Author
MILLER, CHERYL - University Of North Carolina | |
STEELE, SHAUN - University Of North Carolina | |
BRUNTON, JASON - University Of North Carolina | |
JENKINS, RONALD - University Of Michigan | |
Lovullo, Eric | |
TAFT-BENZ, SHARON - University Of North Carolina | |
ROMANCHUK, ARTUR - University Of North Carolina | |
JONES, CORBIN - University Of North Carolina | |
DOTSON, GARRY - University Of Michigan | |
COLLINS, EDWARD - University Of North Carolina | |
KAWULA, THOMAS - University Of North Carolina |
Submitted to: BMC Microbiology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 12/18/2014 Publication Date: 12/31/2014 Citation: Miller, C.N., Steele, S.P., Brunton, J.C., Jenkins, R.J., Lovullo, E.D., Taft-Benz, S.A., Romanchuk, A., Jones, C.D., Dotson, G.D., Collins, E.J., Kawula, T.H. 2014. Extragenic suppressor mutations in RipA disrupt stability and function of LpxA. BMC Microbiology. 14:336. Interpretive Summary: Francisella tularensis is the causative agent of tularemia, an insect vectored disease, and a potential biological weapon. Scientists at University of North Carolina-Chapel Hill, University of Michigan and USDA-ARS, Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida, discovered F. tularensis is able to escape immune detection in humans by modifying a major constituent of its outer membrane. Identifying how the modification is performed presents an additional target for drug development for prevention and therapeutic intervention of tularemia. Technical Abstract: Francisella tularensis is a Gram-negative bacterium that infects hundreds of species including humans, and has evolved unique mechanisms to grow efficiently within a plethora of cell types. We identified a conserved cytoplasmic membrane protein with unknown function, RipA. In F. tularensis RipA is dispensable for growth in defined media, but is required for growth inside host cells. As a means to determine RipA function, we isolated and mapped independent extragenic suppressor mutants in 'ripA that restored growth in host cells. Each suppressor mutation mapped to one of two essential genes, lpxA or glmU, which are involved in lipid A synthesis. We evaluated and characterized one of the suppressor mutations in lpxA (S102). Repairing the base pair mutation back to wild type in lpxA S102 abrogated the suppressor phenotype, demonstrating that the mutation was responsible for the restored intracellular growth of the 'ripA strain. We hypothesize that the mutation in S102 alters the stability of LpxA, thus impacting lipid A synthesis. LpxA is an essential protein, which uses the precursors UDP-N-acetylglucosamine (UDP-GlcNAc), and acyl-ACP to begin lipid A synthesis. We found that the ratio of C:18 to C:16 fatty acids in lipid A was greater in the presence of RipA. Furthermore, LpxA was more abundant in the presence of RipA. lpxA overexpression in the 'ripA strain stopped bacterial division. We also found that the LpxA S102 protein was less stable and therefore less abundant than wild type. These data suggest RipA modulates the activity and abundance of LpxA in F. tularensis as a way to adapt to the host cell environment. |