Genetic regulation of virulence in Francisella tularensis

Date
2010
Authors
Rodriguez, Stephen A.
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Abstract

Francisella tularensis is the causal agent of the zoonotic disease tularemia, and a potential biological weapon. Because studying the molecular mechanisms of pathogenesis has been difficult due to a lack of techniques for targeted gene disruption, we have adapted a gene knockout system that efficiently targets genes for disruption in F. tularensis. This system utilizes mobile group II introns (targetrons) specifically optimized for F. tularensis. Results showed that this system is effective across F. tularensis subspecies, and enables simultaneous inactivation of duplicate genes, which should prove useful in targeting duplicated virulence genes in the highly virulent strains.

The virulence of F. tularensis is dependent on the genes encoded within the Francisella Pathogenicity Island (FPI). The FPI has recently been shown to encode a secretion system related to Type VI secretion systems found in other pathogenic bacteria. A single FPI is found in F. tularensis spp. novicida, which has low virulence for humans, whereas two identical FPIs are found in the highly virulent F. tularensis spp. tularensis. We utilized FLP recombinase-mediated excision to remove the entire FPI from F. tularensis spp. novicida. The resultant ∆FPI mutant was defective for intramacrophage replication, and was attenuated for virulence in mice. These results emphasize the importance of the FPI to F. tularensis spp. novicida virulence. Moreover, the FLP recombinase-mediated excision technique should prove useful in analyzing the duplication of the FPI in F. tularensis spp. tularensis.

The FPI gene iglE encodes a putative lipoprotein with a predicted lipobox that may be part of the T6SS-like secretion system. We constructed an iglE mutant F. tularensis spp. novicida strain, and demonstrated that iglE contributes to intramacrophage replication. Membrane fractionation of F. tularensis expressing IglE-FLAG demonstrated that this protein is found in the outer membrane (OM), but mutation of the putative site of lipidation (C22G) prevents OM localization of IglE-FLAG. Additionally, OM localization of another FPI protein, IglA, appeared to require IglE, suggesting that IglE may facilitate localization of IglA. Further experiments will clarify the contribution of iglE to the pathogenesis of F. tularensis.

The genetic techniques that have been developed and the investigation of IglE should contribute to deciphering the virulence of F. tularensis.

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Keywords
FPI, Francisella tularensis, lipoprotein, pathogenicity island, secretion system, targetron
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Department
Integrative Biology