| Autor: |
Alsenani TA; Department of Biochemistry, Case Western Reserve Universitygrid.67105.35 School of Medicine, Cleveland, Ohio, USA., Viviani SL; Department of Biochemistry, Case Western Reserve Universitygrid.67105.35 School of Medicine, Cleveland, Ohio, USA., Kumar V; Department of Biochemistry, Case Western Reserve Universitygrid.67105.35 School of Medicine, Cleveland, Ohio, USA., Taracila MA; Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio, USA.; Department of Medicine, Case Western Reserve Universitygrid.67105.35 School of Medicine, Cleveland, Ohio, USA., Bethel CR; Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio, USA., Barnes MD; Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio, USA., Papp-Wallace KM; Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio, USA., Shields RK; University of Pittsburghgrid.21925.3dgrid.471408.e, Department of Medicine, Division of Infectious Diseases, Pittsburgh, Pennsylvania, USA., Nguyen MH; University of Pittsburghgrid.21925.3dgrid.471408.e, Department of Medicine, Division of Infectious Diseases, Pittsburgh, Pennsylvania, USA., Clancy CJ; University of Pittsburghgrid.21925.3dgrid.471408.e, Department of Medicine, Division of Infectious Diseases, Pittsburgh, Pennsylvania, USA.; Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, Pennsylvania, USA., Bonomo RA; Department of Biochemistry, Case Western Reserve Universitygrid.67105.35 School of Medicine, Cleveland, Ohio, USA.; Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio, USA.; Department of Medicine, Case Western Reserve Universitygrid.67105.35 School of Medicine, Cleveland, Ohio, USA.; Department of Molecular Biology and Microbiology, Case Western Reserve Universitygrid.67105.35 School of Medicine, Cleveland, Ohio, USA.; Department of Pharmacology, Case Western Reserve Universitygrid.67105.35 School of Medicine, Cleveland, Ohio, USA.; Department of Proteomics and Bioinformatics, Case Western Reserve Universitygrid.67105.35 School of Medicine, Cleveland, Ohio, USA.; Senior Clinical Scientist Investigator, CWRU-Cleveland VAMC Center for Antimicrobial Resistance and Epidemiology (Case VA CARES), Cleveland, Ohio, USA., van den Akker F; Department of Biochemistry, Case Western Reserve Universitygrid.67105.35 School of Medicine, Cleveland, Ohio, USA. |
| Abstrakt: |
Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) present a global clinical threat, as these β-lactamases confer resistance to carbapenems and oxyimino-cephalosporins. Recent clinically identified KPC variants with substitutions at Ambler position D179, located in the Ω loop, are resistant to the β-lactam/β-lactamase inhibitor combination ceftazidime-avibactam, but susceptible to meropenem-vaborbactam. To gain insights into ceftazidime-avibactam resistance conferred by D179N/Y variants of KPC-2, crystal structures of these variants were determined. The D179N KPC-2 structure revealed that the change of the carboxyl to an amide moiety at position 179 disrupted the salt bridge with R164 present in wild-type KPC-2. Additional interactions were disrupted in the Ω loop, causing a decrease in the melting temperature. Shifts originating from N179 were also transmitted toward the active site, including ∼1-Å shifts of the deacylation water and interacting residue N170. The structure of the D179Y KPC-2 β-lactamase revealed more drastic changes, as this variant exhibited disorder of the Ω loop, with other flanking regions also being disordered. We postulate that the KPC-2 variants can accommodate ceftazidime because the Ω loop is displaced in D179Y or can be more readily displaced in D179N KPC-2. To understand why the β-lactamase inhibitor vaborbactam is less affected by the D179 variants than avibactam, we determined the crystal structure of D179N KPC-2 in complex with vaborbactam, which revealed wild-type KPC-2-like vaborbactam-active site interactions. Overall, the structural results regarding KPC-2 D179 variants revealed various degrees of destabilization of the Ω loop that contribute to ceftazidime-avibactam resistance, possible substrate-assisted catalysis of ceftazidime, and meropenem and meropenem-vaborbactam susceptibility. |
