Functional characterisation of Burkholderia pseudomallei biotin protein ligase: a toolkit for anti-melioidosis drug development

Bond, Thomas E.H., Sorenson, Alanna E., and Schaeffer, Patrick M. (2017) Functional characterisation of Burkholderia pseudomallei biotin protein ligase: a toolkit for anti-melioidosis drug development. Microbiological Research, 199. pp. 40-48.

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Abstract

Burkholderia pseudomallei (Bp) is the causative agent of melioidosis. The bacterium is responsible for 20% of community-acquired sepsis cases and 40% of sepsis-related mortalities in northeast Thailand, and is intrinsically resistant to aminoglycosides, macrolides, rifamycins, cephalosporins, and nonureidopeni-cillins. There is no vaccine and its diagnosis is problematic. Biotin protein ligase (BirA) which is essential for fatty acid synthesis has been proposed as a drug target in bacteria. Very few bacterial BirA have been characterized, and a better understanding of these enzymes is necessary to further assess their value as drug targets. BirA within the Burkholderia genus have not yet been investigated. We present for the first time the cloning, expression, purification and functional characterisation of the putative Bp BirA and orthologous B. thailandensis (Bt) biotin carboxyl carrier protein (BCCP) substrate. A GFP-tagged Bp BirA was produced and applied for the development of a high-throughput (HT) assay based on our differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP) principle as well as an electrophoretic mobility shift assay. Our biochemical data in combination with the new HT DSF-GTP and biotinylation activity assay could facilitate future drug screening efforts against this drug-resistant organism.

Item ID: 50388
Item Type: Article (Research - C1)
ISSN: 1618-0623
Keywords: differential scanning fluorometry (DSF), biotin protein ligase (BirA), green fluorescent protein (GFP), biotin carboxyl carrier protein (BCCP), melioidosis, electrophoretic mobility shift assay (EMSA)
Date Deposited: 20 Sep 2017 08:29
FoR Codes: 06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060107 Enzymes @ 50%
06 BIOLOGICAL SCIENCES > 0605 Microbiology > 060502 Infectious Agents @ 50%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 50%
92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920109 Infectious Diseases @ 50%
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