A spontaneous mutation in kdsD, a biosynthesis gene for 3 Deoxy-D-manno-Octulosonic Acid, occurred in a ciprofloxacin resistant strain of Francisella tularensis and caused a high level of attenuation in murine models of tularemia

Abstract : Francisella tularensisis a gramnegative facultative intracellular bacterial pathogen that can infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacinis one of the antibiotics recommended for post-exposure prophylaxisof tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agentsand to better understand the threat, we sought to characterize an existing ciprofloxacinresistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to determine additional genetic alterations that occurred during the selection process. The sequence of the CipR strain showed additional mutations which likely occurred spontaneously during the selection process. Of particular interest was a frameshift mutationwithin kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D50 manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, it shared many phenotypic characteristics with the CipR strain, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and severe attenuation in multiple modelsof murine tularemia. This study demonstrates that the KdsD enzyme is an attractive therapeutic target for developing novel medical countermeasures.