SHV-129: A Gateway to Global Suppressors in the SHV β-Lactamase Family?

Abstract

Enzymes are continually evolving in response to environmental pressures. In order to increase enzyme fitness, amino acid substitutions can occur leading to a changing function or an increased stability. These evolutionary drivers determine the activity of an enzyme and its success in future generations in response to changing conditions such as environmental stressors or to improve physiological function allowing continual persistence of the enzyme. With recent warning reports on antibiotic resistance and multidrug resistant bacterial infections, understanding the evolution of β-lactamase enzymes, which are a large contributor to antibiotic resistance, is increasingly important. Here, we investigated a variant of the SHV β-lactamase identified from a clinical isolate of Escherichia coli in 2011 (SHV-129, G238S-E240K-R275L-N276D) to identify the first instance of a global suppressor substitution in the SHV β-lactamase family. We have used this enzyme to show that several evolutionary principles are conserved in different class A β-lactamases, such as active site mutations reducing stability and requiring compensating suppressor substitutions in order to ensure evolutionary persistence of a given β-lactamase. However, the pathway taken by a given β-lactamase in order to reach its evolutionary peak under a given set of conditions is likely different. We also provide further evidence for a conserved stabilizing substitution among class A β-lactamases, the back to consensus M182T substitution. In addition to expanding the spectrum of β-lactamase activity to include the hydrolysis of cefepime, the amino acid substitutions found in SHV-129 provide the enzyme with an excess of stability, which expands the evolutionary landscape of this enzyme and may result in further evolution to potentially include resistance to carbapenems or β-lactamase inhibitors.

Keywords: antibiotic resistance; global suppressors; protein evolution; β-lactamase.

Fig. 1.

(A) Overlay of the ribbon structure of TEM-1 (protein database (PDB) ID: 1ZG4, yellow), TEM-52 (PDB ID: 1M40, red), SHV-1 (PDB ID: 1SHV, green), and SHV-2 (PDB ID: 1N9B, blue). (B) Ribbon diagram of SHV-1 (PDB ID: 1SHV) with amino acid differences with TEM-1 identified, S70 is in yellow. (C) Ribbon diagram of SHV-1 (PDB ID: 1SHV) with the location of amino acid differences with TEM-1 marked in orange. (D) Active site overlay of TEM-1 (PDB ID: 1ZG4, yellow), TEM-52 (PDB ID: 1M40, red), SHV-1 (PDB ID: 1SHV, green), and SHV-2 (PDB ID: 1N9B, blue). The amino acids studied in this article are also shown. Overall, the active site amino acids are very similar between these proteins. The amino acid alignment of these four β-lactamases is also shown below with shading at the site of amino acid differences illustrated in B and C.

Fig. 2.

Proposed evolutionary mutagenesis scheme for SHV-129 including a separate path testing only our two hypothesized suppressor substitutions (R275L and N276D). G238S is SHV-2, G238S-E240K is SHV-5, and G238S-E240K-R275L-N276D is SHV-129.

Fig. 3.

(A) Immunoblot of variants leading to SHV-129 showing the various expression levels of these proteins in whole cell bacterial preparations. (B) Immunoblot of variants leading to SHV-129 showing the various expression levels of these proteins in soluble fraction preparations. SHV-2 is G238S, SHV-5 is G238S-E240K, and SHV-129 is G238S-E240K-R275L-N276D.

Fig. 4.

(A) Overall CD structure of SHV-1 showing the secondary structure elements of this wild-type β-lactamase. (B) Overall CD structure of the various SHV enzymes showing similarities in α-helical and β-sheet structure with the exception of the T182M variant. (C) Melting curves of the various SHV enzymes showing stabilization of the SHV-129 enzyme and destabilization of T182M, SHV-2 (G238S), and SHV-5 (G238S-E240K) β-lactamases. The R275L-N276D protein does not completely unfold.