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Exploring the role of mutations in the signal peptides of VIM-2, NDM-1, and IMP-1 in the development of advantageous phenotypes in Escherichia coli

University of British Columbia

Metallo-β-lactamases (MBLs) are powerful enzymes conferring antibiotic resistance to various pathogens. They are actively disseminated in a plethora of pathogenic organisms via horizontal gene transfer, and raise clinical concerns. Interestingly, a predominant number of pathogens that favor MBL expression belong to γ-proteobacteria such as Pseudomonas aeruginosa, implying biases in the host distribution. A previous study in the Tokuriki lab also revealed restricted MBL gene expression in phylogenetically distanced bacterial species. However, it is still obscure how the enzymes adapt to bacterial hosts. This thesis aims to understand mutations in the signal peptides of MBLs contributing to the adaptive phenotype development in Escherichia coli. A series of dose-response curve assays revealed a highly positive correlation between the sequenced individual variants and their respective population where a considerably narrow phenotypic diversity is observed. When the phenotypic diversity is in a limited range at a high ampicillin concentration, the variability in mutational effects associated with phenotypic variations may be confined by fitness costs, resulting in the development of similar phenotypes. This suggests that the sequence profiles of the variants are a reasonable representation of the mutational population. A bioinformatic analysis of the sequence profiles of NDM-1, VIM-2, and IMP-1 variants reveals that mutation-driven changes in translational steps are moderately correlated with minimum inhibitory concentration (MIC), implying that the mutations in the signal peptides are associated with adaptive phenotypes elevating MBL gene expression. In addition, I synthesized a new broad host range vector using three individual plasmid vectors by the Golden Gate assembly technique to analyze mutational effects of MBLs in Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida. The newly synthesized plasmid vector is compatible with the listed bacterial species enabling future research. Overall, these results in the thesis will help understand how the mutations in the signal peptide of MBLs, in part, promote adaptive phenotypes in E. coli to elevate MBL gene expression, and may help develop future therapeutic strategies.

Text

2021-04-27

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/77995

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/