MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival

Abstract

Francisella tularensis is able to survive and grow within macrophages, a trait that contributes to pathogenesis. Several genes have been identified that are important for intramacrophage survival, including mglA and iglC. F. tularensis is also able to survive within amoebae. It is shown here that F. tularensis mglA and iglC mutant strains are not only defective for survival and replication within the macrophage-like cell line J774, but also within Acanthamoebae castellanii. Moreover, these strains are highly attenuated for virulence in mice, suggesting that a common mechanism underlies intramacrophage and intraamoebae survival and virulence. A 2D gel analysis of cell extracts of wild-type and mglA mutant strains revealed that at least seven prominent proteins were at low levels in the mglA mutant, and one MglA-regulated protein was identified as the IglC protein. RT-PCR analysis demonstrated reduced transcription of iglC and several other known and suspected virulence genes in the mglA mutant. Thus, MglA regulates the transcription of virulence factors of F. tularensis that contribute to intramacrophage and intraamoebae survival.

Fig. 1.

(A) Survival of F. tularensis strains within the J774 macrophage-like cell line. Strains U112 (WT), KKF34 (mglA), and KKF24 (iglC) were inoculated at a multiplicity of infection of ≈1:1 to J774 cells, and intracellular bacteria were enumerated at 24 h. The assay was performed in triplicate (see Materials and Methods). Bacterial inocula ranged from 5.1 to 6.5 × 10⁵ cfu. (B) Survival of F. tularensis strains within A. castellanii. Strains U112 (WT), KKF34 (mglA), and KKF24 (iglC) were inoculated at an multiplicity of infection of ≈10:1 to A. castellanii, and intracellular bacteria were enumerated at 10 days. The assay was performed in triplicate (see Materials and Methods). Bacterial inocula ranged from 1.3 to 2.1 × 10⁶ cfu.

Fig. 2.

2D analysis of protein expression in F. tularensis strains. Whole-cell lysates of strains U112 (WT), KKF34 (mglA), and KKF24 (iglC) were separated by 2D gel electrophoresis. The putative MglA-regulated spot identified by MALDI-TOF from the wild-type sample is indicated by arrows and corresponds to IglC.

Fig. 3.

RT-PCR analysis of MglA-regulated genes. (A) Diagram of the F. tularensis iglC locus. Genes surrounding iglC are shown as arrows (not drawn to scale) oriented in the direction of transcription; the region between iglD and pdpA contains 11 additional ORFs and is not shown. (B) RT-PCR analysis of the iglC locus. RT-PCR analysis (see Materials and Methods) was performed on total RNA prepared from F. tularensis strains U112 (WT), KKF34 (mglA), and KKF24 (iglC). PCR primers are specific to internal coding sequences of pdpD, iglA, iglC, iglD, pdpA, and tul4.