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BIOLOGICAL SCIENCES / MICROBIOLOGY
Deletion of TolC orthologs in Francisella tularensis identifies roles in multidrug resistance and virulence

Horacio Gil^*,, Gabrielle J. Platz^*, Colin A. Forestal^*, Michael Monfett^*, Chandra Shekhar Bakshi, Timothy J. Sellati, Martha B. Furie^*, Jorge L. Benach^*, and David G. Thanassi^*,

*Center for Infectious Diseases, Stony Brook University, Stony Brook, NY 11794-5120; and Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY 12208

Edited by Emil R. Unanue, Washington University School of Medicine, St. Louis, MO, and approved July 6, 2006 (received for review March 30, 2006)

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, tolC and a gene we term ftlC, whose products have high homology with the Escherichia coli TolC protein. TolC 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 tolC or ftlC 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 tolC nor ftlC was required for replication of the live vaccine strain in murine bone marrow-derived macrophages. However, deletion of tolC, but not ftlC, caused a significant attenuation of virulence in a mouse model of tularemia that could be complemented by addition of tolC in trans. Thus, tolC 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.

multidrug efflux | type I secretion | bacterial pathogenesis

Author contributions: H.G., M.B.F., J.L.B., and D.G.T. designed research; H.G., G.J.P., C.A.F., and M.M. performed research; C.S.B. and T.J.S. contributed new reagents/analytic tools; H.G., G.J.P., C.A.F., M.B.F., J.L.B., and D.G.T. analyzed data; and H.G. and D.G.T. wrote the paper.

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. DQ394299 (F. tularensis subspecies novicida tolC) and DQ394298 (F. tularensis subspecies novicida ftlC)].

To whom correspondence should be addressed. E-mail: dthanassi{at}ms.cc.sunysb.edu

© 2006 by The National Academy of Sciences of the USA

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