Contributions of Listeria monocytogenes sigmaB and PrfA to expression of virulence and stress response genes during extra- and intracellular growth.

Listeria monocytogenes sigmaB and PrfA are pleiotropic regulators of stress response and virulence gene expression. Quantitative RT-PCR (qRT-PCR) was used to measure transcript levels of sigmaB- and PrfA-dependent genes in exponential-phase L. monocytogenes wild-type and DeltasigB strains as well as in bacteria exposed to environmental stresses (0.3 M NaCl or growth to stationary phase) or present in the vacuole or cytosol of human intestinal epithelial cells. Stationary-phase or NaCl-exposed L. monocytogenes showed sigmaB-dependent increases in opuCA (10- and 17-fold higher, respectively) and gadA transcript levels (77- and 14-fold higher, respectively) as compared to non-stressed, exponential-phase bacteria. While PrfA activity, as reflected by plcA transcript levels, was up to 95-fold higher in intracellular L. monocytogenes as compared to non-stressed bacteria, sigmaB activity was only slightly higher in intracellular than in non-stressed bacteria. Increased plcA transcript levels, which were similar in both host cell vacuole and cytosol, were associated with increases in both prfA expression and PrfA activity. qRT-PCR assays were designed to measure expression of prfA from each of its three promoter regions. Under all conditions, readthrough transcription from the upstream plcA promoter was very low. The relative contribution to total prfA transcription from the sigmaA-dependent P1prfA promoter ranged from approximately 17 % to 30 %, while the contribution of the P2prfA region, which appears to be transcribed by both sigmaA and sigmaB, ranged from approximately 70 % to 82 % of total prfA transcript levels. In summary (i) sigmaB is primarily activated during environmental stress and does not contribute to PrfA activation in intracellular L. monocytogenes and (ii) the partially sigmaB-dependent P2prfA promoter region contributes the majority of prfA transcripts in both intra- and extracellular bacteria.