Deletion of TolC orthologs in Francisella tularensis identifies roles in multidrug resistance and virulence

The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia. Interest in this zoonotic pathogen has increased due to its classification as a category A agent of bioterrorism, but little is known about the molecular mechanisms underlying its virulence, and especially what secretion systems and virulence factors are present. In this study, we characterized two genes in the F. tularensis genome, toIC and a gene we term fltC, whose products have high homology with the Escherichia coli ToIC protein. ToIC functions as the outer membrane channel component for both type I secretion and multidrug efflux systems. We constructed deletion mutations of these genes in the F. tularensis live vaccine strain by allelic replacement. Deletion of either toIC or WC caused increased sensitivity to various antibiotics, detergents, and dyes, indicating both genes are involved in the multidrug resistance machinery of F. tularensis. Complementation of the deletion mutations in trans restored drug resistance. Neither toIC nor fltC was required for replication of the live vaccine strain in murine bone marrow-derived macrophages. However, deletion of toIC, but not fltC, caused a significant attenuation of virulence in a mouse model of tularemia that could be complemented by addition of toIC in trans. Thus, toIC is a critical virulence factor of F. tularensis in addition to its role in multidrug resistance, which suggests the presence of a functional type I secretion system.