Please use this identifier to cite or link to this item: http://hdl.handle.net/10397/91532
Title: Structural and functional studies of antibiotic resistance enzymes
Authors: Cheng, Qipeng
Degree: Ph.D.
Issue Date: 2021
Abstract: Multidrug-resistant pathogens have become increasingly common and pose a serious health threat to human health. Antibiotic resistance is caused by several cellular mechanisms, among them, enzyme-mediated resistance is becoming important and prevalent in Gram-positive and Gram-negative bacteria. In this study,it showed that the TEM-1 β-lactamase has evolved to become active on the extended generation cephalosporins through two distinct evolution routes: one began with the Gly238Ser change and the other originated with the Arg164Ser substitution. These two evolution routes underlie the markedly increased prevalence of bacterial resistance to β-lactams in the past few decades. For ESBLs surveillance, a CTX-M-199 was isolated, which is encoded by conjugated mcr-1-bearing IncI2 plasmid and conferred resistance to tazobactam, and sulbactam, while the T130 residue in the CTX-M-199 was found to contribute to inhibitor resistance. Using mass spectrometric and crystallographic approaches, we showed that CTX-M- 64 (S130T) did not cause any conformational change or exert any effect on its ability to hydrolyze β-lactam substrates. However, binding of sulbactam to the active site of CTX-M-64 (S130T) led to the conformational changes in the active site, which weakened the binding of the sulbactam trans-enamine intermediate (TSL) to the active site and hindered the formation of the inhibitor-enzyme complex: a covalent acrylic acid (AKR)-T130 bond, thereby resulting in inhibitor resistance in CTX-M-64 (S130T). Besides, we identified and characterized, both genetically and functionally, a novel carbapenemase known as VMB-1, which is encoded by a gene (blaVMB-1) located in an integron-bearing, highly transmissible IncC type plasmid, namely pVB1796. Sequence alignment analysis showed that VMB-1 shared the structurally identical active site with subclass B1 MBLs. Importantly, pVB1796 was found to be efficiently transferred from Vibrio to other Gram-negative bacterial pathogens via conjugation.
MCR-1 is a mobilized pEtN transferase that modifies the pEtN moiety of lipid A, conferring resistance to colistin. In this study, we determined the crystal structure of the catalytic domain of MCR-1 (MCR-1-ED). Two interlaced molecules with different phosphorylation status of the residue T285 could give rise to two functional statuses of MCR-1 depending on the physiological conditions. MCR-1 possesses an enzymatic site equipped with zinc-binding residues. In addition, two zinc ions were found to mediate intermolecular interactions between MCR-1-ED molecules in one asymmetric unit and allowing the protein to form an oligomer. Recently, new variants of Tet(X2), namely Tet(X3~6), which are responsible for causing resistance to tigecycline among members of Enterobacteriaceae. In this study, we identified essential residues in TetX variants that mediated the evolution of the tigecycline-inactive Tet(X2) enzyme to the active forms of Tet(X3) and Tet(X4). Based on their amino acid sequences and functional features, we classified TetX variants into three groups, namely Tet(X2), Tet(X3), and Tet(X4). Using this new classification system, we found that variants of the tet(X2) gene originated from Bacteroidetes, with some variants evolving further to tet(X3) and being acquired by Enterobacteriaceae. In summary, in this study, we gain a deep understanding of the evolution, function, and structure of antibiotic resistance enzymes. Based on the new knowledge generated in these works we shall attempt to restore the clinical application value of currently available antibiotics by developing potential inhibitors of these enzymes.
Subjects: Anti-infective agents
Antibiotics
Drug resistance in microorganisms
Microbial enzymes
Hong Kong Polytechnic University -- Dissertations
Pages: xvi, 188 pages : color illustrations
Appears in Collections:Thesis

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