Mutational Events in Cefotaximase Extended-Spectrum β-Lactamases of the CTX-M-1 Cluster Involved in Ceftazidime Resistance

The widespread use of oxyimino-cephalosporin antibiotics drives the evolution of the CTX-M family of ␤-lactamases that hydrolyze these drugs and confer antibiotic resistance. Clinically isolated CTX-M enzymes carrying the P167S or D240G active siteassociated adaptive mutation have a broadened substrate profile that includes the oxyimino-cephalosporin antibiotic ceftazidime. The D240G substitution is known to reduce the stability of CTX-M-14 ␤-lactamase, and the P167S substitution is shown here to also destabilize the enzyme. Proteins are marginally stable entities, and second-site mutations that stabilize the enzyme can offset a loss in stability caused by mutations that enhance enzyme activity. Therefore, the evolution of antibiotic resistance enzymes can be dependent on the acquisition of stabilizing mutations. The A77V substitution is present in CTX-M extendedspectrum ␤-lactamases (ESBLs) from a number of clinical isolates, suggesting that it may be important in the evolution of antibiotic resistance in this family of ␤-lactamases. In this study, the effects of the A77V substitution in the CTX-M-14 model enzyme were characterized with regard to the kinetic parameters for antibiotic hydrolysis as well as enzyme expression levels in vivo and protein stability in vitro. The A77V substitution has little effect on the kinetics of oxyimino-cephalosporin hydrolysis, but it stabilizes the CTX-M enzyme and compensates for the loss of stability resulting from the P167S and D240G mutations. The acquisition of global stabilizing mutations, such as A77V, is an important feature in ␤-lactamase evolution and a common mechanism in protein evolution.T he introduction of new antibiotics into clinical use fuels the evolution of enzymes causing drug resistance. These enzymes gain the ability to inactivate antibiotics more efficiently by acquiring point mutations in and around their active sites that increase catalytic activity (1). However, the introduction of new gain-offunction mutations within highly organized active sites often comes at a stability cost to the enzymes (2-6). Therefore, enzymes can absorb only a limited number of destabilizing mutations before they unfold and lose function (7). This limitation in the evolution of catalytic efficiency is often overcome by the acquisition of global stabilizing mutations that offset the incremental loss in stability due to the primary mutation. This compensation mechanism allows the enzymes to continue a mutational trajectory, resulting in increased resistance.The role of global stabilizing mutations in enzyme evolution has been widely studied (4,8,9). In recent decades, the selection of mutant ␤-lactamase enzymes with expanded substrate specificity has occurred for the plasmid-mediated TEM-and SHV-type ␤-lactamases to give rise to extended-spectrum ␤-lactamases (ESBLs) (10, 11). These enzymes arise due to mutations resulting in substitutions near the active site that increase hydrolysis of oxyimino-cephalosporins such as cefotaxime and ceftazidime. The substitutions that increa...

Section: Antibiotic Susceptibility and Fitness Of Ctx-m-14 ␤-Lactamasesupporting

confidence: 80%

mentioning

confidence: 99%

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Characterization of the Global Stabilizing Substitution A77V and Its Role in the Evolution of CTX-M β-Lactamases

Patel

Fryszczyn

Palzkill

2015

“…1A and 5B). The catalytic activity of CTX-M-132 and CTX-M-123 toward different ␤-lactams was similar except toward ampicillin, suggesting these residues showed either a neutral effect or an antagonistic effect between some of them, such as the case of P 167 S and D 240 G reported by Novais et al, on the catalytic activity of these two CTX-Ms (16). Nonetheless, it is almost impossible to identify which ones within these 9 residues play important roles on the catalytic activity unless individual mutations are introduced into the background of CTX-M-132.…”

Section: Resultsmentioning

confidence: 91%

Residues Distal to the Active Site Contribute to Enhanced Catalytic Activity of Variant and Hybrid β-Lactamases Derived from CTX-M-14 and CTX-M-15

Chiou

Zeng

et al. 2015

A variety of CTX-M-type extended-spectrum ␤-lactamases (ESBLs), including hybrid ones, have been reported in China that are uncommon elsewhere. To better characterize the substrate profiles and enzymatic mechanisms of these enzymes, we performed comparative kinetic analyses of both parental and hybrid CTX-M enzymes, including CTX-M-15, -132, -123, -64, -14 and -55, that are known to confer variable levels of ␤-lactam resistance in the host strains. All tested enzymes were susceptible to serine ␤-lactamase inhibitors, with sulbactam exhibiting the weakest inhibitory effects. CTX-M-55, which differs from CTX-M-15 by one substitution, A 77 V, displayed enhanced catalytic activity (k cat /K m ) against expanded-spectrum cephalosporins (ESCs). CTX-M-55 exhibits higher structure stability, most likely by forming hydrophobic interactions between A 77 V and various key residues in different helices, thereby stabilizing the core architecture of the helix cluster, and indirectly contributes to a more stable active site conformation, which in turn shows higher catalytic efficiency and is more tolerant to temperature change. Analyses of the hybrids and their parental prototypes showed that evolution from CTX-M-15 to CTX-M-132, CTX-M-123, and CTX-M-64, characterized by gradual enhancement of catalytic activity to ESCs, was attributed to introduction of different substitutions to amino acids distal to the active site of CTX-M-15. Similarly, the increased hydrolytic activities against cephalosporins and sensitivity to ␤-lactamase inhibitors, clavulanic acid and sulbactam, of CTX-M-64 were partly due to the amino acids that were different from CTX-M-14 and located at both the C and N termini of CTX-M-64. These data indicate that residues distal to the active site of CTX-Ms contributed to their enhanced catalytic activities to ESCs.T he rapid dissemination of CTX-M-type extended-spectrum ␤-lactamases (ESBLs) in Enterobacteriaceae poses a huge threat to both human and animal health. To date, more than 150 different CTX-M-type ESBLs have been identified (http://www .lahey.org/studies/other.asp#table1). Based on the genetic relatedness, CTX-Ms can be divided into six clusters, including CTX-M-1, -2, -8, -9, and -25 and the KLUC groups (1). More than 95% identity is often observed within the same cluster, whereas less than 90% identity is detectable between clusters (2). Several hybrids of the CTX-M-1 and -9 groups, namely, CTX-M-64, CTX-M-123, CTX-M-137, and CTX-M-132, have also been identified recently (3-5). Formation of these hybrid CTX-M enzymes was suggested to be the result of recombination between bla CTX-M-15 and bla CTX-M-14 , the two most dominant variants detectable worldwide (1, 6). However, the genetic environments of bla CTX-M-123 and bla CTX-M-64 were highly similar to that of bla CTX-M-55 identified in pHN1122-1, the second-most prevalent bla CTX-M variant in China (7). This suggests that recombination may take place between bla CTX-M-14 and bla CTX-M-55 , producing various hybrid enzymes. Interestingly, all three p...

“…Several hybrid CTX-M ␤-lactamases (CTX-M-64, CTX-M-123, CTX-M-137, and CTX-M-132), presumably resulting from recombination between bla CTX-M- 15 and bla CTX-M-14 , which are the most common variants worldwide, have been reported in recent years (4)(5)(6)(7). Among these hybrid enzymes, CTX-M-64, which contained the N-and C-terminal portion of CTX-M-15 and the middle fragment of CTX-M-14, exhibited even higher catalytic activity than its parental prototypes (4).…”

mentioning

confidence: 99%

Comparative Characterization of CTX-M-64 and CTX-M-14 Provides Insights into the Structure and Catalytic Activity of the CTX-M Class of Enzymes

Chiou

Zeng

et al. 2016

c Clinical isolates producing hybrid CTX-M ␤-lactamases, presumably due to recombination between the bla CTX-M-15 and bla CTX-M-14 elements, have emerged in recent years. Among the hybrid enzymes, CTX-M-64 and CTX-M-14 display the most significant difference in catalytic activity. This study aims to investigate the mechanisms underlying such differential enzymatic activities in order to provide insight into the structure/function relationship of this class of enzymes. Sequence alignment analysis showed that the major differences between the amino acid composition of CTX-M-64 and CTX-M-14 lie at both the N and C termini of the enzymes. Single or multiple amino acid substitutions introduced into CTX-M-64 and CTX-M-14 were found to produce only minor effects on hydrolytic functions; such a finding is consistent with the notion that the discrepancy between the functional activities of the two enzymes is not the result of only a few amino acid changes but is attributable to interactions between a unique set of amino acid residues in each enzyme. This theory is supported by the results of the thermal stability assay, which confirmed that CTX-M-64 is significantly more stable than CTX-M-14. Our data confirmed that, in addition to the important residues located in the active site, residues distal to the active site also contribute to the catalytic activity of the enzyme through stabilizing its structural integrity.T he utilization of extended-spectrum cephalosporins for the treatment of bacterial infections has been extremely effective since the 1980s. However, such effectiveness has gradually been eroded as a result of the dissemination of resistance elements that encode a variety of ␤-lactamases, especially the extended-spectrum ␤-lactamases (ESBLs). Having emerged as a result of the extensive usage of ␤-lactam antibiotics in humans and animals, the class A serine enzymes of the CTX-M-type ESBLs have become the most common type of plasmid-mediated ESBL enzymes produced by drug-resistant organisms. Sharing less than 40% amino acid sequence homology with the TEM-and SHV-type enzymes, the CTX-M enzymes can be subclassified into six groups (CTX-M-1, -2, -8, -9, -25, and the KLUC groups) (1, 2).Unlike other class A ␤-lactamases, the evolution of genetic elements encoding CTX-M enzymes is known to be mediated by random mutations and recombination between different resistance genes, in particular those encoding the CTX-M-1 and CTX-M-9 enzymes (3). Several hybrid CTX-M ␤-lactamases (CTX-M-64, CTX-M-123, CTX-M-137, and CTX-M-132), presumably resulting from recombination between bla CTX-M-15 and bla CTX-M-14 , which are the most common variants worldwide, have been reported in recent years (4-7). Among these hybrid enzymes, CTX-M-64, which contained the N-and C-terminal portion of CTX-M-15 and the middle fragment of CTX-M-14, exhibited even higher catalytic activity than its parental prototypes (4).The CTX-M-type enzymes degrade a wide range of substrates, including penicillin and the first, second, and third generation of ceph...