Identification of an orphan response regulator required for Francisella virulence and transcription of pathogenicity island genes

The Center for Microbial Interface Biology; Department of Molecular Virology, Immunology and Medical Genetics; and Department of Internal Medicine, Division of Infectious Diseases, The Ohio State University, Columbus, OH 43210, USA; Battelle Memorial Institute, Columbus, OH 43210, USA; Department of Medicine, University of Washington, HSB T-293, Box 357710, 1959 Pacific Street N.E., Seattle, WA 98195, USA; Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, 30602, USA

^* To whom correspondence should be addressed. Email: gunn.43{at}osu.edu.

	Abstract

Francisella tularensis is a Category A agent of biowarfare/biodefense. Little is known about the regulation of virulence gene expression in Francisella spp. Comparatively few regulatory factors exist in Francisella, including those belonging to two-component systems (TCS). However, orphan members of typical TCS can be identified. To determine if orphan TCS members affect Francisella gene expression, a gene encoding a product with high similarity to the Salmonella PmrA response regulator (FTT1557c, FNU0663.2) was deleted in F. novicida (a model organism for F. tularensis). The F. novicida pmrA mutant was defective in survival/growth within human and murine macrophage cell lines and was 100% defective in virulence in mice at a dose up to 10⁸ CFU. In addition, the mutant strain demonstrated increased susceptibility to antimicrobial peptide killing, but no differences were observed in the lipid A of the mutant versus the parental strain, as has been observed with pmrA mutants in other microbes. The F. novicida pmrA mutant was 100% protective as a single dose vaccine when challenged with 10⁶ CFU of F. novicida, but did not protect against Type A Schu S4 wild type challenge. DNA microarray analysis identified 65 genes regulated by PmrA. The majority of these genes were located in the region surrounding pmrA or within the Francisella pathogenicity island (FPI). These FPI genes are also regulated by MglA, but MglA does not regulate pmrA nor does PmrA regulate MglA. Thus, the orphan response regulator PmrA is an important factor in controlling virulence in F. novicida and a pmrA mutant strain is an effective vaccine against homologous challenge.