Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Harlander, J.
In-host evolution of Burkholderia pseudomallei.
[ Diplomarbeit/Master Thesis (UNI) ] Universität Graz; 2024. pp.57.
- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Wagner-Lichtenegger Sabine
- Altmetrics:
- Abstract:
- Burkholderia pseudomallei is a gram-negative rod-shaped bacterium responsible for the severe disease melioidosis. B. pseudomallei has the capability to infect both non-phagocytic and phagocytic cells and has evolved several mechanisms to evade the host immune system. A previous study identified a B. pseudomallei isolate, which showed altered survival within macrophages compared to its isogenic wildtype after passage through human macrophages. The aim of this study was to further characterize the virulence of this isolate (E8_evolved) and to elucidate the underlying mechanisms. B. pseudomallei E8_evolved exhibited increased intracellular invasion and cytotoxicity compared to the isogenic wildtype. This suggests a potentially increased invasion or improved phagosomal escape, potentially leading to a higher bacterial load early in the infection. To determine whether pyroptosis is responsible for the observed increase in cytotoxicity, western blot analyses were performed. The cleavage product of gasdermin D, an indicator of pyroptosis, was detected at a similar intensity in E8_evolved and the wildtype strain. Next caspase-1 and caspase-4, which activate gasdermin D during canonical or non-canonical inflammasome activation, were investigated. While caspase-1 cleavage was similar in both strains, the caspase-4 cleavage product showed increased intensity in E8_evolved indicating increased activation. As no phosphorylated MLKL was detected, necroptosis can be excluded as a potential cause of cell lysis. Additionally, it was observed that the increased cytotoxicity is not associated with elevated production and secretion of the cytokines TNF-α, IL-1α, and IL-1β. Specifically, the concentration of the cytokine TNF-α is reduced in the mutant strain three hours post-infection. To explore changes at the DNA level, a whole genome sequencing analysis of the mutant strain was conducted. This analysis revealed two mutations in the potential T3SS regulatory protein HpaA, specifically in the amino acids VAL25 and ARG28. However, since this protein remains uncharacterized, further research is required to fully understand its role and validate the potential impact of these mutations. Taken together, the mutant exhibits enhanced invasion and bacterial loads, potentially attributable to mutations in the putative T3SS regulatory protein HpaA.