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Identification and partial characterization of a transposon insertion mutant of Burkohderia multivorans with reduced resistence to polymyxin B

University of British Columbia

Burkholderia multivorans is an antibiotic resistant opportunistic pathogen that is being isolated with an increased frequency from immunocompromised patients. B. multivorans can cause serious lung infections in patients with cystic fibrosis but the mechanism of pathogenesis is not understood. As well, B. multivorans causes lung infections in chronic granulomatous disease patients. Neutrophils from these patients are unable to elicit an oxidative burst and are therefore dependent on nonoxidative killing mechanisms for destroying phagocytosed microbes. B. multivorans is resistant to in vitro killing by chronic granulomatous disease neutrophils and is resistant to killing by cationic peptides which are a major component of neutrophil nonoxidative killing. The purpose of these studies was to understand how B. multivorans resists killing by cationic peptides. A transposon insertion mutant of B. multivorans (26D7) was created that had increased susceptibility to polymyxin B, colistin and the cationic peptide CP26 (a cecropin-melittin hybrid). In order to determine the genetic nature of this mutation, a cosmid library o f B. multivorans strain ATCC 17616 was constructed and screened by Southern analysis using a probe specific for the DNA adjacent to the transposon insertion site. Sequence analysis of the DNA at the site of transposon insertion in mutant 26D7 showed four open reading frames (ORJFs). ORF2 showed protein sequence similarity to several glucosyltransferases. ORF3 had protein sequence similarity to methyltransferases and was the site of the transposon insertion in mutant 26D7. Phenotypic analysis of mutant 26D7 showed that the outer membrane of this mutant was more permeable to 1-N-phenylnaphthylamine in the presence of polymyxin than the parent strain; however, like the parent strain, the mutant remained incapable of binding dansylated polymyxin. No difference was seen in the lipopolysaccharide ladders. TLC analysis revealed one fewer glycolipid and a possible shift in one of the phosphatidyl ethanolamine spots in the mutant. The results presented here suggested that a group of genes were identified that encoded for proteins possibly involved in lipid modification, important for resistance to cationic peptides. Further studies may identify the role of lipid modification in this important phenotype of the Burkholderia cepacia complex.

Thesis/Dissertation

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2009-07-16

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http://hdl.handle.net/2429/10915

Microbiology and Immunology

University of British Columbia

UBCV

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