Investigating the Lytic Transglycosylases of Burkholderia pseudomallei

Peptidoglycan is a mesh like structure that is an integral part of the bacterial cell wall. Comprised of glycan chains interconnected with short peptide chains, it is responsible for protecting the bacteria from hydrostatic pressures as well as providing a scaffold for many cell wall spanning structures. Tight regulation of the synthesis, maintenance and turnover of peptidoglycan is essential to maintaining integrity of bacteria. One family of enzymes involved in the regulated cleavage of peptidoglycan are lytic transglycosylases (Ltgs). These proteins are highly conserved in bacteria and function in the restructuring of peptidoglycan during growth and division but also in the insertion of large macromolecular structures including secretion systems and flagella. One class of Ltgs, called resuscitation-promoting factors (Rpf) have also been implicated in resuscitation of dormant Actinobacteria. Burkholderia pseudomallei is the causative agent of melioidosis, a tropical disease prevalent particularly in South East Asia and Northern Australia which claims the lives of an estimated 89,000 people per year. Based on sequence homology to Ltgs of E. coli, we have identified 5 putative Ltgs (LtgA-E) encoded by genes on chromosome 1 of Burkholderia pseudomallei strain K96243. This study aimed to understand the role of these proteins in B. pseudomallei biology and investigate their capacity as drug targets. Using a range of approaches I have shown the muralytic activity of four of these Ltgs. The X-ray crystal structure of LtgE was solved and the catalytic site identified using site directed mutagenesis. In complementary studies single and multiple deletion mutants of B. pseudomallei have been generated. Excision of single or multiple ltg genes from B. pseudomallei resulted in mutants with severely altered cellular morphology (increased cell length and defects in cell division), motility and reduced biofilm formation. ΔltgEBDC had a small increase in susceptibility to carbenicillin, doxycycline and ceftazidime. ΔltgB, ΔltgD, ΔltgE were all attenuated in the BALB/c mice model of melioidosis. In addition, I began to develop a model for the generation of non culturable Burkholderia. Incubation in NaCl concentrations >2.5% (w/v) for 7 days resulted in a complete loss of culturability on solid media. Live/dead staining however, revealed a substantial population of seemingly viable bacteria. Attempts to resuscitate these into actively growing cells however were unsuccessful. Given the role in virulence, the assays available and optimised for use with B. pseudomallei Ltgs and the X-ray crystal structure of LtgE, it would appear that Ltgs are good candidates for novel drug targets and that the tools are now in place for high throughput screening of Ltg inhibitors.