12 research outputs found

    Antimicrobial sensing coupled with cell membrane remodeling mediates antibiotic resistance and virulence in Enterococcus faecalis.

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    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-resistant Enterococcus 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 the Caenorhabditis elegans model, 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

    Mechanism Elucidation and Inhibitor Discovery against Serine and Metallo-Beta-Lactamases Involved in Bacterial Antibiotic Resistance

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    The emergence and proliferation of Gram-negative bacteria expressing β-lactamases is a significant threat to human health. β-Lactamases are enzymes that degrade the β-lactam antibiotics (e.g., penicillins, cephalosporins, monobactams, and carbapenems) that we use to treat a diverse range of bacterial infections. Specifically, β-lactamases catalyze a hydrolysis reaction where the β-lactam ring common to all β-lactam antibiotics and responsible for their antibacterial activity, is opened, leaving an inactive drug. There are two groups of β-lactamases: serine enzymes that use an active site serine residue for β-lactam hydrolysis and metalloenzymes that use either one or two zinc ions for catalysis. Serine enzymes are divided into three classes (A, C, and D), while there is only one class of metalloenzymes, class B. Clavulanic acid, sulbactam, and tazobactam are β-lactam-based BLIs that demonstrate activity against class A and C β-lactamases; however, they have no activity against the class A KPC and MBLs, NDM and VIM. Avibactam and vaborbactam are novel BLIs approved in the last two years that have activity against serine carbapenemases (e.g., KPC), but do not inhibit MBLs. The overall goals of this project were to use X-ray crystallography to study the catalytic mechanism of serine β-lactamases with β-lactam antibiotics and to understand the mechanisms behind the broad-spectrum inhibition of class A β-lactamases by avibactam and vaborbactam. This project also set out to find novel inhibitors using molecular docking and FBDD that would simultaneously inactivate serine β-lactamases and MBLs commonly expressed in Gram-negative pathogenic bacteria. The first project involved examining the structural basis for the class A KPC-2 β-lactamase broad-spectrum of activity that includes cephalosporins and carbapenems. Three crystal structures were solved of KPC-2: (1) an apo-structure at 1.15 Å; (2) a complex structure with the hydrolyzed cephalosporin, cefotaxime at 1.45 Å; and (3) a complex structure with the hydrolyzed penem, faropenem at 1.40 Å. These complex structures show how alternative conformations of Ser70 and Lys73 play a role in the product release step. The large and shallow active site of KPC-2 can accommodate a wide variety of β-lactams, including the bulky oxyimino side chain of cefotaxime and also permits the rotation of faropenem’s 6-alpha-1R-hydroxyethyl group to promote carbapenem hydrolysis. Lastly, the complex structures highlight that the catalytic versatility of KPC-2 may expose a potential opportunity for drug discovery. The second project focused on understanding the stability of the BLI, avibactam, against hydrolysis by serine β-lactamases. A 0.83 Å crystal structure of CTX-M-14 bound by avibactam revealed that binding of the inhibitor impedes a critical proton transfer between Glu166 and Lys73. This results in a neutral Glu166 and neutral Lys73. A neutral Glu166 is unable to serve as a general base to activate the catalytic water for the hydrolysis reaction. Overall, this structure suggests that avibactam can influence the protonation state of catalytic residues. The third project centered on vaborbactam, a cyclic boronic acid inhibitor of class A and C β-lactamases, including the serine class A carbapenemase KPC-2. To characterize vaborbactam inhibition, binding kinetic experiments, MIC assays, and mutagenesis studies were performed. A crystal structure of the inhibitor bound to KPC-2 was solved to 1.25 Å. These data revealed that vaborbactam achieves nanomolar potency against KPC-2 due to its covalent and extensive non-covalent interactions with conserved active site residues. Also, a slow off-rate and long drug-target residence time of vaborbactam with KPC-2 strongly correlates with in vitro and in vivo activity. The final project focused on discovering dual action inhibitors targeting serine carbapenemases and MBLs. Performing molecular docking against KPC-2 led to the identification of a compound with a phosphonate-based scaffold. Testing this compound using a nitrocefin assay confirmed that it had micromolar potency against KPC-2. SAR studies were performed on this scaffold, which led to a nanomolar inhibitor against KPC-2. Crystal structures of the inhibitors complexed with KPC-2 revealed interactions with active site residues such as Trp105, Ser130, Thr235, and Thr237, which are all important in ligand binding and catalysis. Interestingly, the phosphonate inhibitors that displayed activity against KPC-2, also displayed activity against the MBLs NDM-1 and VIM-2. Crystal structures of the inhibitors complexed with NDM-1 and VIM-2 showed that the phosphonate group displaces a catalytic hydroxide ion located between the two zinc ions in the active site. Additionally, the compounds form extensive hydrophobic interactions that contribute to their activity against NDM-1 and VIM-2. MIC assays were performed on select inhibitors against clinical isolates of Gram-negative bacteria expressing KPC-2, NDM-1, and VIM-2. One phosphonate inhibitor was able to reduce the MIC of the carbapenem, imipenem 64-fold against a K. pneumoniae strain producing KPC-2. The same phosphonate inhibitor also reduced the MIC of imipenem 4-fold against an E. coli strain producing NDM-1. Unfortunately, no cell-based activity was observed for any of the phosphonate inhibitors when tested against a P. aeruginosa strain producing VIM-2. Ultimately, this project demonstrated the feasibility of developing cross-class BLIs using molecular docking, FBDD, and SAR studies

    Molecular Basis of Substrate Recognition and Product Release by the \u3cem\u3eKlebsiella pneumoniae\u3c/em\u3e Carbapenemase (KPC-2)

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    Carbapenem-resistant Enterobacteriaceae are resistant to most β-lactam antibiotics due to the production of the Klebsiella pneumoniae carbapenemase (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

    Beta-lactamase inhibitors, formulations, and uses thereof

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    Provided herein are heterocyclic compounds and pharmaceutical formulations that can be used to treat bacterial infections. Also provided herein are methods of making and using the heterocyclic compounds and pharmaceutical formulations

    Molecular Basis of Substrate Recognition and Product Release by the <i>Klebsiella pneumoniae</i> Carbapenemase (KPC-2)

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    Carbapenem-resistant <i>Enterobacteriaceae</i> are resistant to most β-lactam antibiotics due to the production of the <i>Klebsiella pneumoniae</i> carbapenemase (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

    Heteroaryl Phosphonates as Noncovalent Inhibitors of Both Serine- and Metallocarbapenemases

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    Gram-negative pathogens expressing serine β-lactamases (SBLs) and metallo-β-lactamases (MBLs), especially those with carbapenemase activity, threaten the clinical utility of almost all β-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
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