Despite major advances in the β-lactamase inhibitor field, certain enzymes remain refractory to inhibition by agents recently introduced. Most important among these are the class B (metallo) enzyme NDM-1 of Enterobacteriaceae and the class D (OXA) enzymes of Acinetobacter baumannii. Continuing the boronic acid program that led to vaborbactam, efforts were directed toward expanding the spectrum to allow treatment of a wider range of organisms. Through key structural modifications of a bicyclic lead, stepwise gains in spectrum of inhibition were achieved, ultimately resulting in QPX7728 (35). This compound displays a remarkably broad spectrum of inhibition, including class B and class D enzymes, and is little affected by porin modifications and efflux. Compound 35 is a promising agent for use in combination with a β-lactam antibiotic for the treatment of a wide range of multidrug resistant Gram-negative bacterial infections, by both intravenous and oral administration.

Carbapenem-resistant Enterobacteriaceae (CRE) are resistant to most β-lactam antibiotics due to production of the KPC-2 class A β-lactamase. Here we present the first product complex crystal structures of KPC-2 with β-lactam antibiotics, containing hydrolyzed cefotaxime and faropenem. They provide experimental insights into substrate recognition by KPC-2 and its unique cephalosporinase/carbapenemase activity. These structures also represent the first product complexes for a wild type serine β-lactamase, elucidating the product release mechanism of these enzymes in general.

Bacteria have developed several evolutionary strategies to protect their cell membranes (CMs) from the attack of antibiotics and antimicrobial peptides (AMPs) produced by the innate immune system, including remodeling of phospholipid content and localization. Multidrug-resistantEnterococcus faecalis,an opportunistic human pathogen, evolves resistance to the lipopeptide daptomycin and AMPs by diverting the antibiotic away from critical septal targets using CM anionic phospholipid redistribution. The LiaFSR stress response system regulates this CM remodeling via the LiaR response regulator by a previously unknown mechanism. Here, we characterize a LiaR-regulated protein, LiaX, that senses daptomycin or AMPs and triggers protective CM remodeling. LiaX is surface exposed, and in daptomycin-resistant clinical strains, both LiaX and the N-terminal domain alone are released into the extracellular milieu. The N-terminal domain of LiaX binds daptomycin and AMPs (such as human LL-37) and functions as an extracellular sentinel that activates the cell envelope stress response. The C-terminal domain of LiaX plays a role in inhibiting the LiaFSR system, and when this domain is absent, it leads to activation of anionic phospholipid redistribution. Strains that exhibit LiaX-mediated CM remodeling and AMP resistance show enhanced virulence in theCaenorhabditis elegansmodel, an effect that is abolished in animals lacking an innate immune pathway crucial for producing AMPs. In conclusion, we report a mechanism of antibiotic and AMP resistance that couples bacterial stress sensing to major changes in CM architecture, ultimately also affecting host–pathogen interactions.

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are a diverse family of enzymes that are rapidly becoming the predominant cause of bacterial resistance against βlactam antibiotics in many regions of the world. OXA-48, an atypical member of CHDLs, is one of the most frequently observed in the clinic and exhibits a unique substrate profile. We applied Xray crystallography to OXA-48 complexes with multiple β-lactam antibiotics to elucidate this enzyme's carbapenemase activity and its preference of imipenem over meropenem and other substrates such as cefotaxime. In particular, we obtained acyl-enzyme complexes of OXA-48 with imipenem, meropenem, faropenem, cefotaxime, and cefoxitin, and a product complex with imipenem. Importantly, the product complex captures a key reaction milestone with the newly generated carboxylate group still in the oxyanion hole, and represents the first such complex with a wild-type serine β-lactamase. A potential hydrogen bond is observed between the two carboxylate groups from the product and the carbamylated Lys73, representing the stage immediately after the breakage of the acyl-enzyme bond where the product carboxylate would be neutral. The placement of the product carboxylate also illustrates the approximate transient location of the deacylation water that has long eluded structural characterization in class D β-lactamases. Additionally, comparing the product complex with the acyl-enzyme intermediates provides new insights into the various mechanisms by which specific side chain groups hinder the access of the deacylation water to the acyl-enzyme linkage, especially in meropenem. Taken together, these data offer valuable information on the substrate specificity of OXA-48 and the catalytic mechanism of CHDLs.

Serine active-site β-lactamases hydrolyze β-lactam antibiotics through the formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most β-lactamases owing to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared with the common TEM-1 β-lactamase. Furthermore, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase β-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 β-lactamase.