Examining the consequences of constitutive PrfA activation and of long-term stationary phase survival strategies on the fitness of Listeria monocytogenes inside and outside of the host

The central virulence transcriptional activator, PrfA, mediates the transition of Listeria monocytogenes from an environmental bacterium to a human pathogen. L. monocytogenes is an intracellular pathogen and, as such, possesses factors that allow it to invade cells, escape the phagosome, replicate in the cytosol, and spread into neighboring cells. PrfA regulates the expression of the genes that encode the factors required for these pathogenic processes. While PrfA itself is regulated via activation upon entry of L. monocytogenes into the cytosol of an infected host cell, the mechanism of PrfA activation is unknown. However, missense mutations within the prfA allele (prfA* mutations) result in constitutive activation of PrfA. The focus of the research presented in this dissertation is to better understand the factors and mechanisms required by L. monocytogenes to live the lifestyles of an environmental bacterium and a human pathogen.;Because little is known about the physiological consequences of PrfA activation outside of the host cell, mono-culture growth experiments and mixing experiments were used to assess the fitness of prfA* mutants. Constitutive activation of PrfA was found to decrease the fitness of L. monocytogenes in many growth conditions. Specifically, constitutive PrfA activation was found to impair utilization of the PTS carbon source glucose and to increase sensitivity to osmotic and acid stresses. Interestingly, constitutive activation of PrfA was found to enhance utilization of glycerol, a non-PTS carbon source. Constitutive PrfA activation was also found to enhance L. monocytogenes virulence following both intravenous and intragastric infections of mice.;L. monocytogenes was found to possess a survival strategy for long-term stationary growth, as demonstrated by its ability to express the GASP phenotype. During long-term stationary growth, L. monocytogenes acquired stable genetic mutations that increased its fitness during this growth phase. Intravenous infections of mice showed that these mutations do not appear to affect the fitness of L. monocytogenes inside of a host.;These findings emphasize the importance of proper PrfA regulation and long-term stationary growth to maintaining the environmental and pathogenic lifestyles of L. monocytogenes.