Impact of Organic Acids on the Stress Response and Virulence of Listeria Monocytogenes

<p>Cells of L. monocytogenes will be habituated to lactic and acetic acids following the procedures in place in the Carpenter and Broadbent laboratories The strains of L. monocytogenes will be FSL R2-499, serotype 1/2 a, and FSL N1-277, serotype 4b because of their previously demonstrated capacity to mount an inducible resistance to both acid and bile in response to organic acids, and because they represent two serotypes that commonly become virulent in humans. Cells will be harvested during log phase growth from control or organic acid-habituated L. monocytogenes cultures, and their RNA will be isolated and converted to cDNA. Real-time quantitative PCR (Q-PCR) will be performed to quantify expression of several genes of interest including: objective 1 - LisRK , sigma B, glutamate decarboxylase, arginine deaminase, bile salt hydrolase, bile exclusion factor; objective 2 - ActA, listeriolysin, internalins. The virulence of the habituated cells will also be determined using the Galleria mellonella wax worm model using the standard methods. Briefly, 50 ul of 10E4-7 CFU L. monocytogenes, both control and habituated, will be injected into the haemocel of the wax worms (20 per treatment). The larvae will be placed in petri plates and incubated at 37C, and their survival will be monitored for 7 days post injection. The larvae will be considered dead when they show no movement in response to touch. Experiments will be repeated three times and differences in % survival at any time point will be compared using a paired student's t test. Values will be considered significantly different when P values are less than 0.05.</p>