In vivo negative selection screen identifies genes required for Francisella virulence

doi:10.1073/pnas.0609675104

In vivo negative selection screen identifies genes required for Francisella virulence

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305

Edited by E. Peter Greenberg, University of Washington School of Medicine, Seattle, WA, and approved February 12, 2007 (received for review November 2, 2006)

Abstract

Francisella tularensis subverts the immune system to rapidly grow within mammalian hosts, often causing tularemia, a fatal disease. This pathogen targets the cytosol of macrophages where it replicates by using the genes encoded in the Francisella pathogenicity island. However, the bacteria are recognized in the cytosol by the host's ASC/caspase-1 pathway, which is essential for host defense, and leads to macrophage cell death and proinflammatory cytokine production. We used a microarray-based negative selection screen to identify Francisella genes that contribute to growth and/or survival in mice. The screen identified many known virulence factors including all of the Francisella pathogenicity island genes, LPS O-antigen synthetic genes, and capsule synthetic genes. We also identified 44 previously unidentified genes that were required for Francisella virulence in vivo, indicating that this pathogen may use uncharacterized mechanisms to cause disease. Among these, we discovered a class of Francisella virulence genes that are essential for growth and survival in vivo but do not play a role in intracellular replication within macrophages. Instead, these genes modulate the host ASC/caspase-1 pathway, a previously unidentified mechanism of Francisella pathogenesis. This finding indicates that the elucidation of the molecular mechanisms used by other uncharacterized genes identified in our screen will increase our understanding of the ways in which bacterial pathogens subvert the immune system.

Footnotes

^†To whom correspondence should be addressed at:
Stanford University, 299 Campus Drive, Fairchild Building, Room D041, Stanford, CA 94305.
E-mail: dmonack{at}stanford.edu
Author contributions: A.B. and T.H. contributed equally to this work; D.S.W. and D.M.M. designed research; D.S.W., A.B., T.H., J.J.M., and K.C. performed research; D.S.W., A.B., T.H., and D.M.M. analyzed data; and D.S.W. and D.M.M. wrote the paper.
↵*Present address: Department of Microbiology and Immunology, University of California, 600 16th Street, MC 2200, San Francisco, CA 94143.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0609675104/DC1.
Abbreviations:

CI,

competitive index;

FPI,

Francisella pathogenicity island;

SAM,

significance analysis of microarrays.
Freely available online through the PNAS open access option.