Year

2000

Degree Name

Doctor of Philosophy

Department

Department of Biological Sciences

Abstract

Bordetella bronchiseptica is a Gram-negative respiratory pathogen of a wide range of mammals. B. bronchiseptica infections are of key interest to the veterinary and agricultural industries. Lipopolysaccharide (LPS) is the major component of the outer cellular membrane and is hence integral to the implementation of many cellular processes throughout the life cycle. A genetic locus responsible for LPS core and biosynthesis in B. bronchiseptica has been recently identified. However discovery of all the genes responsible for the production of the LPS inner core thusfar evaded researchers. A novel genetic locus, encoding phosphoglucomutase (PGM) is described here. The pgm gene of B. bronchiseptica is required for the biosynthesis of the LPS inner core molecule. Genetic analysis of pgm identified gene as approximately 1.2 kb in length that shares high amino acid identity with PGM of various other bacterial species. The pgm gene forms part of an operon which also encompasses the gene encoding phosphoglucose isomerase. B. pertussis was also found to contain a homologous gene sharing 97% identity with that of B. bronchiseptica. PGM is an enzyme responsible for the conversion of glucose to the high energy nucleotide sugar, UDP-glucose, which is subsequently incorporated into the LPS inner core, being only the second residue following the lipid Aketodeoxyoctulosonic acid (KDO) moiety. Insertion mutations of the wild type B. bronchiseptica strain BB7865 and the bvg strain BB7866, which disrupted LPS biosynthesis was created and characterised (BB7S65pgm and BB7S66pgm). Functional assays for PGM revealed that enzyme activity is expressed in both bvg-positive and bvg-negative strains of B. bronchiseptica and is substantially reduced BB7S65pgm and BB7866pgm. Silver stained tricine SDS PAGE analysis of purified LPS from the mutants revealed a profile severely modified from that of the parental strains. Complementation of the mutated pgm gene with that from BB7865 restored the wild type LPS phenotype. To test the invasive ability of these LPS deficient strains, in vitro survival assays were performed with the J774.A1 mouse macrophage-like cell line. The results of these assays demonstrated that the mutants were significantly reduced in their ability to invade (2 h, 40% reduction) and survive (24 h, 60% reduction) post-infection. Many other phenotypes were tested and shown to be significantly altered by this mutation. One such phenotype is motility. Only low levels of motility were achieved by either the bvg-positive or the bvg-negative mutant, indicating a possible role for LPS as an important "anchor" for the flagella filament. The inability to produce wild type LPS also resulted in a reduced bacterial resistance to oxidative stress and a higher susceptibility to the antimicrobial peptide cecropin P. In contrast, the activities of two enzymes thought to play important intracellular survival roles, ie. superoxide dismutase and acid phosphatase, were unaffected in the PGM deficient strain, thereby implicating LPS as playing a protective role during intracellular survival. Complementation of the mutated pgm gene with pgm from BB7865 completely restored the wild type condition as to resistance to oxidative stress and cecropin P, and invasion and survival within J774.A1 cells in vitro. To investigate the role of LPS in pathogenesis, BB7865pgm was directly compared to the wild type strain BB7865, in its ability to cause respiratory infection. BB7865pgm was shown to be significantly attenuated in its ability to survive in the murine respiratory tract following intranasal inoculation, being effectively cleared from the lungs within 4 days. The wild type strain in contrast was shown to persist for at least 35 days-post infection. These results for first time not only determine a gene for inner core biosynthesis, but also demonstrate the importance of the lipopolysaccharide molecule in the virulence of B. bronchiseptica.

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