Studies on the intracellular life of the melioidosis pathogen Burkholderia pseudomallei
Zainal Abidin, Nurhamimah
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Melioidosis, caused by the environmental Gram negative bacillus Burkholderia pseudomallei, is an emerging infectious disease affecting both animals and humans. B. pseudomallei has the ability to enter the host cell and escape from the phagosome. Once in the cytoplasm, the pathogen proliferates and expresses a virulence-associated protein known as BimA which polymerises cellular actin at the pole of the bacterium to promote its movement inter- and intracellularly, a process known as actin-based motility. This actin-based motility is also used as a strategy to evade host immune responses and survive intracellularly. In the first part of the thesis, we demonstrate that a B. pseudomallei ΔbimA mutant displays impaired intracellular survival compared to the isogenic parent strain in BALB/C bone-marrow derived macrophages (BMDMs), notably at later time points post-infection. Macrophages are the key innate immune cells that control B. pseudomallei in vivo and in vitro, and BALB/C mice provide an excellent model of acute human melioidosis. We also have determined that in BMDMs, the ΔbimA mutant is able to escape from the phagosome and enters the cytosol where it is unable to form actin tails. We used targeted, hypothesis-driven experiments to identify potential cell-autonomous innate mechanism/s of killing the mutant. First, we speculated that BimA mediates escape from autophagy. However our studies, including LC3-conversion assays, and bacterial co-localisation studies, failed to demonstrate a role for autophagy in clearance of the ΔbimA mutant from infected BMDMs. In the second part of this thesis, we investigated the role of Toll-like Receptors (TLR) in recognition and elimination of B. pseudomallei. MyD88 (Myeloid differentiation primary-response gene 88) and TRIF (TIR-domain-containing adaptor protein inducing IFNβ) are the main adaptor proteins involved in TLR signalling. We utilised the gene silencing technique using short interfering RNAs (siRNAs) to knockdown MyD88 transcript, and in a separate experiment used MyD88- or TRIF-blocking peptides. In addition, we investigated the involvement of canonical and non-canonical inflammasome pathways in cell-autonomous immunity of the BMDMs. However, none of these pathways were shown to be involved in clearance of the ΔbimA mutant from infected BMDMs. Finally we took an unbiased approach by microarray to characterise the global host transcriptome in BALB/C BMDMs upon B. pseudomallei infection, and to identify specific responses to the ΔbimA mutant. Analyses performed at the gene level revealed that several interferon signalling-related pathways are activated in cells infected with either the WT or ΔbimA mutant strains. A number of other pro-inflammatory mediators that are commonly seen in general inflammatory infections, such as IL-1α, IL-1β, IL-12β, and IL-6, were also upregulated. Interestingly, the cytoplasmic RNA sensors RIG-1 and MDA-5, thought primarily to be involved in the detection of RNA viruses, were also induced upon B. pseudomallei infection. Very few pathways were associated with a specific macrophage response to the ΔbimA mutant, indicating that an as yet undescribed pathway may play a role in sensing and eliminating the ΔbimA mutant. We conclude that actin-based motility mediates escape of B. pseudomallei from macrophage intracellular killing through a novel pathway which has yet to be unravelled.