Identification of a Putative Chaperone Involved in Stress Resistance and Virulence in Francisella tularensis

Abstract

Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. This facultative intracellular bacterium replicates in vivo mainly inside macrophages and therefore has developed strategies to resist this stressful environment. Here, we identified a novel genetic locus that is important for stress resistance and intracellular survival of F. tularensis. In silico and transcriptional analyses suggest that this locus (genes FTL_0200 to FTL_0209 in the live vaccine strain [LVS]) constitutes an operon controlled by the alternative sigma factor 32 . The first gene, FTL_0200, encodes a putative AAA؉ ATPase of the MoxR subfamily. Insertion mutagenesis into genes FTL_0200, FTL_0205, and FTL_0206 revealed a role for the locus in both intracellular multiplication and in vivo survival of F. tularensis. Deletion of gene FTL_0200 led to a mutant bacterium with increased vulnerability to various stress conditions, including oxidative and pH stresses. Proteomic analyses revealed a pleiotropic impact of the ⌬FTL_0200 deletion, supporting a role as a chaperone for FTL_0200. This is the first report of a role for a MoxR family member in bacterial pathogenesis. This class of proteins is remarkably conserved among pathogenic species and may thus constitute a novel player in bacterial virulence.

Characterization of Tetratricopeptide Repeat-Like Proteins in Francisella tularensis and Identification of a Novel Locus Required for Virulence

“…Although we observed differences in the levels of intracellular replication between these two cell lines, the inFTS_1680 mutant showed significantly reduced replication in both cell lines, implying its importance for optimal intracellular replication. Phenotypic differences in intracellular proliferation within differing phagocytic cell lines have been observed with other attenuated Francisella mutants, as previously reported (5,6,34).…”

“…3A). In contrast to our studies with inFTS_0201, it has previously been reported that intraperitoneal infection of BALB/c mice with a disrupted FTL_0205 gene (FTS_0201 ortholog) in the F. tularensis LVS strain revealed a strong attenuation (6). A possible explanation for the discrepancy in the reported virulence between the inFTS_0201 and inFTL_0205 mutant strains could be the variable number of NKD repeats existing in these orthologs, the clear differences in the challenge model, or simply that within the FSC200 strain other virulence factors mitigate the effect of FTS_0201 disruption, similarly to data published by Meibom et al (35).…”

“…For oxidative stress experiments, stationary-phase bacteria from F. tularensis strain FSC200 or inFTS_1680 were diluted 1:10 into fresh Chamberlain's medium and exposed to 0.03% H 2 O 2 . Samples were harvested at 0, 20, and 40 min postexposure and viable bacteria enumerated by serial dilution on McLeod agar plates (5,6).…”

“…These include genes located in a 34-kb Francisella pathogenicity island (FPI), genes responsible for the presence of a noninflammatory lipopolysaccharide, protective capsule, and siderophores, and genes encoding proteins involved in resistance to various stress conditions (5)(6)(7)(8)(9)(10)(11)(12).…”

Antioxidant Defenses of Francisella tularensis Modulate Macrophage Function and Production of Proinflammatory Cytokines

“…We have also demonstrated that the sodBC mutant of F. tularensis LVS, which carries a point mutation in the sodB gene and a clean in-frame deletion of the sodC gene, exhibits enhanced sensitivity toward ROS and RNS, attenuated survival in macrophages, and virulence in mice (30). In addition to these primary antioxidant enzymes, a moxR family ATPase encoded by the FTL_0200 gene and two additional genes present on the moxR locus of F. tularensis LVS are required for resistance against oxidative and pH stresses (31). Additionally, the ohr gene of F. novicida and F. tularensis LVS is required for resistance to organic hydroperoxides (32).…”