Regulation of virulence gene transcripts by the Francisella novicida orphan response regulator PmrA: role of phosphorylation and evidence of MglA/SspA interaction

Abstract

Francisella tularensis subsp. 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 subsp. tularensis are devoid of classical, tandemly arranged TCS genes, but orphaned members, such as that encoding the response regulator PmrA, have been identified. In the F. novicida model system, previous work has shown that a pmrA mutant shows decreased expression of FPI genes, 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. With regard to virulence gene induction, PmrA coprecipitates with the FPI transcription factors MglA and SspA, which bind RNA polymerase. Together, these data suggest 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.

FIG. 1.

EMSA. PmrA binding. Mobility shift assays were used to determine if purified proteins bound to regulated gene promoters. (A) His-PmrA binds to the pmrA (12 ng; lanes 1 to 6) and pdpD (12 ng; lanes 7 to 12) promoters in a dose-dependent manner. (B) His-PmrA binding to the pmrA promoter is affected by specific competitor DNA but not nonspecific competitor DNA. Each lane contains 12 ng of labeled pmrA promoter. (C) His-PmrA binding to the pdpD promoter is affected by specific but not nonspecific competitor DNA. Each lane contains 12 ng of labeled pdpD promoter.

FIG. 2.

EMSA. PmrA phosphorylation. Phosphorylating His-PmrA increases binding to regulated promoters. His-MglA does not bind to either the pdpD or the pmrA promoter, the combined addition of His-PmrA and His-MglA to promoters does not result in a supershift, and His-PmrA(D51A) retains its ability to bind regulated promoters. Lanes 1 to 9, 12 ng of labeled pmrA promoter and 1 μg of each indicated protein. Lanes 10 to 18, 12 ng of labeled pdpD promoter and 150 nanomoles of each indicated protein. -P indicates that the protein was treated with acetyl phosphate.

FIG. 3.

Phosphotransfer from CheA. (A) PmrA but not PmrA(D51A) is phosphorylated by the enteric histidine kinase CheA. Each lane consists of 0.1 ng phosphorylated CheA and 1 μg His-PmrA or His-PmrA(D51A). (B) Duplicate reaction mixtures were stained for total protein to demonstrate the presence of His-PmrA and His-PmrA(D51A) throughout the reaction.

FIG. 4.

Phosphotransfer from membrane fractions. (A) Francisella membrane fractions phosphorylate His-PmrA but not His-PmrA(D51A), and the putative histidine kinase KdpD is primarily responsible for phosphorylating His-PmrA. Each lane consists of 5 μg of membrane fractions (lane designations above panel A) and 1 μg His-PmrA or His-PmrA(D51A) (lane designations directly above panel B). (B) Western blot analysis detecting PmrA in duplicate phosphotransfer reaction mixtures. (C) His-PmrA shows enhanced phosphorylation by the ΔkdpE mutant and F. tularensis Schu4 membrane fractions. WT, wild type.

FIG. 5.

RT-PCR analysis of iglC in an F. novicida ΔpmrA strain complemented with PmrA or PmrA(D51A). Expression of iglC, which is within the PmrA-regulated FPI, is significantly reduced in an F. novicida ΔpmrA strain compared to wild-type (WT) bacteria. Complementation of an F. novicida ΔpmrA strain with PmrA resulted in restored expression of iglC, while complementation with PmrA(D51A) failed to restore iglC expression. *, P < 0.05.

FIG. 6.

PmrA(D51A) is important for intramacrophage replication and mouse virulence. (A) PmrA D51 is required for replication within PMA-induced (10 ng/ml) THP-1 cells at a multiplicity of infection of 50:1. Closed circles, F. novicida wild type; open circles, F. novicida ΔpmrA mutant; open squares, F. novicida ΔpmrA pKK214pgroEL [pmrA]; closed squares, F. novicida ΔpmrA pKK214pgroEL [pmrA D51A]; closed triangles, F. novicida ΔkdpD strain. (B) PmrA D51 is required for maximal virulence of F. novicida in mice. Bacteria were given intranasally to groups of five anesthetized female 6- to 8-week-old BALB/c mice at a dose of 1 × 10³ or 1 × 10⁶ CFU. Closed circles, 1 × 10³ F. novicida ΔpmrA pKK214pgroEL [pmrA]; closed squares with solid line, 1 × 10³ F. novicida ΔpmrA pKK214pgroEL [pmrA D51A]; closed squares with dashed line, 1 × 10⁶ F. novicida ΔpmrA pKK214pgroEL [pmrA D51A].

FIG. 7.

Coprecipitation. PmrA, MglA, and SspA coprecipitate. Western blot analysis of soluble lysates to which His-PmrA or His-MglA was added and precipitated with His-bind resin. (A) Anti-PmrA sera. (B) Anti-MglA sera. (C) Anti-His monoclonal antibody. Lane 1, His-PmrA added to an F. novicida ΔpmrA soluble fraction and precipitated with His-bind resin; lane 2, His-MglA added to an F. novicida ΔmglA soluble fraction and precipitated with His-bind resin; lane 3, His-SspA from F. novicida with chromosomally encoded His-tagged sspA precipitated with His-bind resin.