IDENTIFICATION OF LerC, A SINGLE-DOMAIN RESPONSE REGULATOR WHICH CONNECTS THE PmrAB TWO COMPONENT SYSTEM AND THE LetAS-RsmYZ-CSRA REGULATORY CASCADE IN LEGIONELLA PNEUMOPHILA

Legionella pneumophila, is an intracellular human pathogen that utilizes the Icm/Dot type-IVB secretion system to translocate a large cohort of effector proteins into host cells. The PmrAB two-component system and the LetAS-RsmYZ-CsrA cascade were shown before to regulate the expression of effector-encoding genes (EEGs) in L. pneumophila. The LetAS-RsmYZ-CsrA regulatory cascade includes the sensor histidine kinase LetS which phosphorylates and thus activates the response regulator LetA; which in turn activates the expression of the two sRNAs RsmY and RsmZ; which bind the CsrA translational repressor thus relieving the repression from its target mRNAs. In this study we characterized LerC, a single domain response regulator which is unique to Legionella. We found that lerC expression is activated by PmrA at the transcriptional level, and repressed by CsrA at the translational level, linking LerC to the two major EEGs regulatory systems. Next, we found that LerC is required for maximal expression of CsrA-regulated EEGs and the sRNAs RsmYZ. Furthermore, we compared the effect of a lerC deletion mutant to the effect of double deletion mutants of lerC and either letA or letS on CsrA-regulated EEGs, and found no additive effect. Moreover, we showed using site-directed mutagenesis of the CsrA binding sites, that the effect of LerC on CsrA-regulated EEGs occurs via CsrA. Finally, using a bacterial two-hybrid system, we showed that LerC directly interacts with LetS in its Hpt domain. This interaction was found to be independent of the LerC conserved aspartic acid in its putative phosphorylation site. This study shows for the first time that the Legionella LerC protein functions as a connector between the two major regulatory systems of EEGs.