Protein secretion by the type IV pilus machinery in Francisella tularensis

ABSTRACT Francisella tularensis is a highly virulent, Gram-negative bacterial pathogen that causes the zoonotic disease tularemia. F. tularensis infects a variety of host cells and replicates intracellularly while evading and interfering with host immune responses. The molecular mechanisms that facilitate the intracellular replication and virulence of F. tularensis are poorly understood. The Francisella genome contains a set of pil genes that code for the assembly of surface fibers termed type IV pili (T4P). T4P are major bacterial virulence determinants but the function of the pil system during F. tularensis infection and intracellular growth is unclear. T4P are closely related to the type II secretion pathway and the pil system of a related Francisella species, F. novicida , was shown to function in protein secretion as well as pilus assembly. To identify proteins secreted by F. tularensis , we analyzed the F. tularensis Live Vaccine Strain (LVS) using bio-orthogonal non-canonical amino acid tagging (BONCAT). Using BONCAT in conjunction with proteomics, we identified candidate proteins secreted by the wild-type LVS, as well as candidate proteins whose extracellular abundance decreased in the absence of the PilF ATPase or the PilE4 pilus subunit. Using epitope tagging of selected candidates, we validated T4P-mediated secretion of the ChiA and ChiD chitinases and the KatG catalase by the LVS. These results further our understanding of the pil system and protein secretion pathways in F. tularensis . IMPORTANCE Francisella tularensis is a highly virulent Gram-negative bacterial pathogen and the causative agent of tularemia. F. tularensis lacks secretion systems utilized by other intracellular bacterial pathogens but contains pil genes that encode for type IV pili (T4P) and may also function in protein secretion. T4P are observed on the surface of all Francisella spp. but pil -mediated protein secretion has only been reported for F. novicida , which is not normally pathogenic in humans. In this study, we used bio-orthogonal non-canonical amino acid tagging to identify proteins secreted by F. tularensis , for which there is limited information. We demonstrate that the F. tularensis pil system is capable of protein secretion and validate T4P-medeated secretion of the ChiA and ChiD chitinases and the KatG catalase. These results will facilitate investigation of Francisella virulence mechanisms and may provide targets for therapeutic intervention.