Francisella tularensis tularensis Schu S4 Disulfide bond formation protein B, (DsbB), but not an RND-type Efflux pump, is required for virulence

Department of Medicine, and Department of Microbiology, University of Virginia, Charlottesville, VA, 22908

^* To whom correspondence should be addressed. Email: bjm2r{at}virginia.edu.

	Abstract

Francisella tularensis subsp. tularensis is a highly virulent bacterium that is a CDC Select Agent. Despite advancements in the understanding of its biology, details pertaining to virulence are poorly understood. In previous work we identified a transposon insertion mutant in the locus FTT0107c that was defective in intracellular survival in HepG2 and J77A.1 cells. Here we report that this mutant was also highly attenuated in vivo. The locus FTT0107c is predicted to encode an ortholog of Disulfide Bond Formation B (DsbB). This designation was confirmed by complementation of an E. coli dsbB mutant. This dsbB mutant of Schu S4 was highly attenuated in mice, but unlike what has been reported in F. novicida, intranasal immunization with a sublethal dose did not induce protection against wild-type challenge. dsbB was found to be transcribed in an operon with acrA and acrB which encode an RND-type efflux pump. However, this pump did not make a significant contribution to virulence because strains with nonpolar deletions in acrA and acrB behaved like wild-type Schu S4 with respect to intracellular growth and in vivo virulence. This result is in contrast to a report that an acrB mutant of Live Vaccine Strain of F. tularensis has decreased virulence in mice. Overall these results demonstrate key differences between the virulence requirements of Schu S4 and less virulent subspecies of Francisella. We have shown DsbB is a key participant in intracellular growth and virulence, and our results suggest that there are critical virulence factors that contain disulfide bonds.