RESUMEN
Invasive cardiovascular procedures which include heart transplantations, congenital heart surgery, coronary artery bypass grafts, cardiac valve repair and replacement, and interventional cardiac electrophysiology procedures represent common mechanisms to treat a variety of cardiovascular diseases across the globe. The majority of these invasive approaches employ antibiotics as a regular and obligatory feature of the invasive procedure. Although the growing incidence of bacterial resistance to currently used antibiotics threatens to curtail the use of all interventional surgical techniques, it remains an underappreciated threat within the arsenal of cardiovascular therapies. It is reasonable to expect that the continued overuse of antibiotics and the frequent management of coronavirus disease 2019 (COVID-19) infected patients with high doses of antibiotics will inevitably accentuate the rise of multidrug resistance. The purpose of this article is to heighten awareness of the role of bacterial infections in cardiovascular disease, the use of antibiotics in today's cardiovascular surgical theaters, the threat facing cardiovascular surgery should multidrug resistance continue to rise unabated, and the development of new antibiotic platforms to solve this problem.
Asunto(s)
Infecciones Bacterianas , COVID-19 , Bacterias , Resistencia a Múltiples Medicamentos , Humanos , SARS-CoV-2RESUMEN
The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is the major Na+ pump in aerobic pathogens such as Vibrio cholerae. The interface between two of the NQR subunits, NqrB and NqrD, has been proposed to harbor a binding site for inhibitors of Na+-NQR. While the mechanisms underlying Na+-NQR function and inhibition remain underinvestigated, their clarification would facilitate the design of compounds suitable for clinical use against pathogens containing Na+-NQR. An in silico model of the NqrB-D interface suitable for use in molecular dynamics simulations was successfully constructed. A combination of algorithmic and manual methods was used to reconstruct portions of the two subunits unresolved in the published crystal structure and validate the resulting structure. Hardware and software optimizations that improved the efficiency of the simulation were considered and tested. The geometry of the reconstructed complex compared favorably to the published V. cholerae Na+-NQR crystal structure. Results from one 1 µs, three 150 ns and two 50 ns molecular dynamics simulations illustrated the stability of the system and defined the limitations of this model. When placed in a lipid bilayer under periodic boundary conditions, the reconstructed complex was completely stable for at least 1 µs. However, the NqrB-D interface underwent a non-physiological transition after 350 ns.
Asunto(s)
Proteínas Bacterianas/química , Simulación de Dinámica Molecular , Complejos Multienzimáticos/química , NAD(P)H Deshidrogenasa (Quinona)/química , Vibrio cholerae/enzimología , Proteínas Bacterianas/genética , Complejos Multienzimáticos/genética , NAD(P)H Deshidrogenasa (Quinona)/genética , Vibrio cholerae/genéticaRESUMEN
The genome of Vibrio cholerae contains three structural genes for the NhaP-type cation-proton antiporter paralogues, Vc-NhaP1, Vc-NhaP2, and Vc-NhaP3, mediating exchange of K+ and or Na+ for protons across the membrane. Based on phenotypic analysis of chromosomal Vc-NhaP1, Vc-NhaP2, and Vc-NhaP3 triple deletion mutants, we suggest that Vc-NhaP paralogues are primarily K+/H+ antiporters and might play a role in the acid tolerance response of V. cholerae as it passes through the gastric acid barrier of the stomach. Comparison of the biochemical properties of Vc-NhaP isoforms revealed that Vc-NhaP2 was the most active among all three paralogues. Therefore, the Vc-NhaP2 antiporter is a plausible therapeutic target for developing novel inhibitors targeting these ion exchangers. Our structural and mutational analysis of Vc-NhaP2 identified a putative cation-binding pocket formed by antiparallel extended regions of two transmembrane segments (TMSs V and XII) along with TMS VI. Molecular dynamics simulations suggested that the flexibility of TMSs V and XII is crucial for intramolecular conformational events in Vc-NhaP2. In this study, we developed putative Vc-NhaP2 inhibitors from amiloride analogs. Molecular docking of the modified amiloride analogs revealed promising binding properties. The four selected drugs potentially interacted with functionally important amino acid residues located on the cytoplasmic side of TMS VI, the extended chain region of TMSs V and XII, and the loop region between TMSs VIIII and IX. Molecular dynamics simulations revealed that binding of the selected drugs can potentially destabilize Vc-NhaP2 and alter the flexibility of functionally important TMS VI. This work presents the utility of in silico approaches for the rational identification of potential targets and drugs that could target NhaP2 cation proton antiporters to control V. cholerae. The goal was to identify potential drugs that could be validated in future experiments.
Asunto(s)
Vibrio cholerae , Amilorida , Antiportadores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cationes/metabolismo , Simulación del Acoplamiento Molecular , Protones , Intercambiadores de Sodio-Hidrógeno/genética , Vibrio cholerae/genéticaRESUMEN
The transmembrane K+/H+ antiporters of NhaP type of Vibrio cholerae (Vc-NhaP1, 2, and 3) are critical for maintenance of K+ homeostasis in the cytoplasm. The entire functional NhaP group is indispensable for the survival of V. cholerae at low pHs suggesting their possible role in the acid tolerance response (ATR) of V. cholerae. Our findings suggest that the Vc-NhaP123 group, and especially its major component, Vc-NhaP2, might be a promising target for the development of novel antimicrobials by narrowly targeting V. cholerae and other NhaP-expressing pathogens. On the basis of Vc-NhaP2 in silico structure modeling, Molecular Dynamics Simulations, and extensive mutagenesis studies, we suggest that the ion-motive module of Vc-NhaP2 is comprised of two functional regions: (i) a putative cation-binding pocket that is formed by antiparallel unfolded regions of two transmembrane segments (TMSs V/XII) crossing each other in the middle of the membrane, known as the NhaA fold; and (ii) a cluster of amino acids determining the ion selectivity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Antiportadores de Potasio-Hidrógeno/metabolismo , Vibrio cholerae/metabolismo , Proteínas Bacterianas/química , Cólera/microbiología , Humanos , Simulación de Dinámica Molecular , Antiportadores de Potasio-Hidrógeno/química , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Vibrio cholerae/químicaRESUMEN
The genome of Vibrio cholerae encodes three cation-proton antiporters of NhaP-type, Vc-NhaP1, 2, and 3. To examine physiological roles of Vc-NhaP antiporters, triple ΔnhaP1ΔnhaP2ΔnhaP3 and single ΔnhaP3 deletion mutants of V. cholerae were constructed and characterized. Vc-NhaP3 was, for the first time, cloned and biochemically characterized. Activity measurements on the inside-out membrane vesicle experimental model defined Vc-NhaP3 as a potassium-specific cation-proton antiporter. While elimination of functional Vc-NhaP3 resulted in only minor growth defect in potassium-rich medium at pH 6.0, the triple Vc-NhaP mutant demonstrated severe growth defects at both low and high [K+] at pH 6.0 and failed to grow at high [K+] in mildly alkaline (pH 8.0 and 8.5) media, as well. Expressed from a plasmid, neither of the Vc-NhaP paralogues was able to complement the severe potassium-sensitive phenotype of the triple deletion mutant completely. Vc-NhaP1 provided much better complementation at acidic pH compared to Vc-NhaP2, despite the fact that Vc-NhaP2 showed much higher antiport activity in sub-bacterial vesicles. In mildly alkaline pH only Vc-NhaP2 complemented the potassium-sensitive phenotype of the triple deletion mutant. Taken together, these data suggest that in vivo all three isoforms operate in concert, contributing to K+ resistance of V. cholerae. We suggest that the Vc-NhaP paralogue group might play a role in passing gastric acid barrier by ingested V. cholerae cells.
Asunto(s)
Antiportadores/genética , Antiportadores/metabolismo , Vibrio cholerae/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Medios de Cultivo/química , Eliminación de Gen , Concentración de Iones de Hidrógeno , Potasio/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMEN
The search for new nontraditional targets is a high priority in antibiotic design today. Bacterial membrane energetics based on sodium ion circulation offers potential alternative targets. The present work identifies the Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR), a key respiratory enzyme in many microbial pathogens, as indispensible for the Chlamydia trachomatis infectious process. Infection by Chlamydia trachomatis significantly increased first H+ and then Na+ levels within the host mammalian cell. A newly designed furanone Na+-NQR inhibitor, PEG-2S, blocked the changes in both H+ and Na+ levels induced by Chlamydia trachomatis infection. It also inhibited intracellular proliferation of Chlamydia trachomatis with a half-minimal inhibitory concentration in the submicromolar range but did not affect the viability of mammalian cells or bacterial species representing benign intestinal microflora. At low nanomolar concentrations (IC50 value = 1.76 nmol/L), PEG-2S inhibited the Na+-NQR activity in sub-bacterial membrane vesicles isolated from Vibrio cholerae. Taken together, these results show, for the first time, that Na+-NQR is critical for the bacterial infectious process and is susceptible to a precisely targeted bactericidal compound in situ. The obtained data have immediate relevance for many different diseases caused by pathogenic bacteria that rely on Na+-NQR activity for growth, including sexually transmitted, pulmonary, oral, gum, and ocular infections.
Asunto(s)
Antibacterianos/farmacología , Chlamydia trachomatis/efectos de los fármacos , Diseño de Fármacos , Furanos/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Furanos/síntesis química , Furanos/química , Células HEK293 , Células HeLa , Homeostasis/efectos de los fármacos , Humanos , NADH NADPH Oxidorreductasas/antagonistas & inhibidoresRESUMEN
To examine the possible physiological significance of Mrp, a multi-subunit cation/proton antiporter from Vibrio cholerae, a chromosomal deletion Δmrp of V. cholerae was constructed and characterized. The resulting mutant showed a consistent early growth defect in LB broth that became more evident at elevated pH of the growth medium and increasing Na+ or K+ loads. After 24 h incubation, these differences disappeared likely due to the concerted effort of other cation pumps in the mrp mutant. Phenotype MicroArray analyses revealed an unexpected systematic defect in nitrogen utilization in the Δmrp mutant that was complemented by using the mrpA'-F operon on an arabinose-inducible expression vector. Deletion of the mrp operon also led to hypermotility, observable on LB and M9 semi-solid agar. Surprisingly, Δmrp mutation resulted in wild-type biofilm formation in M9 despite a growth defect but the reverse was true in LB. Furthermore, the Δmrp strain exhibited higher susceptibility to amphiphilic anions. These pleiotropic phenotypes of the Δmrp mutant demonstrate how the chemiosmotic activity of Mrp contributes to the survival potential of V. cholerae despite the presence of an extended battery of cation/proton antiporters of varying ion selectivity and pH profile operating in the same membrane.
Asunto(s)
Antiportadores/metabolismo , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/genética , Eliminación de Gen , Vibrio cholerae/metabolismo , Antiportadores/genética , Proteínas Bacterianas/genética , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Concentración de Iones de Hidrógeno , Operón , Potasio/metabolismo , Sodio/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/crecimiento & desarrolloRESUMEN
In this work, we report the phenotypic and biochemical effects of deleting the C-terminal cytoplasmic portion of the NhaP2 cation/proton antiporter from Vibrio cholerae. While the deletion changed neither the expression nor targeting of the Vc-NhaP2 in an antiporter-less Escherichia coli strain, it resulted in a changed sensitivity of the host to sodium ions at neutral pH, indicating an altered Na(+) transport through the truncated variant. When assayed in inside-out sub-bacterial vesicles, the truncation was found to result in greatly reduced K(+)/H(+) and Na(+)/H(+) antiport activity at all pH values tested and a greater than fivefold decrease in the affinity for K(+) (measured as the apparent K m) at pH 7.5. Being expressed in trans in a strain of V. cholerae bearing a chromosomal nhaP2 deletion, the truncated nhaP2 gene was able to complement its inability to grow in potassium-rich medium at pH 6.0. Thus the residual K(+)/H(+) antiport activity associated with the truncated Vc-NhaP2 was still sufficient to protect cells from an over-accumulation of K(+) ions in the cytoplasm. The presented data suggest that while the cytoplasmic portion of Vc-NhaP2 is not involved in ion translocation directly, it is necessary for optimal activity and substrate binding of the Vc-NhaP2 antiporter.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cationes/metabolismo , Citoplasma/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Vibrio cholerae/metabolismo , Proteínas Bacterianas/genética , Clonación Molecular/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Potasio/metabolismo , Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Vibrio cholerae/genéticaRESUMEN
Na(+)/H(+) antiporters are ubiquitous membrane proteins that play a central role in the ion homeostasis of cells. In this study, we examined the possible role of Na(+)/H(+) antiport in Yersinia pestis virulence and found that Y. pestis strains lacking the major Na(+)/H(+) antiporters, NhaA and NhaB, are completely attenuated in an in vivo model of plague. The Y. pestis derivative strain lacking the nhaA and nhaB genes showed markedly decreased survival in blood and blood serum ex vivo. Complementation of either nhaA or nhaB in trans restored the survival of the Y. pestis nhaA nhaB double deletion mutant in blood. The nhaA nhaB double deletion mutant also showed inhibited growth in an artificial serum medium, Opti-MEM, and a rich LB-based medium with Na(+) levels and pH values similar to those for blood. Taken together, these data strongly suggest that intact Na(+)/H(+) antiport is indispensable for the survival of Y. pestis in the bloodstreams of infected animals and thus might be regarded as a promising noncanonical drug target for infections caused by Y. pestis and possibly for those caused by other blood-borne bacterial pathogens.
Asunto(s)
Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Virulencia/genética , Yersinia pestis/genética , Yersinia pestis/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Femenino , Ratones , Peste/genética , Peste/metabolismo , Peste/microbiología , Eliminación de Secuencia/genética , Ovinos/sangre , Ovinos/microbiologíaRESUMEN
Vibrio cholerae has adapted to a wide range of salinity, pH and osmotic conditions, enabling it to survive passage through the host and persist in the environment. Among the many proteins responsible for bacterial survival under these diverse conditions, we have identified Vc-NhaP1 as a K(+)(Na(+))/H(+) antiporter essential for V. cholerae growth at low environmental pH. Deletion of the V. cholerae nhaP1 gene caused growth inhibition when external potassium was either limited (100 mM and below) or in excess (400 mM and above). This growth defect was most apparent at mid-exponential phase, after 4-6 h of culture. Using a pH-sensitive GFP, cytosolic pH was shown to be dependent on K(+) in acidic external conditions in a Vc-NhaP1-dependent manner. When functionally expressed in an antiporterless Escherichia coli strain and assayed in everted membrane vesicles, Vc-NhaP1 operated as an electroneutral alkali cation/proton antiporter, exchanging K(+) or Na(+) ions for H(+) within a broad pH range (7.25-9.0). These data establish the putative V. cholerae NhaP1 protein as a functional K(+)(Na(+))/H(+) antiporter of the CPA1 family that is required for bacterial pH homeostasis and growth in an acidic environment.
Asunto(s)
Proteínas Bacterianas/metabolismo , Antiportadores de Potasio-Hidrógeno/metabolismo , Potasio/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Vibrio cholerae/crecimiento & desarrollo , Proteínas Bacterianas/genética , Clonación Molecular , Citoplasma/fisiología , Eliminación de Gen , Homeostasis , Concentración de Iones de Hidrógeno , Antiportadores de Potasio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/genética , Vibrio cholerae/genética , Vibrio cholerae/fisiologíaRESUMEN
Na+/H+ antiporters are integral membrane proteins that exchange Na+ for H+ across the cytoplasmic or organellar membranes of virtually all living cells. They are essential for control of cellular pH, volume homeostasis, and regulation of Na+ levels. Na+/H+ antiporters have become increasingly characterized and are now becoming important drug targets. The recently identified NhaP family of Na+/H+ antiporters, from the CPA1 superfamily, contains proteins with a surprisingly broad collective range of transported cations, exchanging protons for alkali cations such as Na+, Li+, K+, or Rb+ as well as for Ca2+ and, possibly, NH4+. Questions about ion selectivity and the physiological impact of each particular NhaP antiporter are far from trivial. For example, Vc-NhaP2 from Vibrio cholerae has recently been shown to function in vivo as a specific K+/H+ antiporter while retaining the ability to exchange H+ for Na+ and bind (but not exchange with H+) Li+ in a competitive manner. These and other findings reviewed in this communication make antiporters of the NhaP type attractive systems to study intimate molecular mechanisms of cation exchange. In an evolutionary perspective, the NhaP family seems to be a phylogenetic entity undergoing active divergent evolution. In this minireview, to rationalize peculiarities of the cation specificity in the NhaP family, the "size-exclusion principle" and the idea of "ligand shading" are discussed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cationes/metabolismo , Protones , Intercambiadores de Sodio-Hidrógeno/metabolismo , Vibrio cholerae/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Evolución Molecular , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Filogenia , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/clasificación , Intercambiadores de Sodio-Hidrógeno/genética , Vibrio cholerae/químicaRESUMEN
The existence of bacterial K(+)/H(+) antiporters that prevent the overaccumulation of potassium in the cytoplasm was predicted by Peter Mitchell almost 50 years ago. The importance of K(+)/H(+) antiport for bacterial physiology is widely recognized, but its molecular mechanisms remain underinvestigated. Here, we demonstrate that a putative Na(+)/H(+) antiporter, Vc-NhaP2, protects cells of Vibrio cholerae growing at pH 6.0 from high concentrations of external K(+). Resistance of V. cholerae to Na(+) was found to be independent of Vc-NhaP2. When assayed in inside-out membrane vesicles derived from antiporter-deficient Escherichia coli, Vc-NhaP2 catalyzed the electroneutral K(+)(Rb(+))/H(+) exchange with a pH optimum of approximately 7.75 with an apparent K(m) for K(+) of 1.62 mM. In the absence of K(+), it exhibited Na(+)/H(+) antiport, albeit rather weakly. Interestingly, while Vc-NhaP2 cannot exchange Li(+) for protons, elimination of functional Vc-NhaP2 resulted in a significantly higher Li(+) resistance of V. cholerae cells growing at pH 6.0, suggesting the possibility of Vc-NhaP2-mediated Li(+)/K(+) antiport. The peculiar cation specificity of Vc-NhaP2 and the presence of its two additional paralogues in the same genome make this transporter an attractive model for detailed analysis of the structural determinants of the substrate specificity in alkali cation exchangers.
Asunto(s)
Proteínas Bacterianas/metabolismo , Hidrógeno/metabolismo , Potasio/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Vibrio cholerae/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Biocatálisis , Clonación Molecular , Concentración de Iones de Hidrógeno , Homología de Secuencia de Aminoácido , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/aislamiento & purificación , Especificidad por SustratoRESUMEN
All living cells routinely expel Na(+) ions, maintaining lower concentration of Na(+) in the cytoplasm than in the surrounding milieu. In the vast majority of bacteria, as well as in mitochondria and chloroplasts, export of Na(+) occurs at the expense of the proton-motive force. Some bacteria, however, possess primary generators of the transmembrane electrochemical gradient of Na(+) (sodium-motive force). These primary Na(+) pumps have been traditionally seen as adaptations to high external pH or to high temperature. Subsequent studies revealed, however, the mechanisms for primary sodium pumping in a variety of non-extremophiles, such as marine bacteria and certain bacterial pathogens. Further, many alkaliphiles and hyperthermophiles were shown to rely on H(+), not Na(+), as the coupling ion. We review here the recent progress in understanding the role of sodium-motive force, including (i) the conclusion on evolutionary primacy of the sodium-motive force as energy intermediate, (ii) the mechanisms, evolutionary advantages and limitations of switching from Na(+) to H(+) as the coupling ion, and (iii) the possible reasons why certain pathogenic bacteria still rely on the sodium-motive force.
Asunto(s)
Sodio/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Sitios de Unión , Transporte Biológico Activo , Metabolismo Energético , Modelos Moleculares , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón/metabolismo , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Escherichia coli is a convenient host for the expression of proteins, but the heterologous production of large membrane protein complexes often is hampered by the lack of specific accessory genes required for membrane insertion or cofactor assembly. In this study we introduce the non-pathogenic and fast-growing Vibrio natriegens as a suitable expression host for membrane-bound proteins from Vibrio cholerae. We achieved production of the primary Na+ pump, the NADH:quinone oxidoreductase (NQR), from V. cholerae in an active state, as indicated by increased overall NADH:quinone oxidoreduction activity of membranes from the transformed V. natriegens, and the sensitivity toward Ag+, a specific inhibitor of the NQR. Complete assembly of V. cholerae NQR expressed in V. natriegens was demonstrated by BN PAGE followed by activity staining. The secondary transport system Mrp from V. cholerae, another membrane-bound multisubunit complex, was also produced in V. natriegens in a functional state, as demonstrated by in vivo Li+ transport. V. natriegens is a promising expression host for the production of membrane protein complexes from Gram-negative pathogens.
RESUMEN
The recent breakthrough in structural studies on Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae creates a perspective for the systematic design of inhibitors for this unique enzyme, which is the major Na+ pump in aerobic pathogens. Widespread distribution of Na+-NQR among pathogenic species, its key role in energy metabolism, its relation to virulence in different species as well as its absence in eukaryotic cells makes this enzyme especially attractive as a target for prospective antibiotics. In this review, the major biochemical, physiological and, especially, the pharmacological aspects of Na+-NQR are discussed to assess its 'target potential' for drug development. A comparison to other primary bacterial Na+ pumps supports the contention that NQR is a first rate prospective target for a new generation of antimicrobials. A new, narrowly targeted furanone inhibitor of NQR designed in our group is presented as a molecular platform for the development of anti-NQR remedies.
Asunto(s)
Complejo I de Transporte de Electrón/antagonistas & inhibidores , Complejo I de Transporte de Electrón/metabolismo , Vibrio cholerae/enzimología , Antibacterianos/metabolismo , Sistemas de Liberación de Medicamentos , Descubrimiento de Drogas , Inhibidores Enzimáticos/metabolismoRESUMEN
The Vc-NhaD is an Na(+)/H(+) antiporter from Vibrio cholerae belonging to a new family of bacterial Na(+)/H(+) antiporters, the NhaD family. In the present work we mutagenized five conserved Asp and Glu residues and one conserved Thr residue to Ala in order to identify amino acids that are critical for the antiport activity. All mutations fall into two distinct groups: (i) four variants, Glu(100)Ala, Glu(251)Ala, Glu(342)Ala, and Asp(393)Ala, did not abolish antiport activity but shifted the pH optimum to more alkaline pH, and (ii) variants Asp(344)Ala, Asp(344)Asn, and Thr(345)Ala caused a complete loss of both Na(+)/H(+) and Li(+)/H(+) antiport activity whereas the Asp(344)Glu variant exhibited reduced Na(+)/H(+) and Li(+)/H(+) antiport activity. This is the first mutational analysis of the antiporter of NhaD type and the first demonstration of Thr residue being indispensable for Na(+)/H(+) antiport. We discuss the possible role of Asp(344) and Thr(345) in the functioning of Vc-NhaD.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/metabolismo , Vibrio cholerae/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Ácido Aspártico/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión/genética , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Genes Bacterianos , Transporte Iónico , Litio/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Treonina/química , Transformación Genética , Vibrio cholerae/genética , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismoRESUMEN
The diuretic drug amiloride and its numerous derivatives are competitive inhibitors of mammalian Na(+)/H(+) antiporters and other eukaryotic antiporters. Most prokaryotic antiporters, including the major NhaA family of enterobacteria, are resistant to these compounds. We show that 2-aminoperimidine (AP), a guanidine-containing naphthalene derivative with some similarity to amiloride, acts as a specific inhibitor of NhaA from Escherichia coli. Similar concentrations (IC(50) of 0.9 muM) inhibit the proton motive force dependent Na(+)(Li(+))/H(+) exchange reaction in inside-out sub-bacterial vesicles (at 10 mM NaCl, pH 8) as well as the initial rate of (22)Na(+)/Na(+) exchange mediated by pure NhaA in proteoliposomes. The inhibitor is specific to NhaA type antiporters, so AP is a new tool to study the mechanism and roles of NhaA antiporters of enterobacteria as well as the molecular basis of inhibition by an amiloride-like compound.
Asunto(s)
Proteínas de Escherichia coli/antagonistas & inhibidores , Quinazolinas/farmacología , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Amilorida/análogos & derivados , Membrana Celular/fisiología , Proteolípidos/efectos de los fármacos , Proteolípidos/fisiología , Quinazolinas/química , Sodio/metabolismoRESUMEN
The mrp operon from Vibrio cholerae encoding a putative multisubunit Na(+)/H(+) antiporter was cloned and functionally expressed in the antiporter-deficient strain of Escherichia coli EP432. Cells of EP432 expressing Vc-Mrp exhibited resistance to Na(+) and Li(+) as well as to natural bile salts such as sodium cholate and taurocholate. When assayed in everted membrane vesicles of the E. coli EP432 host, Vc-Mrp had sufficiently high antiport activity to facilitate the first extensive analysis of Mrp system from a Gram-negative bacterium encoded by a group 2 mrp operon. Vc-Mrp was found to exchange protons for Li(+), Na(+), and K(+) ions in pH-dependent manner with maximal activity at pH 9.0-9.5. Exchange was electrogenic (more than one H(+) translocated per cation moved in opposite direction). The apparent K(m) at pH 9.0 was 1.08, 1.30, and 68.5 mM for Li(+), Na(+), and K(+), respectively. Kinetic analyses suggested that Vc-Mrp operates in a binding exchange mode with all cations and protons competing for binding to the antiporter. The robust ion antiport activity of Vc-Mrp in sub-bacterial vesicles and its effect on bile resistance of the heterologous host make Vc-Mrp an attractive experimental model for the further studies of biochemistry and physiology of Mrp systems.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ácidos y Sales Biliares/metabolismo , Cationes , Protones , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Vibrio cholerae/metabolismo , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Litio/metabolismo , Membranas/metabolismo , Sodio/metabolismo , Vibrio cholerae/genéticaRESUMEN
Vc-NhaD is a Na(+)/H(+) antiporter from Vibrio cholerae with a sharp maximum of activity at pH approximately 8.0. NhaD homologues are present in many bacteria as well as in higher plants. However, very little is known about structure-function relations in NhaD-type antiporters. In this work 14 conserved polar residues associated with putative transmembrane segments of Vc-NhaD have been screened for their possible role in the ion translocation and pH regulation of Vc-NhaD. Substitutions S150A, D154G, N155A, N189A, D199A, T201A, T202A, S389A, N394G, S428A, and S431A completely abolished the Vc-NhaD-mediated Na(+)-dependent H(+) transfer in inside-out membrane vesicles. Substitutions T157A and S428A caused a significant increase of apparent K(m) values for alkali cations, with the K(m) for Li(+) elevated more than that for Na(+), indicating that Thr-157 and Ser-428 are involved in alkali cation binding/translocation. Of six conserved His residues, mutation of only His-93 and His-210 affected the Na(+)(Li(+))/H(+) antiport, resulting in an acidic shift of its pH profile, whereas H93A also caused a 7-fold increase of apparent K(m) for Na(+) without affecting the K(m) for Li(+). These data suggest that side chains of His-93 and His-210 are involved in proton binding and that His-93 also contributes to the binding of Na ions during the catalytic cycle. These 15 residues are clustered in three distinct groups, two located at opposite sides of the membrane, presumably facilitating the access of substrate ions to the third group, a putative catalytic site in the middle of lipid bilayer. The distribution of these key residues in Vc-NhaD molecule also suggests that transmembrane segments IV, V, VI, X, XI, and XII are situated close to one another, creating a transmembrane relay of charged/polar residues involved in the attraction, coordination, and translocation of transported cations.