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Effects of Asp-179 mutations in TEMpUC19 beta-lactamase on susceptibility to beta-lactams
S B Vakulenko1, M Tóth, P Taibi
1Department of Medicine, Wayne State University, Detroit, Michigan 48202, USA.
Abstract:
To examine the effect of disruption of the salt bridge (between Arg-164 and Asp-179 [numbering of Ambler et al. (Biochem J. 267:269-272, 1991)]) that anchors the conserved omega-loop in class A beta-lactamases, we obtained mutant enzymes with each of the 19 other amino acid residues replacing Asp-179 in the TEM beta-lactamase encoded by pUC19 and studied the level of resistance to various beta-lactams conferred by each enzyme. All mutations of Asp-179 compromised the level of resistance to ampicillin, but most of them enhanced resistance to ceftazidime. In contrast, mutations of Asp-179 generally impaired the low levels of resistance to cefepime and aztreonam. One might expect to find clinical isolates with mutant TEM beta-lactamases with replacements of Asp-179 that express an expanded spectrum of resistance to beta-lactams including ceftazidime.
Insights
Disrupting a key salt bridge in TEM beta-lactamase mutants altered antibiotic resistance. Mutations enhanced ceftazidime resistance but reduced ampicillin, cefepime, and aztreonam resistance.
Area of Science:
- Biochemistry
- Microbiology
- Drug Resistance
Background:
- Class A beta-lactamases are crucial in bacterial antibiotic resistance.
- The conserved omega-loop, anchored by a salt bridge, is vital for beta-lactamase function.
- TEM beta-lactamase is a significant enzyme conferring resistance to beta-lactam antibiotics.
Purpose of the Study:
- To investigate the impact of disrupting the Arg-164/Asp-179 salt bridge on TEM beta-lactamase activity.
- To determine how mutations at Asp-179 affect resistance to various beta-lactam antibiotics.
- To predict the potential for clinical isolates with altered TEM beta-lactamases.
Main Methods:
- Site-directed mutagenesis was used to replace Asp-179 with 19 different amino acid residues in TEM beta-lactamase.
- Mutant enzymes were expressed using the pUC19 plasmid.
- The level of resistance to ampicillin, ceftazidime, cefepime, and aztreonam was evaluated for each mutant.
Main Results:
- All Asp-179 mutations compromised ampicillin resistance.
- Most mutations enhanced resistance to ceftazidime.
- Mutations generally impaired resistance to cefepime and aztreonam.
Conclusions:
- Disruption of the Asp-179 salt bridge significantly alters TEM beta-lactamase substrate specificity.
- Mutant TEM beta-lactamases with Asp-179 replacements may confer an expanded resistance spectrum, particularly to ceftazidime.
- Clinical isolates with such mutant enzymes could pose a challenge in antibiotic treatment strategies.