2 research outputs found
Studies on the intracellular life of the melioidosis pathogen Burkholderia pseudomallei
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