The identification and characterization of microbial FK506-binding proteins

Leigh-Cooper, Jesse (2017) The identification and characterization of microbial FK506-binding proteins. Honours thesis, Murdoch University.

Abstract

FK506-binding proteins (FKBPs) represent a diverse family of enzymes which typically exhibit peptidyl-prolyl cis-trans isomerase (PPIase) activity, catalysing a rate limiting step of protein folding. Prior studies have identified FKBP members BPSL0918 and the macrophage infectivity potentiator (MIP) as virulence determinants of the tropical pathogen Burkholderia pseudomallei. Due to the strong association with virulence, a range of small molecule inhibitors have been developed to interact with the PPIase domain of B. pseudomallei MIP. Targeting the shared PPIase domain of FKBPs may allow for the inhibition of multiple FKBPs, potentially enhancing drug efficacy. However, the biological roles of the remaining B. pseudomallei FKBPs remains unknown.

This study aimed to identify and experimentally validate FKBPs as potential anti-virulence targets for the purpose of investigating the potential for multi-target inhibition using MIP inhibitors from previous studies. Through phylogenetic analysis 58 putative FKBPs were identified within the 16 bacterial taxa studied. BPSL2254, a putative SlyD-like FKBP closely related to the virulence associated BPSL0918, was recombinantly expressed and purified from E. coli. To investigate PPIase activity for the subsequent assessment of inhibitors, purified BPSL2254 was used in protease-coupled assays in which no PPIase activity was detected.

The closely-related environmental saprophyte B. thailandensis encodes BTH_I1911 gene product, which was determined to share 98% amino acid sequence identity with BPSL2254. B. thailandensis was used as a surrogate model featuring an in-frame deletion of the BTH_I1911 gene. The contribution of BTH_I1911 to virulence was assessed through a range of in vitro assays to evaluate potential as an anti-virulence target. The role of BTH_I1911 in the capacity of B. thailandensis to invade J774A.1 macrophages and form biofilm remains inconclusive. However, the BTH_I1911 null strain appeared to exhibit filamentation after incubation in M9 minimal media. Further study is required to conclusively determine the enzymatic activities of BPSL2254, which in turn may facilitate assessment of inhibitors. Investigation of putative FKBPs identified here may lead to the identification of novel virulence determinants. Further study including the use of in vitro assays and the infection of animal models are required to elucidate the role of BTH_I1911 as a virulence determinant.

Item Type:	Thesis (Honours)
Murdoch Affiliation(s):	School of Veterinary and Life Sciences
Supervisor(s):	Sarkar-Tyson, Mitali, Coombs, Geoffrey and Kahler, Charlene
URI:	http://researchrepository.murdoch.edu.au/id/eprint/40176

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