Characterisation of Burkholderia pseudomallei type III secretion system III components.
thesisposted on 23.02.2017 by Treerat, Puthayalai
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In many intracellular pathogens, the type III secretion system (TTSS) plays an important role in virulence by secreting effector molecules directly across the host cell membrane. These effectors subsequently interact with, and alter, host signalling pathways for the benefit of the pathogen. Burkholderia pseudomallei, an intracellular pathogen that is the causative agent of the potentially fatal disease melioidosis, utilises a TTSS for its survival and replication in both phagocytic and non-phagocytic cells. Although this pathogen contains three TTSS gene clusters, the Type III Secretion System 3 (TTSS3) is critical for bacterial infectivity and pathogenesis. , However, to date, only BopE, BopA and BopC are characterised effectors of the TTSS-3. This research aimed to identify and characterise the putative TTSS3 proteins BapA, BapB and BapC with regard to their possible functions as bacterial effectors involved in either modulation of host cell functions for bacterial survival, replication or escape from host endosomal vacuoles, or the secretion of the other TTSS3 effectors. By using a double cross-over allelic exchange approach, bapA, bapB, bapC and double bapBC mutant strains were generated and assayed for their in vivo and in vitro phenotypes. Competitive growth assays in BALB/c mice showed reduced growth of each of the mutants compared to the wild-type. Furthermore, all showed reduced virulence in the acute mouse infection model, indicating possible roles in bacterial virulence. Complementation was attempted but was unsuccessful. Therefore, independent mutants were constructed. The independent mutants were all tested for virulence in the BALB/c acute model but only the bapA_2 mutant showed reduced virulence compared to the wild-type strain. These data suggest that BapA likely plays a minor role in virulence, although successful complementation is required to conclusively prove this. To determine whether BapA, BapB and BapC were secreted effectors, the TC-FlAsHTM labelling technique was used to monitor the secretion of tetracysteine-tagged fusion proteins. It was demonstrated that BapA and BapC are secreted in vitro. These proteins were secreted in a TTSS3-dependant manner as they were not secreted by mutant B. pseudomallei expressing a non-functional TTSS3. To further investigate any potential involvement of BapA, BapB and BapC in the TTSS3 secretion process, the well-characterised TTSS3 effector BopE was used as a marker to examine TTSS secretion in each of the mutant strains compared to the wild-type and the bopE mutant. The level of transcription of bopE was also assessed in certain strains in order to determine if there was any difference in the transcriptional regulation of this gene. It was demonstrated that, although BapA, BapB and BapC are not required for TTSS function, BapB appears to be necessary for normal secretion of BopE. Therefore, this study defines BapA and BapC as B. pseudomallei TTSS-3 effectors, and BapB as a possible regulator of BopE secretion that may play a role in the pathogenicity of B. pseudomallei.