Regulation of stress resistance and virulence genes in Acinetobacter baumannii



 DESCRIPTION (provided by applicant): Acinetobacter baumannii is a strictly aerobic, gram-negative coccobacillus that has recently emerged as a significant opportunistic human pathogen. This organism is most commonly associated with nosocomial infections and is generally considered to be an opportunistic human pathogen, although some community acquired infections are reported. Diseases caused by A. baumannii include skin and soft tissue infections, infections of the urinary tract, as well as more invasive diseases such as endocarditis, sepsis, osteomyelitis and pneumonia. A. baumannii infections are further complicated by the increasing prevalence of multidrug resistant (MDR) strains. At present, little is known about A. baumannii virulence mechanisms. Currently proposed virulence determinants include the production of siderophores, elaboration of capsular polysaccharides, biofilm production, the bacterial outer membrane protein A (OmpA), as well as several other candidate proteins of unknown function. There is even less information about how virulence-related genes are regulated. To discover A. baumannii virulence genes, we took advantage of an invertebrate insect model that has been used to study a variety of infectious agents, the larva of the wax moth, Galleria mellonella. We conducted a forward genetic screen using a large pool of transposon insertion mutations in the genome of a MDR pathogenic strain of A. baumannii, AB5075. By sequencing the insertion sites of the transposons in bacteria grown in rich media and from infected larvae, we identified more than 180 genes that appear to be specifically required for survival in larvae and dispensable for survival and growth in rich media. Seventeen of the genes encode products that are annotated as either DNA binding proteins or other types of regulatory proteins. In addition, 11 genes predicted to encode proteins related to resistance to osmotic stress were found. We created deletions of the genes annotated as trehalose synthetase (otsB), potassium channels (trkAH), and a mechanosensitive channel (mscS). These mutants also exhibited decreased survival in larva hemolymph compared to wild-type A. baumannii AB5075. This proposed work includes construction of deletion mutations of the 28 genes (17 regulators and 11 stress resistance) and testing their phenotypes in the Galleria model. The ability of the corresponding wild-type genes to complement mutants that exhibit a phenotype will be evaluated. Transcriptional profiling by RNAseq will be performed on several mutants that exhibit decreased virulence in the Galleria larva model. This work will result in important new knowledge about the genetic basis of virulence in an emerging human pathogen.