The Major Pilin Subunit Homolog PilE4 is Essential for the Expression of a Novel Type IV Pilus by Francisella tularensis

Francisella tularensis is a facultative intracellular bacterium and the causative agent of tularemia. Transmission of this bacterium is zoonotic in nature; however, the low infectious dose and ease of dissemination via aerosol release have raised awareness concerning weaponization of this organism, and led to its classification as a Category A select agent. Prior to the availability of the virulent F. tularensis Schu S4 genome sequence, virulence factors utilized by this pathogen remained largely unknown. In silica analysis of the Schu S4 genome sequence facilitated the identification of genes homologous to those involved in the assembly and expression of type IV pili, an extracellular virulence factor utilized in host cell attachment by many bacterial pathogens. F. tularensis subspecies encode six genes, designated pilEI - pilE6, with similarity to previously characterized major pilin subunit proteins. F. tularensis spp. holarctica, and its derivative LVS, have deletions or nucleotide substitutions within one or more of pilEI - pilE3 which result in absent (PilEl) or truncated (PilE2, PilE3) protein products; however, the expression of fibrous extracellular appendages remains unaffected by these mutations. This report encompasses the characterization of the remaining pilin subunit proteins PilE4, PilE5, and PilE6 in both the virulent Schu S4 and avirulent LVS strains. Using allelic exchange, we demonstrate that the absence of PilE4 results in the abrogation of pilus expression, implicating this protein as the major pilin subunit in both strains; absence of PilE5 or PilE6 does not affect pilus expression in either strain. These derivative LVS mutants all demonstrate increased adherence in vitro, though only the absence of PilE5 or PilE6 leads to attenuated virulence in viva. Notably, the pilT::HimarFT mutant, with its abrogated pilus expression comparable to that of the PilE4 mutant, demonstrates the same trends in vitro and in vivo as the PilE5 and PilE6 mutants. Similar phenotypic alterations to adherence are observed in corresponding Schu S4 mutant derivative strains; however, in this background these mutations do not impact virulence in vivo. We speculate that, in the more complete Schu S4 background, the activities of one or more of PilEl - PilE3 may compensate for the absence of PilE4, PilE5 and/or PilE6, while the absence of PilEl - PilE3 in the LVS background precludes such functional complementation. Taken together, the data suggest that neither PilE4, nor the pilus structure it comprises, contribute significantly to the virulence of F. tularensis subspecies in the model systems tested. Our findings support PilE4 as the putative structural subunit of the fibrous extracellular appendage that has been designated a type IV pilus, but suggest that piliation itself is not a prerequisite for attachment to and invasion of host cells. It seems apparent that PilE5 and PilE6 function independently of PilE4, perhaps exerting their effects within the context of the proposed type II secretion system. Further characterization of these proteins will provide a better understanding of their role in Francisella pathogenesis, in addition to their more specific role(s) within protein transport system(s) of F. tularensis subspecies.