Antibiotic resistance protein : functional characterization and drug development

Abstract

Resistance to every major class of antibiotic has been reported. Mobile antimicrobial determinants are known to be disseminated efficiently even among different bacterial species, leading to emergence of multidrug-resistant strains. The growing proportion of such superbugs recorded among clinical strains has seriously compromised the efficacy of antimicrobial therapy and limited therapeutic options. To tackle this issue and develop new generation antimicrobial compounds, we aimed to delineate the molecular mechanism of action of specific resistance-encoding determinants as well as that of an effective yet currently poorly characterized antibiotic, daptomycin. The first part of the study involves characterization of the structure and function of representative antimicrobial determinants, namely the Qnr proteins, which are responsible for quinolone resistance, and the CTX-M β-lactamases which confer cephalosporins resistance. The second part of this work involves detailed investigation of the mode of action of daptomycin and development and characterization of daptomycin analogues. Firstly, we have identified a novel qnr variant, named as qnrVC7, from a Vibrio food isolate. Our data confirm its role in expression of quinolone resistance by cloning this gene into E.coli system and subsequently testing the difference between the degree of quinolone susceptibilities of the parental and transformant strains. MIC data showed that QnrVC7 exhibited lower protection activity than other Qnr proteins. The reduced protective activity was found to be associated with amino acid T152 located at the i-2 position on face 4 of the tandem repeat loops by sequence comparison and mutational analysis. Mutagenesis was then performed for all residues located at the i(-2) position of face 4 to further characterize their functional importance. The i(-2) position of face 4 on different coils comprised amino acid residues of different nature. Our data showed that substitutions by other amino acids at specific sites resulted in reduction in protective activity of QnrVC7, whereas specific substitutions led to increased protective activity. These findings improved our understanding on the detailed structural organization and functional requirements of Qnr proteins. Next, DNA shuffling of fragments of the blaCTX-M-14 and blaCTX-M-15 genes was performed to investigate the molecular events driving the evolution of the CTX-M-type β-lactamases. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet hybrids that were active possessed fewer amino acid substitutions than those that were inactive, suggesting that point mutations in the constructs rather than reshuffling of the fragments of the two target genes would more likely cause disruption of CTX-M activity. Mutagenesis study identified certain point mutations that significantly suppress CTX-M activity. Further analysis of the structure-function relationship of a range of mutant enzymes showed that a large number of mutations could render the enzyme unstable and inactive, suggesting that proper functioning of the enzyme has a very stringent structural requirement. Our findings also suggest that the distal pockets could also contribute to the activity of the enzymes and may be regarded as alternative targets for inhibitor development. In the second part of the study, we attempted to identify and characterize the potential target of the antibiotic daptomycin. Although daptomycin was thought to mainly interact with membrane lipid, characterization of daptomycin analogs by MIC assay, fluorescent binding assay and ITC assay showed that daptomycin may have a protein target. A universal stress protein, Usp2, was then identified by pull down assay. The interaction between Usp2 and daptomycin was confirmed by ITC assay and fluorescence labelling assay. Antibody blocking assay showed that the anti-Usp2 antibody would cause reduced activity of daptomycin; consistently, over-expression of Usp2 was found to confer protection against daptomycin activity and improve the survival rate of the test organisms upon daptomycin treatment. Functional tests performed on Usp2 provide further evidence which confirms its role as a target of daptomycin. Finally, a daptomycin analogue, termed 'kynomycin' was identified and found to exhibit significantly enhanced antibacterial activity to MRSA and VRE when compared to daptomycin itself. The superior antimicrobial efficacy of kynomycin was demonstrated by in vitro time killing assay, SYTOX green staining assay, in vivo wax worm model and different mouse infection models. The enhanced potency of kynomycin was found to be due to its high affinity towards cell membrane and its ability to cause increased membrane permeability and damage. Our data showed that kynomycin exhibited lower cytoxicity than daptomycin. It can be a promising candidate of next generation of daptomycin that acts against daptomycin resistant pathogens.