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Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase

Abstract

Carbapenems, 'last-resort' β-lactam antibiotics, are inactivated by zinc-dependent metallo-β-lactamases (MBLs). The host innate immune response withholds nutrient metal ions from microbial pathogens by releasing metal-chelating proteins such as calprotectin. We show that metal sequestration is detrimental for the accumulation of MBLs in the bacterial periplasm, because those enzymes are readily degraded in their nonmetallated form. However, the New Delhi metallo-β-lactamase (NDM-1) can persist under conditions of metal depletion. NDM-1 is a lipidated protein that anchors to the outer membrane of Gram-negative bacteria. Membrane anchoring contributes to the unusual stability of NDM-1 and favors secretion of this enzyme in outer-membrane vesicles (OMVs). OMVs containing NDM-1 can protect nearby populations of bacteria from otherwise lethal antibiotic levels, and OMVs from clinical pathogens expressing NDM-1 can carry this MBL and the blaNDM gene. We show that protein export into OMVs can be targeted, providing possibilities of new antibacterial therapeutic strategies.

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Figure 1: Zn(II) deprivation reduces bacterial antibiotic resistance and causes degradation of MBLs in the Escherichia coli periplasm.
Figure 2: The cellular localization of MBLs is determined by their N-terminal region.
Figure 3: Membrane anchoring protects NDM-1 from degradation upon Zn(II) deprivation.
Figure 4: Membrane-anchoring favors secretion of NDM-1 into OMVs.
Figure 5: NDM-1-carrying OMVs provide carbapenem resistance to susceptible bacteria.
Figure 6: Membrane anchoring assures long-term survival of NDM-1.

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Acknowledgements

We thank J. Spencer (University of Bristol), H. Menzella (University of Rosario) and R. Rasia (IBR, CONICET-UNR) for providing plasmids pET26-blaNDM-1, pTGR11 and pET28-TEV, respectively, A. Corso (Administración Nacional de Laboratorios e Instituos de Salud) for providing clinical strains, and A. Viale (IBR) for GroEL antibodies. This research was supported by grants from the US National Institutes of Health (NIH; R01AI100560) to A.J.V. and R.A.B., and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) to A.J.V. R.A.B. acknowledges support from NIH under award numbers R01AI072219, R01AI063517 and R01AI100560, and funds and/or facilities provided by the Louis Stokes Cleveland Department of Veterans Affairs Medical Center and the VISN 10 Geriatric Research, Education and Clinical Care Center (VISN 10) of the Department of Veterans Affairs. E.M.N. thanks support from the Kinship Foundation (Searle Scholars Program) and MIT Department of Chemistry.

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L.J.G. and G.B. performed the microbiological, molecular biology and biochemical experiments. T.G.N. purified calprotectin. L.J.G., G.B., T.G.N., E.M.N., R.A.B. and A.J.V. analyzed and discussed data. L.J.G., G.B., R.A.B. and A.J.V. wrote the paper, and all authors discussed the results and commented on the manuscript.

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Correspondence to Alejandro J Vila.

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González, L., Bahr, G., Nakashige, T. et al. Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase. Nat Chem Biol 12, 516–522 (2016). https://doi.org/10.1038/nchembio.2083

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