Role of BapA type III effector in the intracellular lifecycle of Burkholderia pseudomallei / Choh Leang Chung.

Burkholderia pseudomallei is a Gram-negative bacterium that causes the fatal disease, melioidosis. Treatment of melioidosis is difficult as the bacterium is intrinsically resistant to multiple antibiotics and there is a risk of recurrence despite prolonged and adequate antimicrobial therapy. To facilitate the development of effective prevention and eradication strategies, there is a need to gain further molecular insights into the molecular pathogenesis and intracellular lifecycle of this pathogen. B. pseudomallei utilises the type III secretion systems (T3SSs) to translocate effector proteins (T3SEs) directly into host cell cytoplasm to establish an intracellular infection to subvert the host immune systems. Herein, the putative T3SE protein, BapA, was characterised using bioinformatic analyses, in vitro and in vivo infection models and basic protein analyses. Bioinformatic analyses predicted the presence of a T3SE translocation signal in the first 100 amino acid sequence at the N-terminal of BapA amino acid sequence, indicating with a high probability that BapA is a T3SE of B. pseudomallei. Based on secondary structure prediction, de novo protein structure prediction models and intrinsically unstructured region prediction, the amino acid sequence after 320th amino acid at the C-terminal of BapA was predicted to be intrinsically unfolded. This may confer mechanistic roles in BapA function(s). The functional roles of BapA in the pathogenesis of B. pseudomallei were experimentally dissected by utilising a bapA knockout strain (ΔbapA) derived from the parental strain, B. pseudomallei K96243. In the in vitro infection assays using human cell lines, ΔbapA was significantly attenuated in adherence and invasion efficiencies onto and into A549 human lung epithelial cells, respectively. Phagocytosis of ΔbapA by U937 human macrophages was significantly reduced. A substantial decrease in the intracellular replication rate of ΔbapA was also observed in the U937 cells but not in A549 cells. In addition, the capacity of ΔbapA for cell-to-cell spread was significantly reduced without any defects in its actin-based intracellular motility. Collectively, it is rational to speculate that BapA may have essential role(s) in host cell entry into the host cell cytoplasm from the extracellular environment or from an infected neighbouring cell via actin-rich protrusion. These entry steps are the key steps required for B. pseudomallei to initiate its intracellular lifecycle and infection. The E. coli-expressed, purified recombinant BapA protein was analysed using SDS-PAGE. The intrinsically unstructured nature of BapA was demonstrated by the aberrant mobility of recombinant BapA on the SDS-PAGE. Presence of these dynamic regions in BapA may have a role in forming macromolecular assemblies by confering great flexibility and capacity in protein-protein or protein-ligand interactions. Thus, the intrinsically unstructured regions were hypothesised to confer the capability for BapA to exert its function in the different stages of B. pseudomallei intracellular lifecycle, as demonstrated in the current study. In conclusion, intrinsically unstructured BapA performs important roles in the intracellular entry of B. pseudomallei. Since cellular entry is an essential phase in the intracellular lifecycle, BapA T3SE is important in the pathogenesis of B. pseudomallei