| Author: | Wong, Tsz Fung |
| Title: | Study of resistance mechanism of TEM-type extended spectrum β-lactamases (ESBLs) by mass spectrometry |
| Advisors: | Yao, Zhongping (ABCT) |
| Degree: | Ph.D. |
| Year: | 2022 |
| Subject: | Drug resistance Drug resistance in microorganisms Antibiotics Mass spectrometry Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | xxii, 192 pages : color illustrations |
| Language: | English |
| Abstract: | Antibiotics resistance has long been a big worldwide health concern, and expression of β-lactamases is the most common strategy for bacteria to elude the attack of antibiotics. Since the discovery of the first TEM-type β-lactamase, its various variants have been found. G238S, E104K and M182T, which are three point mutations commonly found in variants, either with single or in combination with other point mutation(s), are responsible for conferring the extra hydrolytic power towards new generation antibiotics, e.g., cefotaxime. Substantial efforts have been made in the past few decades to study the properties and structures of these extended spectrum β-lactamases (ESBLs). X-ray crystallography has been extensively utilized to investigate ESBLs and it can provide valuable information of static structures for β-lactamases, particularly their intra-molecular interactions. However, there is still a lack of comprehensive study for the conformational dynamics of TEM-type ESBLs. Therefore, in chapters 1 - 3 of this thesis, individual and interactive effects of the three point mutations on the conformational dynamics of TEM-type β-lactamases were investigated with the help of hydrogen/deuterium exchange-mass spectrometry (HDX-MS). Six TEM-type β-lactamases, TEM-1, TEM-19 (G238S), TEM-17 (E104K), TEM-15 (G238S/E104K), TEM-20 (G238S/M182T) and TEM-52 (G238S/E104K/M182T), were included in this study. Conformational changes among the apo-TEM-type β-lactamases, cefotaxime-bound TEM-type β-lactamases and those due to cefotaxime binding were comprehensively compared. The comparison between apo-enzymes reflected that G238S caused more significant enhancement in flexibility than E104K. Despite the catalytic efficiency enhancement towards cefotaxime due to E104K, the conformation did not undergo any significant changes compared with the wild type TEM-1. However, the synergistic effect of G238S and E104K caused an acute enhancement in flexibility. Apart from key mutations like G238S and E104K, the reported global suppressor M182T, a secondary point mutation, optimized the flexibility of mutants in order to compensate for the severe conformational changes caused by key mutations. Upon the cefotaxime binding, key regions such as the region near S70,Ω-loop, SDN loop and β-3 strand underwent significant conformational changes and became rigid, which could be attributed to the direct interaction of these regions to cefotaxime. Two allosteric regions were first discovered and discussed in this thesis. H5-H6 region, which was far away from the active site and not directly involved in the cefotaxime hydrolysis, was found to relate to the M182T stabilization during binding. The flexibility enhancement of Ω-loop was compensated by a more compact H5-H6 in M182T-containing mutants and hence thermostabilities of mutants were increased. Therefore, this region could be a potential allosteric region as a target for novel antibiotics and inhibitors. Moreover, one edge of the β-sheet, which was also not directly involved in the antibiotics binding, was found to become more flexible particularly upon cefotaxime binding to G238S-containing mutants. However, the edge had insignificant flexibility changes or became slightly compact than TEM-1 after mutants incorporating M182T. This could be a result of compensating for destabilization effect of the flexibility enhancement for the active site related β-3 strand to accommodate the bulky side chain of cefotaxime and promote the effective interaction to the cefotaxime in the binding site. These findings on conformational dynamics changes of TEM-type ESBLs upon single or combined point mutations, particularly in the two allosteric regions, provided new insights into the resistance mechanism of ESBLs and design of new inhibitors targeting non-active site regions. The occurrence of coronavirus disease in 2019 (COVID-19) has caused the unprecedented global health threat. Rapid detection of infected cases is an important step to combat the disease. Currently, nucleic acid detection approach is regarded as the golden method for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19, with serological detection as the alternative approach. However, these approaches suffer from certain drawbacks including time-consuming and labor-intensive steps, false positive or negative results, etc. In chapter 4, a targeted proteomics-based approach has been attempted for detection of SARS-CoV-2 based on the detection of specific peptides from the SARS-CoV-2 nucleocapsid (N) protein. Four specific peptides were obtained with a 3-minute tryptic digestion of the N protein, and successfully eluted within 3 minutes by liquid chromatography and detected by mass spectrometry using multiple reaction monitoring, with a limit of detection of the specific peptides down to low ppb level in saliva, demonstrating the possibility of the targeted proteomics approach for rapid detection of SARS-CoV-2. |
| Rights: | All rights reserved |
| Access: | open access |
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