Molecular determinants of Listeria virulence: regulation and pathogen-host cell interactions

Listeria monocytogenes, the causative agent of Listeriosis, is a severe foodborne pathogen that can be ubiquitously found in the environment and also invading mammalian cells. Its ability to survive in the environment and in processing plants is of serious concern for the food industry. The continuous emergence of foodborne outbreaks associated with a high mortality is a serious threat to public health. Understanding how the environmental cues enhance the adaptation of L. monocytogenes during saprophytic life is of valuable importance to prevent the infection. To sense and trigger the transition from saprophytism to parasitism, L. monocytogenes integrates a series of signals by means of the transcription factor PrfA. Outside the host, the genes under the control of PrfA are downregulated to maintain the bacterial fitness and are strongly activated during infection. In order to invade the mammalian cells, this pathogen utilises a family of proteins named Internalins (Inls). The Inls promote internalisation, triggering host cell defences. Therefore, avoiding recognition during intracellular infection facilitates cytosolic replication and spread of the bacteria in the host. In this MSc thesis I aimed to shed light on a novel regulatory mechanism that causes the repression of the L. monocytogenes PrfA system. This is part of a wider study, where previous chemical studies conducted in our laboratory determined that the hydrophobic resin Amberlite-XAD causes an upregulation of the listerial virulence in L. monocytogenes via sequestration of phenylalanine (Phe) from the culture medium (BHI). To this end, I defined the effect of Amberlite in the PrfA system during growth in BHI and tested the repressor effect of Phe in a chemical defined media (IMM). I assessed the impact of Phe on PrfA regulon expression during culture in vitro using lux reporter strains and explored the implication of the nutritional regulator CodY. In addition, I also directly measured the effect on virulence gene transcription on L. monocytogenes strains that lack the PrfA positive feedback loop of L. monocytogenes to better understand the mechanism by which the PrfA system is repressed. For these experiments, RNA extraction and QRT-PCR were performed at different stages of the exponential growth of bacteria. The data are consistent with the hypothesis that Phe, an abundant plant metabolite, acts as an environmental signal molecule to repress the PrfA system when L. monocytogenes is living as a soil saprophyte, in a similar way to the plant-derived beta-glucoside sugar cellobiose does. Furthermore, during this MSc thesis I worked in unravelling novel host-pathogen interactors of potential importance during Listeria infection. I conducted an analysis of previous high-throughput yeast two-hybrid screens with the aim of identifying potential host cell proteins likely to interact with the PrfA regulated Inls of L. monocytogenes. The work conducted in this second part of my MSc focussed on establishing and standardising protocols to investigate the co-localization of PrfA-regulated Inl-expressing bacteria and ubiquitin using different combinations of stainings, immunofluorescence and the construction of fluorescent listerias

Chitin Attenuates Expression of Listeria monocytogenes Virulence Genes in vitro

International audienceExternal signals are crucial for bacteria to sense their immediate environment and fine-tune gene expression accordingly. The foodborne pathogen Listeria monocytogenes senses a range of environmental cues in order to activate or deactivate the virulence-inducing transcriptional factor PrfA during transition between infectious and saprophytic lifecycles. Chitin is an abundant biopolymer formed from linked β-(1–4)-N-acetyl-D-glucosamine residues associated with fungi, the exoskeleton of insects and often incorporated into foods as a thickener or stabilizer. L. monocytogenes evolved to hydrolyse chitin, presumably, to facilitate nutrient acquisition from competitive environments such as soil where the polymer is abundant. Since mammals do not produce chitin, we reasoned that the polymer could serve as an environmental signal contributing to repression of L. monocytogenes PrfA-dependent expression. This study shows a significant downregulation of the core PrfA-regulon during virulence-inducing conditions in vitro in the presence of chitin. Our data suggest this phenomenon occurs through a mechanism that differs from PTS-transport of oligosaccharides generated from either degradation or chitinase-mediated hydrolysis of the polymer. Importantly, an indication that chitin can repress virulence expression of a constitutively active PrfA∗ mutant is shown, possibly mediated via a post-translational modification inhibiting PrfA∗ activity. To our knowledge, this is the first time that chitin is reported as a molecule with anti-virulence properties against a pathogenic bacterium. Thus, our findings identify chitin as a signal which may downregulate the virulence potential of the pathogen and may provide an alternative approach toward reducing disease risk