is a potential lifethreatening disease with a total mortality rate of 20-30 %. The development of listeriosis may lead to meningitis and septicemia or other invasive diseases. Pregnant women are of increased risk of developing listeriosis and a materno-fetal infection commonly lead to spontaneous abortion or still-birth.Regulation of gene expression, and specifically virulence gene expression, is essential for pathogenic bacteria to be equipped for handling counteractions from the host as well as thriving in the often hostile environment. In pathogenic Listeria, virulence gene expression is under the control of the global virulence gene regulator PrfA. The expression of prfA is highly regulated at the transcriptional, post-transcriptional and post- translational level. We have identified a novel type of post-transcriptional regulation of prfA-mRNA by a trans-acting riboswitch element (SreA). By binding to the leader region of prfA-mRNA, SreA negatively regulates the expression of prfA. To our knowledge, this is the first description of a cis-acting riboswitch capable of functioning as a small RNA in trans, regulating targets on distant sites.To date, there have been around 100 sRNAs identified in Listeria monocytogenes, but experimental data is still limited. We have characterized a blood induced sRNA, Rli38, which is important for full virulence during oral infection of mice. Our data suggest that Rli38 regulates the expression of at least two proteins; OppD (Oligopeptide transport protein) and IsdG (heme degrading monooxygenase). Both of these proteins have been implicated in the infectious cycle of L.m. We speculate that the virulence phenotype of an ?rli38 mutant is possibly mediated through the effect of these proteins.L.m. is a complex pathogen, able to infect and replicate in a variety of organs and cause several distinctive forms of disease. These qualities of L.m. generate difficulties in simulating human listeriosis in animal models, as entailed by the multitude of models used in the field. In this work, we have evaluated the use of an alternative animal model in studying listeriosis. Our results describe the differentiated virulence potential of wildtype bacteria and a ?prfA mutant strain in the chicken embryo by live/death screening and organ colonization. Large differences in mean time to death were found between wild-type and the ?prfA strain and ?prfA cells displayed a considerable defect in colonization of the embryonal liver. The results presented in this thesis show that the chicken embryo infection model is a valuable and convenient tool in studying end-outcome and organ colonization of Listeria monocytogenes.Taken together, this thesis describes the characterization of two previously unknown sRNAs in the human pathogen Listeria monocytogenes and the use of an alternative infection model for simulating listeriosis.