LiaX is a surrogate marker for cell envelope stress and daptomycin non-susceptibility in Enterococcus faecium.

Daptomycin (DAP) is often used as a first-line therapy to treat vancomycin-resistant Enterococcus faecium infections, but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP minimum inhibitory concentrations (MICs) have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system, and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ. In Enterococcus faecalis, LiaX is surface-exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis, LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium. Here, we found that liaX is essential in E. faecium with an activated LiaFSR system. Unlike E. faecalis, E. faecium LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX enzyme-linked immunosorbent assay (ELISA). We then assessed 86 clinical E. faecium bloodstream isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-resistant clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-susceptible isolates by standard MIC determination also had elevated LiaX ELISAs compared to a well-characterized DAP-susceptible strain. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many E. faecium isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.

LiaX is a surrogate marker for cell-envelope stress and daptomycin non-susceptibility in Enterococcus faecium.

Daptomycin (DAP) is often used as a first line therapy to treat vancomycin-resistant Enterococcus faecium (VR Efm ) infections but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP MICs have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ . In Enterococcus faecalis , LiaX is surface exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis , LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium . Here, we found that liaX is essential in E. faecium ( Efm ) with an activated LiaFSR system. Unlike E. faecalis , Efm LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX ELISA. We then assessed 86 clinical E. faecium BSI isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-R clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-S isolates by standard MIC determination had elevated LiaX ELISAs above the established cut-off. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many Efm isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.

KPC-2 ß-lactamase enables carbapenem antibiotic resistance through fast deacylation of the covalent intermediate.

Serine active-site ß-lactamases hydrolyze ß-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 ß-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 ß-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 ß-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 ß-lactamase.

Structural Basis and Binding Kinetics of Vaborbactam in Class A ß-Lactamase Inhibition.

Class A ß-lactamases are a major cause of ß-lactam resistance in Gram-negative bacteria. The recently FDA-approved cyclic boronate vaborbactam is a reversible covalent inhibitor of class A ß-lactamases, including CTX-M extended-spectrum ß-lactamase and KPC carbapenemase, both frequently observed in the clinic. Intriguingly, vaborbactam displayed different binding kinetics and cell-based activity for these two enzymes, despite their similarity. A 1.0-Å crystal structure of CTX-M-14 demonstrated that two catalytic residues, K73 and E166, are positively charged and neutral, respectively. Meanwhile, a 1.25-Å crystal structure of KPC-2 revealed a more compact binding mode of vaborbactam versus CTX-M-14, as well as alternative conformations of W105. Together with kinetic analysis of W105 mutants, the structures demonstrate the influence of this residue and the unusual conformation of the ß3 strand on the inactivation rate, as well as the stability of the reversible covalent bond with S70. Furthermore, studies of KPC-2 S130G mutant shed light on the different impacts of S130 in the binding of vaborbactam versus avibactam, another recently approved ß-lactamase inhibitor. Taken together, these new data provide valuable insights into the inhibition mechanism of vaborbactam and future development of cyclic boronate inhibitors.

Mechanism of proton transfer in class A ß-lactamase catalysis and inhibition by avibactam.

Gram-negative bacteria expressing class A ß-lactamases pose a serious health threat due to their ability to inactivate all ß-lactam antibiotics. The acyl-enzyme intermediate is a central milestone in the hydrolysis reaction catalyzed by these enzymes. However, the protonation states of the catalytic residues in this complex have never been fully analyzed experimentally due to inherent difficulties. To help unravel the ambiguity surrounding class A ß-lactamase catalysis, we have used ultrahigh-resolution X-ray crystallography and the recently approved ß-lactamase inhibitor avibactam to trap the acyl-enzyme complex of class A ß-lactamase CTX-M-14 at varying pHs. A 0.83-Å-resolution CTX-M-14 complex structure at pH 7.9 revealed a neutral state for both Lys73 and Glu166. Furthermore, the avibactam hydroxylamine-O-sulfonate group conformation varied according to pH, and this conformational switch appeared to correspond to a change in the Lys73 protonation state at low pH. In conjunction with computational analyses, our structures suggest that Lys73 has a perturbed acid dissociation constant (pKa) compared with acyl-enzyme complexes with ß-lactams, hindering its function to deprotonate Glu166 and the initiation of the deacylation reaction. Further NMR analysis demonstrated Lys73 pKa to be ∼5.2 to 5.6. Together with previous ultrahigh-resolution crystal structures, these findings enable us to follow the proton transfer process of the entire acylation reaction and reveal the critical role of Lys73. They also shed light on the stability and reversibility of the avibactam carbamoyl acyl-enzyme complex, highlighting the effect of substrate functional groups in influencing the protonation states of catalytic residues and subsequently the progression of the reaction.

Discovery of Cyclic Boronic Acid QPX7728, an Ultrabroad-Spectrum Inhibitor of Serine and Metallo-ß-lactamases.

Despite major advances in the ß-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 ß-lactam antibiotic for the treatment of a wide range of multidrug resistant Gram-negative bacterial infections, by both intravenous and oral administration.

Structural Basis for Substrate Specificity and Carbapenemase Activity of OXA-48 Class D ß-Lactamase.

Carbapenem-hydrolyzing class D ß-lactamases (CHDLs) are a diverse family of enzymes that are rapidly becoming the predominant cause of bacterial resistance against ß-lactam 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 X-ray crystallography to OXA-48 complexes with multiple ß-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 ß-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 ß-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.

Heteroaryl Phosphonates as Noncovalent Inhibitors of Both Serine- and Metallocarbapenemases.

Gram-negative pathogens expressing serine ß-lactamases (SBLs) and metallo-ß-lactamases (MBLs), especially those with carbapenemase activity, threaten the clinical utility of almost all ß-lactam antibiotics. Here we describe the discovery of a heteroaryl phosphonate scaffold that exhibits noncovalent cross-class inhibition of representative carbapenemases, specifically the SBL KPC-2 and the MBLs NDM-1 and VIM-2. The most potent lead, compound 16, exhibited low nM to low µM inhibition of KPC-2, NDM-1, and VIM-2. Compound 16 potentiated imipenem efficacy against resistant clinical and laboratory bacterial strains expressing carbapenemases while showing some cytotoxicity toward human HEK293T cells only at concentrations above 100 µg/mL. Complex structures with KPC-2, NDM-1, and VIM-2 demonstrate how these inhibitors achieve high binding affinity to both enzyme classes. These findings provide a structurally and mechanistically new scaffold for drug discovery targeting multidrug resistant Gram-negative pathogens and more generally highlight the active site features of carbapenemases that can be leveraged for lead discovery.

Active-Site Druggability of Carbapenemases and Broad-Spectrum Inhibitor Discovery.

Serine and metallo-carbapenemases are a serious health concern due to their capability to hydrolyze nearly all ß-lactam antibiotics. However, the molecular basis for their unique broad-spectrum substrate profile is poorly understood, particularly for serine carbapenemases, such as KPC-2. Using substrates and newly identified small molecules, we compared the ligand binding properties of KPC-2 with the noncarbapenemase CTX-M-14, both of which are Class A ß-lactamases with highly similar active sites. Notably, compared to CTX-M-14, KPC-2 was more potently inhibited by hydrolyzed ß-lactam products (product inhibition), as well as by a series of novel tetrazole-based inhibitors selected from molecular docking against CTX-M-14. Together with complex crystal structures, these data suggest that the KPC-2 active site has an enhanced ability to form favorable interactions with substrates and small molecule ligands due to its increased hydrophobicity and flexibility. Such properties are even more pronounced in metallo-carbapenemases, such as NDM-1, which was also inhibited by some of the novel tetrazole compounds, including one displaying comparable low µM affinities against both KPC-2 and NDM-1. Our results suggest that carbapenemase activity confers an evolutionary advantage on producers via a broad ß-lactam substrate scope but also a mechanistic Achilles' heel that can be exploited for new inhibitor discovery. The complex structures demonstrate, for the first time, how noncovalent inhibitors can be engineered to simultaneously target both serine and metallo-carbapenemases. Despite the relatively modest activity of the current compounds, these studies also demonstrate that hydrolyzed products and tetrazole-based chemotypes can provide valuable starting points for broad-spectrum inhibitor discovery against carbapenemases.

Antibacterial Spectrum of a Tetrazole-Based Reversible Inhibitor of Serine ß-Lactamases.

CTX-M is the most prevalent family of extended-spectrum ß-lactamases. We recently developed a tetrazole-derived noncovalent inhibitor of CTX-M-9. Here, we present the biochemical and microbiological activity of this inhibitor across a representative panel of serine ß-lactamases and Gram-negative bacteria. The compound displayed significant activity against all major subgroups of CTX-M, including CTX-M-15, while it exhibited some low-level inhibition of other serine ß-lactamases. Complex crystal structures with the CTX-M-14 S237A mutant and CTX-M-27 illustrate the binding contribution of specific active-site residues on the ß3 strand. In vitro pharmacokinetic studies revealed drug-like properties and positive prospects for further optimization. These studies suggest that tetrazole-based compounds can provide novel chemotypes for future serine ß-lactamase inhibitor discovery.

Molecular Basis of Substrate Recognition and Product Release by the Klebsiella pneumoniae Carbapenemase (KPC-2).

Carbapenem-resistant Enterobacteriaceae are resistant to most ß-lactam antibiotics due to the production of the Klebsiella pneumoniae carbapenemase (KPC-2) class A ß-lactamase. Here, we present the first product complex crystal structures of KPC-2 with ß-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 ß-lactamase, elucidating the product release mechanism of these enzymes in general.