Exploration into the virulence mechanisms of Listeria
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Date
27/06/2011Author
Bielecka, Magdalena Kamila
Metadata
Abstract
Pathogenic Listeria are the causative agents of listeriosis, a severe food-borne
infection. They are able to invade various non-phagocytic cell types including epithelial
cells. The life cycle of these intracellular parasites involves penetrating into host cells,
rupturing of the phagocytic vacuole, rapidly proliferating in the cytosol, and directly
spreading cell to cell. Each step of the listerial intracellular infection involves activation of
virulence factors dependent on PrfA, the master regulator of Listeria virulence. PrfAmediated
virulence gene activation occurs within host cells by mechanisms that remain
unknown. This thesis explores several aspects of PrfA regulation and its impact in the host-pathogen
interaction.
Methods for assessing PrfA-dependent gene expression were first developed and
standardized, including a highly sensitive and accurate quantitative reverse-transcription
real-time PCR (RT-QPCR), as well as procedures to investigate the correlation with
virulence using cell culture-based assays. These techniques were applied in an investigation
into the structure-function of PrfA. We studied the role of a solvent-accessible pocket
identified in the N-terminal domain of PrfA, homologous to the cyclic nucleotide-binding
(CNB) domain of Crp and other cAMP-regulated proteins, in intracellular virulence gene
activation. Site-directed PrfA mutants were constructed. Our data support the notion that
PrfA activity is allosterically regulated and are consistent with a role for the pocket as
putative binding site for the PrfA-activating allosteric effector. The characterization of
spontaneously occurring PrfA mutations that identified in our laboratory as PrfA*-
suppressor or attenuator mutations, A129T, E173G and C229Y, allowed us to gain
additional insight into PrfA structure-function. The role of the C229Y in sugar-mediated
repression was investigated and found to explain the anomalous phenotype of strain NCTC
7973, a prfA* (G145S) mutant that carries this second mutation and is repressed by
cellobiose but not glucose.
We also carried out experiments to address the intriguing activation of PrfAdependent
virulence genes upon addition of an adsorbent to the culture medium, the socalled
"charcoal effect". Using a chemically defined culture medium and resin, Amberlite™
XAD-4, we provided evidence that the virulence gene activation may involve the
sequestration of a medium component rather than a bacteria-derived autorepressor, as
initially thought.
We also explored the role of PrfA and the sigma factor σB in L. monocytogenes entry
into host cells. ΔsigB mutants in different prfA regulation backgrounds were constructed. We
showed that σB has no major effect on host cell invasion, and that L. monocytogenes
invasiveness is a strictly PrfA-dependent trait. Our results also demonstrate a differential role
of σB in L. monocytogenes serotypes. σB apparently plays no role in stress tolerance in
serotype 4b, whereas it is important in serotype 1/2a for maintenance of bacterial fitness in
stress conditions.
Finally, we investigated the occurrence of apoptosis in Listeria-infected cells and
developed normalized methods to accurately determine and quantify this cellular response in
infected cell monolayers.