| Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infections in cystic fibrosis and other immunocompromised patients and has high innate resistance to antimicrobial peptides. The hypothesis of this thesis is that B. cenocepacia possesses multiple determinants of antimicrobial peptide resistance that can be either structural components of the lipopolysaccharide (LPS) molecule, or LPS-independent.;I show that there are at least three major determinants of B. cenocepacia antimicrobial peptide resistance: the LPS core oligosaccharide; the most highly expressed UDP-glucose dehydrogenase encoding gene, likely for the synthesis of 4-amin0-4-deoxy-L-arabinose (Ara4N) residues located in the lipid A and LPS core oligosaccharide; and the genetic regulon controlled by the alternative sigma factor RpoE and responsible for the extracytoplasmic stress response. Next, an LPS core oligosaccharide mutant (deep-rough mutant) was used to identify secondary mechanisms of B. cenocepacia antimicrobial peptide resistance. Three genes ( ispHBCAL2710, BCAM2739, and mucD) were identified that are required for antimicrobial peptide resistance in the deep-rough mutant but not in the wild-type LPS strain. The two-component response regulator system BCAL2830/BCAL2831 was required for polymyxin B resistance in the deep-rough mutant and also in the wild-type LPS strain but only at very high concentrations of polymyxin B. The deep-rough mutant was selected on increasing concentrations of polymyxin B and regained its ability to grow in high concentrations of polymyxin B. Polymyxin B resistant isolates exhibited alterations in colony morphology, increased adherent growth in the presence of polymyxin B, and increased secreted protease activity.;I propose a two-level model of B. cenocepacia antimicrobial peptide resistance. The first level, which provides for the majority of antimicrobial peptide resistance, consists of the complete LPS core oligosaccharide, lipid A and LPS core oligosaccharide Ara4N sugars, and the RpoE-mediated, extracytoplasmic stress response. The second level of resistance consists of other mechanisms that are individually required to a lesser extent for resistance to antimicrobial peptides. They are required for resistance to only some antimicrobial peptides, or are required for resistance under some conditions but not others. Taken as a whole this second level likely also contributes significantly to the high inherent resistance of B. cenocepacia to antimicrobial peptides.;Keywords: Burkholderia, Burkholderia cenocepacia, antimicrobial peptides, polymyxin B, lipopolysaccharide, deep-rough lipopolysaccharide, molecular genetics, outer membrane, Gram-negative bacteria, extracytoplasmic stress response, 4-amino-4-deoxy-L-arabinose, UDP-glucose dehydrogenase, antimicrobial adjuvant, cystic fibrosis. |
