Regulation of L. monocytogenes PrfA regulon by environmental stimuli in gastrointestinal tract

Abstract

Listeria monocytogenes, a Gram-positive bacterium, is a food-borne pathogen which causes listeriosis. It is an intracellular pathogen invading the epithelial cells where it escapes from the vacuole into the host cytoplasm to replicate, using actin-based motility to move within and between cells. The intracellular life cycle is well documented whereas the time spent in the lumen of the intestine is poorly understood. The aim of this study was to investigate the mechanism by which L. monocytogenes adapts to the environment of the small intestine prior to invasion. Specifically, to determine if the PrfA regulon, that encodes the virulence factors of L. monocytogenes, is switched on by signals within the intestinal lumen. Initially three signals were examined, butyrate, a short chain fatty acid molecule synthesised by bacteria within the gut microbiota, microaerobic conditions (5.5 %v/v oxygen) and serotonin (5-HT), a key neurotransmitter that modulates brain behaviour. 5-HT is secreted by enterochromaffin cells (EC) into the intestinal lumen where it acts to control gut motility, secretion and vasodilation. L. monocytogenes strains with either chromosomal phly::gfp or pactA::gfp transcriptional fusions were grown in MD10 medium with two different source of carbon either aerobically or microaerobically with and without 5 mM butyrate or 100 µM 5-HT and Gfp expression monitored. There was significant induction of the pactA and phly expression in microaerobic versus aerobic conditions. The addition of 5-HT had no effect while butyrate significantly lowered both phly and pactA activity. A prfA mutation abolished detectable transcription from phly and pactA while a sigB mutation led to increased expression from phly regardless of the oxygen concentration or carbon source used in the experiment. In contrast, the transcription from pactA showed a trend of increased activity in a sigB mutation but this increase was only significant in mid-log phase during aerobic growth. Western blot analysis demonstrated that under microaerobic conditions there was increased production of PrfA, LLO and ActA proteins. The RNA-seq analysis showed 27 annotated genes were specifically regulated by microaerobic conditions either up or down including the PrfA regulon virulence factors. Overall, these data indicated that L. monocytogenes PrfA regulon is highly responsive to signals likely to be encountered in the small intestine.

Date of Award	31 Dec 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	David Thornton (Supervisor), Ian Roberts (Supervisor) & Jennifer Cavet (Supervisor)