Molecular determinants of Listeria virulence: regulation and pathogen-host cell interactions
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Date
29/11/2016Item status
Restricted AccessEmbargo end date
31/12/2100Author
Villoria Recio, Miguel
Metadata
Abstract
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.