1. UKM Learning and Research Repository
2. PHD Thesis / Tesis PHD
3. Faculty of Science and Technology / Fakulti Sains dan Teknologi

Title:	Characterisation of transpeptidase and transglycosylase domains of penicillin-binding protein 1c from burkholderia pseudomallei and their response to antibacterial agents
Authors:	Parastoo Tajzadeh (P38425)
Supervisor:	Rahmah Mohamed, Prof. Datuk
Keywords:	Transpeptidase Transglycosylase Burkholderia pseudomallei Antibacterial agents Dissertations, Academic -- Malaysia
Issue Date:	3-Sep-2016
Description:	Burkholderia pseudomallei causes melioidosis, a significant public health threat in endemic regions. The organism is found to persist in some tropical regions such as east Malaysia and north-eastern Thailand. Treatment of melioidosis is intensive, prolonged and the bacteria are gradually progressing to be resistant to drugs of choice. Currently, studying potential drug targets among Burkholderia proteins seems to be essential. Acquiring resistance to β-lactam antibiotics in Burkholderia spp. is mediated by β-lactamase production and modified penicillin-binding proteins (PBPs). These proteins are responsible for biosynthesis, elongation and division of bacterial cell wall that will decrease the affinity to β-lactam antibiotics. Thus, this study was initiated to characterise one of the high molecular weight penicillin-binding proteins (PBP) in Burkholderia pseudomallei using in sillico and physicochemical methods. In silico studies revealed similarities between primary and secondary structures of Pbp1C and PBPs in other bacteria. A recombinant protein technology was assumed to identify Pbp1C, a possible novel antimicrobial target in B. pseudomallei. Transglycosylase (TG) and transpeptidase (TP) domains of Pbp1C, involved in peptidoglycan polymerisation, are considered potential antimicrobial drug targets in other pathogenic bacteria. Subsequently, these potential domains were studied for drug targeting and improving the treatment of melioidosis. These catalytic domains were cloned from clinically isolated Malaysian strain R15. The biophysical characterisation of recombinant TG protein showed stability at pH 8.0 and 66°C, using differential scanning calorimetry (DSC) assay. To assess in vitro affinity of TG to moenomycin A, isothermal titration calorimetric assay (ITC) showed close correlation with a number of pathogenic Gram-positive and Gram- negative bacteria species with Ka value 28×106 mM. Based on the observed DSC and ITC assays, TG domain represents an excellent target for specific inhibitors against B. pseudomallei. TP domain showed high stability at acidic pH of up to 76°C for one hour, using DSC method. This recombinant protein showed antibiotic hydrolysis activity as 73×10-3 μM of nitrocefin within one minute to produce an open-ring form at 30°C in phosphate buffer (pH 7.0). The observed enzyme activity data indicated that this domain could function as a sole β-lactamase in B. pseudomallei which may be involved in resistance mechanism. To determine the chemical affinity of TP domain, Nitrocefin Competition Assay (NCA) was performed. Based on the observed NCA assay, penicillin G and ampicillin could bind more effectively (60%) than ceftazidime to TP active site. This study provides the first look at the molecular details of Pbp1C from B. pseudomallei effective β-lactam-resistant PBP interaction, leading to new insights into the mechanism of β-lactam efficacy against B. pseudomallei.,Certification of Master's/Doctoral Thesis" is not available
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.