Identification of MglA-Regulated Genes Reveals Novel Virulence Factors in Francisella tularensis

Abstract

The facultative intracellular bacterium Francisella tularensis causes the zoonotic disease tularemia. F. tularensis resides within host macrophages in vivo, and this ability is essential for pathogenesis. The transcription factor MglA is required for the expression of several Francisella genes that are necessary for replication in macrophages and for virulence in mice. We hypothesized that the identification of MglAregulated genes in the Francisella genome by transcriptional profiling of wild-type and mglA mutant bacteria would lead to the discovery of new virulence factors utilized by F. tularensis. A total of 102 MglA-regulated genes were identified, the majority of which were positively regulated, including all of the Francisella pathogenicity island (FPI) genes. We mutated novel MglA-regulated genes and tested the mutants for their ability to replicate and induce cytotoxicity in macrophages and to grow in mice. Mutations in MglA-regulated genes within the FPI (pdpB and cds2) as well as outside the FPI (FTT0989, oppB, and FTT1209c) were either attenuated or hypervirulent in macrophages compared to the wild-type strain. All of these mutants exhibited decreased fitness in vivo in competition experiments with wild-type bacteria. We have identified five new Francisella virulence genes, and our results suggest that characterizations of additional MglA-regulated genes will yield further insights into the pathogenesis of this bacterium.

In vivo negative selection screen identifies genes required for Francisella virulence

“…For example, mglA and mglB, which are required for expression of FPI genes and Francisella virulence (10,15,16) were not identified in our screen. A closer examination of our data suggests that the absence of these and other genes that may have been missed by our screen is likely because of the stringent cutoff used during our analysis or variability at a small number of spots on our microarray.…”

“…2) (4,(10)(11)(12)(13)15). The FTT0798 (capsule production) mutant was between 10-and 100-fold less virulent compared with wild-type bacteria, a milder phenotype than that of the FPI mutant.…”

“…Mice were injected s.c. with 2 ϫ 10 5 cfu of the transposon insertion library to mimic the most common route of infection through the skin. Genomic DNA was collected from the input bacteria as well as the bacteria harvested from the spleens of five mice at 48 h after infection, digested, and used to generate probes for hybridization to our Francisella microarray (15). Similar to previous experiments using wild-type bacteria, an average of 5.2 ϫ 10 6 cfu (or Ϸ10 8 cfu/g of spleen) were present per spleen (8).…”

“…Thus far, host cell death has been closely linked with bacterial replication because mutants unable to replicate in macrophages do not induce cell death and mutants which replicate faster than wild-type bacteria induce cell death more rapidly (8,15). The FTT0748 and FTT0584 mutants are the first Francisella mutants described that are hypercytotoxic even though they replicate to similar levels as wild-type bacteria in unstimulated (Fig.…”

“…Some of the genes Francisella uses to cause disease are known, including the genes in the Francisella pathogenicity island (FPI), which are essential for intracellular replication and the induction of cell death in macrophages as well as for bacterial growth in mice (10)(11)(12)(13)(14)(15). The transcription factor MglA regulates all of the genes in the FPI and is also required for replication in macrophages and mice (10,15,16). However, the genes required for many other aspects of Francisella pathogenesis (spread from peripheral to systemic sites, phagosome escape within host cells) remain unknown.…”