Identifying virulence factors and regulators contributing to pathogenesis by the select-agent bacterium burkholderia pseudomallei
Identifying virulence factors and regulators contributing to pathogenesis by the select-agent bacterium burkholderia pseudomallei
Date
2014-08
Authors
Norris, Michael Holt
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[Honolulu] : [University of Hawaii at Manoa], [August 2014]
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Abstract
Many virulence factors contribute to the molecular pathogenesis of Bp, such as type III and VI secretion systems, flagella, toxins, and capsule. A large subset of Bp proteins, particularly those annotated as hypothetical and putative (~35.5% of the genome), are avoided in experiments. Some of these proteins probably contribute to pathogenesis and its' regulation. Whether for a lack of tools or techniques, many questions about the molecular pathogenesis of Bp remain unanswered.
Engineered K96243, 1026b, and MSHR487 Δasd mutants could not grow without the essential cell-wall component diaminopimelic acid (DAP) and were unable to replicate in mice. All strains were excluded from select-agent regulations, providing the research community with helpful BSL-2 surrogates. Intracellular replication and timelapse movies of fluorescent two-color cell-fusion assays demonstrated that B0011 behaved similarly to WT. B0011's utility in virulence factor investigations was demonstrated by testing in the nematode Caenorhabditis elegans. B0011 was also found to kill Tenebrio molitor (yellow mealworm) in a novel feeding assay indicating the biosafe B0011 would be a good surrogate for research of virulence factors. The CDC and Los Alamos have obtained the strains for diagnostics and JCVI for persister cell research.
To identify essential virulence factors, a transposon mutant library was constructed in Bp K96243 Δasd and passaged through macrophages 3 times. gDNA from the passaged pool and the un-passaged pool was isolated and the transposon/genome junctions were enriched using TN-seq. Bacterial genes that were not present in the samples after passaging through macrophages were considered putative virulence factors. 113 genes underwent greater than 1,000-fold negative selection. Of those, many were deficient in some part of the infectious process. 20 were used to infect mice in an intranasal melioidosis model. 10 mutants demonstrated attenuation and represent new virulence factors.
40 hypothetical regulatory genes were deleted and mutants tested for a decreased plaque forming ability in cell monolayers. Nine regulatory mutants were unable to form plaques, indicating a role in virulence regulation and were complemented with ChIP-seq vectors. Nine regulators were characterized by growth and infection in vitro. Of nine, six regulators were used to infect mice and showed attenuation in vivo.
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Ph.D. University of Hawaii at Manoa 2014.
Includes bibliographical references.
Includes bibliographical references.
Keywords
Burkholderia pseudomallei,
pathogenesis,
melioidosis
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Theses for the degree of Doctor of Philosophy (University of Hawaii at Manoa). Molecular Biosciences and Bioengineering.
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