CONTRIBUTIONS OF SIGMA-B AND PRFA TO STRESS RESPONSE AND VIRULENCE GENE EXPRESSION IN LISTERIA MONOCYTOGENES

Abstract

The food borne bacterial pathogen Listeria monocytogenes has several mechanisms for regulating expression of stress response and virulence genes. The alternative sigma factor sigmaB is a global regulator of genes active under environmental stress conditions and during stationary phase growth. I identified a large portion of the genes regulated by sigmaB using a promoter consensus sequence search and microarrays. sigmaB directly controls expression of at least 54 genes. The genes regulated by sigmaB encode proteins with a wide variety of functions, including basic metabolic pathways, membrane solute transporters, and stress resistance. In addition, I found six virulence genes, including bsh and five internalin genes, to be controlled by sigmaB. Another protein that regulates virulence gene expression in L. monocytogenes is PrfA. I measured expression of PrfA-dependent and sigmaB-dependent genes under conditions that activate each regulator, using quantitative reverse transcription-PCR (qRT-PCR). I found that sigmaB is active preferentially under environmental stress conditions, and activity decreases upon internalization of the bacteria by human epithelial cells. Conversely, PrfA is not active under stress conditions, but is highly active intracellularly. Additionally, I used qRT-PCR to determine that sigmaB contributes directly to prfA expression at the P2 promoter region. One gene initially identified in the microarray analysis to be sigmaB-dependent is lmo1433, which is predicted to encode glutathione reductase, an enzyme used by some bacteria to counteract oxidative stress. Characterizations of a strain bearing an in frame polar deletion of lmo1433 (delta-lmo1433), as well as a delta-sigB delta-lmo1433 strain, showed no difference in the strains' abilities to survive oxidative stress when compared to their respective parent strains, although presence of an intact sigB allele was important for survival. Likewise, the delta-lmo1433 and delta-sigB delta-lmo1433 strains did not show a difference in total glutathione reductase activity or intracellular survival and spread, as determined by plaquing ability on mouse L2 cell monolayers, when compared to parent strains.