Burkholderia pseudomallei (Bp) is the causative agent of the potentially fatal disease melioidosis. Bp is a facultative intracellular pathogen that employs a variety of virulence factors, including members of the FK506-binding proteins (FKBPs). FKBPs catalyse the cis-trans isomerisation of proline bonds, a rate-limiting step in protein folding. One member of the FKBP is the Sensitive to Lysis D (SlyD) protein, which has been associated with the virulence of several pathogenic bacteria. In Salmonella enterica serovar Typhimurium, disruption of slyD resulted in a 50% reduction in chick intestine colonisation of the mutant strain compared to the wild type. While in Helicobacter pylori, SlyD is required for nickel incorporation by urease, which is essential for acid resistance and survival in the gastric environment.
This study characterises the role of slyD in the virulence and intracellular lifecycle of Bp by generating a slyD deletion mutant in Bp strain K96243. Infection of A549 epithelial cells and THP-1 differentiated macrophage revealed that the Bps∆slyD mutant has severe adhesion and internalisation defects, which are steps that are essential for the establishment of infection. Additionally, Bps∆slyD showed an increased sensitivity to host cell killing, as seen by the increased susceptibility of the slyD mutant to both 1 mM hydrogen peroxide and pH4 acid stress. The reduction in the bacterial entry to cells and increased sensitivity to host cell killing ultimately resulted in the reduction in Bp-induced cell cytotoxicity at 24 hours post-infection, as determined by the levels of lactate dehydrogenase release.
To further uncover the mechanism of SlyD and how the loss of this protein affected Bp, global proteomics was conducted on the Bps∆slyD mutant. Proteomic analysis revealed a significant reduction in the abundance of Sap1, a surface attachment protein essential in the adhesion of Bp to host cells, and BipD, a translocator tip protein required for vacuolar escape, in the proteome of Bps∆slyD compared to BpsWT explaining the intracellular phenotypes seen. In conclusion, this study uncovers that SlyD is a novel virulence factor of Bp, essential for bacterial adhesion and internalisation and is involved in different stages of the intracellular lifecycle of Bp.