Resuscitation of non-culturable forms of Burkholderia pseudomallei

Microorganisms have an ability to survive hostile conditions for prolonged periods of time. In particular, they sometimes produce dormant cells, which are non-detectable by the standard methods or, in other words, cannot be cultured in conventional media. However, these specialised forms remain virulent and can convert to normal growing cells when incubated under appropriate conditions. Importantly, dormant cells are more resistant to antimicrobial treatment and various stresses and therefore have higher chances of survival in the infected host and transmission to a new host. Resuscitation-promoting factors (Rpf), a family of secreted muralytic enzymes, play an essential role in reactivation of such dormant cells. Many causative agents of life-threatening infectious diseases, including Mycobacterium tuberculosis and Corynebacterium diphtheriae produce Rpfs or related proteins.
Burkholderia pseudomallei, a Gram-negative non-spore-forming bacterium, is the causative agent of melioidosis. Despite the great phylogenetic distance between B. pseudomallei and M. tuberculosis, they display a remarkable similarity in adaptation to the host environment and can both produce non-replicating (non-culturable) forms, which are believed to be associated with latent infections. The resuscitation mechanism of B. pseudomallei is unknown, but we hypothesise that it could be similar to that present in mycobacteria and may involve remodelling of peptidoglycan by lytic transglycosylases (LTG).

The main goals of this project are (1) to establish a reliable protocol for generation of non-culturable B. pseudomallei bacilli; and (2) to investigate their resuscitation by Rpf and LTG.
Experimental approaches will include molecular cloning, expression and purification of recombinant LTGs; characterisation of their enzymatic activities; generation of B. pseudomallei mutants and investigation of their phenotypes in vitro (resuscitation) and in vivo (macrophage infection); peptidoglycan analysis.


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