Date on Master's Thesis/Doctoral Dissertation

5-2016

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Warawa, Jonathan

Committee Co-Chair (if applicable)

Abu-Kwaik, Yousef

Committee Member

Abu-Kwaik, Yousef

Committee Member

Demuth, Donald

Committee Member

Suttles, Jill

Committee Member

Uriarte, Silva

Author's Keywords

Burkholderia pseudomallei; melioidosis; T3SS; IMIT

Abstract

Melioidosis is a fatal infectious disease caused by the Tier 1 Select Agent Burkholderia pseudomallei. Hallmarks of melioidosis include pneumonic disease and prominent septicaemic spread. Both forms of disease are contingent upon the bacterium’s intracellular life cycle and particularly on its ability to escape from host cell phagosomes. Upon encountering a host cell, B. pseudomallei is internalized into membrane-bound vacuoles from which the bacterium must rapidly escape to the cytoplasm in order to replicate and promote its survival. In the host cytoplasm, B. pseudomallei is capable of polymerizing actin for intracellular and intercellular motility and spread, lysing the host cell and perpetuating the cycle of infection. Commonly used intranasal and aerosol models to study respiratory melioidosis result in significant upper respiratory tract colonization, dramatically altering disease progression. Accordingly, we developed an improved lung-specific instillation approach to deliver bacteria directly into mice lungs, coupled with in vivo optical imaging and observed the development of disease that closely resembles human melioidosis in mice. We found that in the absence of upper respiratory tract infection, a capsular polysaccharide (CPS) mutant is only 6.8-fold attenuated. This mutant is unable to spread to secondary sites of infection, consistent with the role of capsule in protecting the bacterium from host antimicrobial activity. Similarly, a type 3 secretion system cluster 3 (T3SS3) structural mutant is spread deficient, yet this mutant is attenuated 290-fold, strongly suggesting that T3SS3 is critical for respiratory melioidosis. Having a strong platform for studying the pathogenesis of B. pseudomallei in a mouse model of lung-specific melioidosis, we used transposon mutagenesis to comprehensively identify virulence factors required for B. pseudomallei lung colonization and spread to the liver and spleen. Notably, T3SS3, capsular polysaccharide and type 6 secretion system cluster 5 (T6SS5) were the major genetic loci required for respiratory melioidosis. A T6SS5 mutant is not attenuated by LD50 estimations using our lung-specific melioidosis mouse model. Yet by competition analysis T6SS5, T3SS3 and CPS mutants were attenuated, substantiating the requirement of these factors for B. pseudomallei infection as previously reported. These results highlight the importance of competition analysis for studying the fitness of distinct virulence determinants. Importantly, T3SS3 was the only virulence determinant attenuated by both LD50 analysis and competition studies, corroborating the critical requirement of this virulence system for respiratory melioidosis. B. pseudomallei is a facultative intracellular cytosolic bacterium and its ability to survive intracellularly is fundamental to mammalian host infection. Upon B. pseudomallei internalization into host cell vacuoles, the bacterium rapidly escapes this compartment by action of the T3SS3. The T3SS3 is required by B. pseudomallei for intracellular survival by translocating protein effectors to the cytoplasm of host cells and mediating the rapid escape of the bacterium from phagosomes of these cells. We hypothesized that effectors act in concert to mediate B. pseudomallei’s rapid escape from phagosomes of host cells by manipulating host signaling pathways and promoting bacterial survival. Using a high-resolution, high-throughput in vivo imaging screening approach we profiled the contributions of five of the six B. pseudomallei putative type 3 effectors to the bacterium’s rapid phagosome escape. Effector mutants exhibited distinct temporal differences in escape, with bopA inactivation resulting in the most pronounced delay in vacuolar rupture, strongly suggesting that BopA directly mediates escape of B. pseudomallei from endocytic vesicles. We confirmed that a previously identified BopA host target, the trafficking particle protein C8 (TRAPPC8), colocalized with Burkholderia containing vacuoles. Small interfering RNA knockdown of this protein strongly suggests that TRAPPC8 is required for vacuolar membrane stabilization. These findings substantiate the significant role of the T3SS3 and provide a paradigm to study B. pseudomallei natural infection processes and potential vaccine and therapeutic targets.

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