Identification of B. pseudomallei &B. mallei adhesins

University Of Toledo Health Sci Campus, Toledo OH

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Abstract

DESCRIPTION (provided by applicant): Burkholderia pseudomallei and Burkholderia mallei cause serious and often fatal infections that generally occur through the aerosol route and which manifest as severe pneumonia and highly fatal bacteremia. The diseases are difficult to diagnose and necessitate a prolonged antibiotic therapy that has a low success rate. There is no vaccine for the organisms and there is concern regarding their use as bioweapons due to their lethality and aerosol infectivity. These Burkholderia species are classified as category B agents of bioterrorism, and the development of preventative as well as novel curative treatments is a priority. One poorly understood aspect of pathogenesis by these bacteria that needs to be explored is how they bind to human mucosal surfaces. Our long-term goals are to study B. pseudomallei and B. mallei adherence to human cells and evaluate the potential of interfering with this adherence as a strategy to reduce the risks of infection. This is a logical approach to preventing disease because adherence is necessary for colonization of the host and precedes invasion by intracellular pathogens such as B. pseudomallei and B. mallei. Thus, we believe that Burholderia adhesins are potential vaccine candidates. In our first aim, we propose to identify the genes encoding adhesins by creating plasmid-based libraries from the DNA of B. pseudomallei and B. mallei and introducing them into a nonadherent Escherichia coil strain. We will enrich for E. coli recombinant clones expressing putative Burkholderia adhesins for human lung cells via consecutive adherence assays and recovery. Adherent clones will contain plasmids encoding putative Burkholderia adhesins and their structural genes will be studied. In our second aim, we propose to identify Burkholderia surface proteins that are adhesins by selectively labeling intact bacteria with a membrane-impermeable biotinylating agent. The labeled proteins will be purified with streptavidin-coated beads, resolved by SDS-PAGE, and analyzed by peptide mass fingerprinting. To conclusively determine whether Burkholderia proteins are candidate adhesins, we will clone their structural genes into a nonadherent E. coli strain and measure the adherence of recombinant clones. We will also disrupt the candidate adhesins structural genes and test whether these mutations affect B. pseudomallei and B. mallei adherence.