Regulation of virulence gene transcripts by the Francisella orphan response regulator PmrA: Role of phosphorylation and evidence of MgIA/SspA interaction

Francisella tularensis subspecies tularensis is the etiologic agent of tularemia and has been designated a category A biothreat agent by the CDC. Tularemia is characterized by replication and dissemination within host phagocytes. Intramacrophage growth is dependent upon the regulation of Francisella Pathogenicity Island (FPI) virulence genes, which is poorly understood. Two-component regulatory systems (TCS) are widely employed by Gram negative bacteria to monitor and respond to environmental signals. Virulent strains of F. tularensis are devoid of classical, tandemnly arranged TCS, but orphaned members, such as the response regulator PmrA, have been identified. In the F. novicida model system, previous work has shown that a pmrA mutant is deficient for intramacrophage growth and is avirulent in the mouse model. Here we determine that phosphorylation aids PmrA binding to regulated promoters pmrA and the FPI encoded pdpD, and KdpD is the histidine kinase primarily responsible for phosphorylation of PmrA at the aspartic acid at position 51 (D51). A strain expressing PmrA D51A retains some DNA binding but exhibits reduced expression of the PmrA-regulon, is deficient for intramacrophage replication and is attenuated in the mouse model. PmrA co-precipitates with the FPI transcription factors MglA and SspA, which bind RNA polymerase. Together this data suggests a model of Francisella gene regulation that includes a TCS consisting of KdpD and PmrA. Once phosphorylated, PmrA binds to regulated gene promoters recruiting free or RNA polymerase bound MglA and SspA to initiate FPI gene transcription.