Structure, function, and regulation of bacterial efflux pumps

Kumar, Nitin
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The continued inappropriate use of antibiotics over the years has led to the development of resistance mechanisms in microorganisms against multiple drugs. One of the most important resistance mechanisms in Gram-negative bacteria is the usage of membrane proteins that act as efflux pumps and help in recognizing and exporting a broad range of drugs from the cell. This dissertation mainly focuses on the structure, function, and regulation of these bacterial efflux pumps. Using X-ray crystallography, we have studied the Mycobacterium tuberculois transcriptional regulator Rv1219c, which controls the expression of the ABC superfamily multidrug efflux pump Rv1217c-Rv1218c. We also quantified the binding of Rv1219c with different ligands and DNA using fluorescence polarization and isothermal titration calorimetry (ITC), respectively. An inner membrane protein HpnN belonging to the RND family in Burkholderia multivorans, is known to be involved in transporting certain pentacyclic lipids called hopanoids towards the outer membrane. To elucidate the structural basis of hopanoid transport, we have determined the crystal structures of HpnN. Further, by using in vivo studies we were able to confirm that HpnN mediates drug resistance in B. multivorans by transporting hopanoids, hence creating a protective layer in the outer membrane. In order to understand the structure and transport dynamics of multidrug efflux pump CmeB in Campylobacter jejuni, also belonging to the resistance nodulation-division (RND) family, we used X-ray crystallography and single-molecule fluorescence resonance energy transfer (sm-FRET) imaging. Using sm-FRET, we were able to see that one of the crystallized forms of CmeB follows a mechanism where each protomer in an RND trimer might be able to work independently, instead of the previously proposed “rotating mechanism”.

Cloning, Crystallization, Expression, Protein, Purification, X-ray