RESUMEN
Awareness of the problem of antimicrobial resistance (AMR) has escalated and drug-resistant infections are named among the most urgent problems facing clinicians today. Our experiments here identify a transporter interactome and portray its essential function in acquisition of antimicrobial resistance. By exposing E. coli cells to consecutive increasing concentrations of the fluoroquinolone norfloxacin we generated in the laboratory highly resistant strains that carry multiple mutations, most of them identical to those identified in clinical isolates. With this experimental paradigm, we show that the MDTs function in a coordinated mode to provide an essential first-line defense mechanism, preventing the drug reaching lethal concentrations, until a number of stable efficient alterations occur that allow survival. Single-component efflux transporters remove the toxic compounds from the cytoplasm to the periplasmic space where TolC-dependent transporters expel them from the cell. We postulate a close interaction between the two types of transporters to prevent rapid leak of the hydrophobic substrates back into the cell. The findings change the prevalent concept that in Gram-negative bacteria a single multidrug transporter, AcrAB-TolC type, is responsible for the resistance. The concept of a functional interactome, the process of identification of its members, the elucidation of the nature of the interactions and its role in cell physiology will change the existing paradigms in the field. We anticipate that our work will have an impact on the present strategy searching for inhibitors of AcrAB-TolC as adjuvants of existing antibiotics and provide novel targets for this urgent undertaking.
Asunto(s)
Proteínas Portadoras/metabolismo , Farmacorresistencia Microbiana , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Medios de Cultivo , Escherichia coli/metabolismoRESUMEN
Transporters essential for neurotransmission in mammalian organisms and bacterial multidrug transporters involved in antibiotic resistance are evolutionarily related. To understand in more detail the evolutionary aspects of the transformation of a bacterial multidrug transporter to a mammalian neurotransporter and to learn about mechanisms in a milieu amenable for structural and biochemical studies, we identified, cloned, and partially characterized bacterial homologues of the rat vesicular monoamine transporter (rVMAT2). We performed preliminary biochemical characterization of one of them, Brevibacillus brevis monoamine transporter (BbMAT), from the bacterium B. brevis. BbMAT shares substrates with rVMAT2 and transports them in exchange with >1H(+), like the mammalian transporter. Here we present a homology model of BbMAT that has the standard major facilitator superfamily fold; that is, with two domains of six transmembrane helices each, related by 2-fold pseudosymmetry whose axis runs normal to the membrane and between the two halves. The model predicts that four carboxyl residues, a histidine, and an arginine are located in the transmembrane segments. We show here that two of the carboxyls are conserved, equivalent to the corresponding ones in rVMAT2, and are essential for H(+)-coupled transport. We conclude that BbMAT provides an excellent experimental paradigm for the study of its mammalian counterparts and bacterial multidrug transporters.
Asunto(s)
Proteínas Bacterianas/química , Monoaminas Biogénicas/química , Brevibacillus/química , Proteínas Portadoras/química , Proteínas de Transporte Vesicular de Monoaminas/química , Secuencia de Aminoácidos , Animales , Arginina/química , Arginina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Monoaminas Biogénicas/metabolismo , Brevibacillus/genética , Brevibacillus/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Farmacorresistencia Bacteriana , Escherichia coli/genética , Escherichia coli/metabolismo , Evolución Molecular , Expresión Génica , Histidina/química , Histidina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología Estructural de Proteína , Relación Estructura-Actividad , Especificidad por Sustrato , Transmisión Sináptica/fisiología , Proteínas de Transporte Vesicular de Monoaminas/genética , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
Vesicular monoamine transporter 2 (VMAT2) catalyzes transport of monoamines into storage vesicles in a process that involves exchange of the charged monoamine with two protons. VMAT2 is a member of the DHA12 family of multidrug transporters that belongs to the major facilitator superfamily (MFS) of secondary transporters. Here we present a homology model of VMAT2, which has the standard MFS fold, that is, with two domains of six transmembrane helices each which are related by twofold pseudosymmetry and whose axis runs normal to the membrane and between the two halves. Demonstration of the essential role of a membrane-embedded glutamate and confirmation of the existence of a hydrogen bond probably involved in proton transport provide experimental evidence that validates some of the predictions inherent to the model. Moreover, we show the essential role of residues at two anchor points between the two bundles. These residues appear to function as molecular hinge points about which the two six transmembrane-helix bundles flex and straighten to open and close the pathways on either side of the membrane as required for transport. Polar residues that create a hydrogen bond cluster form one of the anchor points of VMAT2. The other results from hydrophobic interactions. Residues at the anchor points are strongly conserved in other MFS transporters in one way or another, suggesting that interactions at these locations will be critical in most, if not all, MFS transporters.