Francisella tularensis is a facultative intracellular bacterium and the causative agent of tularemia. Virulence factors for this bacterium, particularly those that facilitate host cell interaction, remain largely uncharacterized. However, genes homologous to those involved in type IV pilus structure and assembly, including six genes encoding putative major pilin subunit proteins, are present in the genome of the highly virulent Schu S4 strain. To analyze the roles of three putative pilin genes in pili structure and function we constructed individual pilE4, pilE5, and pilE6 deletion mutants in both the F. tularensis tularensis strain Schu S4 and the Live Vaccine Strain (LVS), an attenuated derivative strain of F. tularensis holarctica. Transmission electron microscopy (TEM) of Schu S4 and LVS wild-type and deletion strains confirmed that pilE4 was essential for the expression of type IV pilus-like fibers by both subspecies. By the same method, pilE5 and pilE6 were dispensable for pilus production. In vitro adherence assays with J774A.1 cells revealed that LVS pilE4, pilE5, and pilE6 deletion mutants displayed increased attachment compared to wild-type LVS. However, in the Schu S4 background, similar deletion mutants displayed adherence levels similar to wild-type. In vivo, LVS pilE5 and pilE6 deletion mutants were significantly attenuated compared to wild-type LVS by intradermal and subcutaneous murine infection, while no Schu S4 deletion mutant was significantly attenuated compared to wild-type Schu S4. While pilE4 was essential for fiber expression on both Schu S4 and LVS, neither its protein product nor the assembled fibers contributed significantly to virulence in mice. Absent a role in pilus formation, we speculate PilE5 and PilE6 are pseudopilin homologs that comprise, or are associated with, a novel type II-related secretion system in Schu S4 and LVS.
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