Characterization of lipid-bound Metallo-β-Lactamase NDM-1

BAHR, GUILLERMO; VILA, ALEJANDRO J.; GONZÁLEZ, LISANDRO J.

San Miguel de Tucumán

Congreso; III Latin American Federation of Biophysical Societies (LAFeBS) ? IX IberoAmerican Congress of Biophysics ? XLV Reunion Anual SAB 2016; 2016

Sociedad Argentina de Biofísica

The clinical efficacy ofcarbapenems, last resort antibiotics, is threatened by the global disseminationof metallo-β-lactamases (MBLs). The genes encoding these Zn(II)-dependentenzymes are frequently associated with mobile genetic elements, acceleratingtheir transfer among pathogens, and there is no therapeutic inhibitor capableof abolishing their activity. Among MBLs, NDM-1 has shown the fastest andlargest geographical spread, having already been detected in more than 70countries worldwide less than a decade after its discovery.Unlike all other characterized MBLs,which are soluble periplasmic proteins, NDM-1 is covalently bound to a lipidgroup that anchors the enzyme to the inner leaflet of the outer membrane ofGram-negative bacteria. We have recently shown that membrane-anchoringstabilizes NDM-1 within the bacterial cell and allows it to endure low-Zn(II)availability conditions, similar to those encountered during pathogenesis.Furthermore, we demonstrated that the membrane localization enhances secretion ofNDM-1 within outer membrane vesicles in Gram-negative bacteria. These vesiclespossess carbapenemase activity, and are able to protect nearby populations ofotherwise antibiotic-susceptible cells. We propose that membrane anchoring ofNDM-1 constitutes an evolutionary adaptation contributing to its clinicalsuccess.Up to date, all in vitro biochemical and biophysicalcharacterizations of NDM-1 have been performed with the enzyme in a truncatedsoluble form, overlooking its native state and cellular localization. Here wereport the overexpression, solubilization from membranes and purification oflipidated NDM-1, and the initial characterization of this enzyme in detergentmicelles.