RESUMEN
Pseudomonas aeruginosa is a leading cause of nosocomial infections worldwide and notorious for its broad-spectrum resistance to antibiotics. A key mechanism that provides extensive resistance to ß-lactam antibiotics is the inducible expression of AmpC ß-lactamase. Recently, a number of clinical isolates expressing mutated forms of AmpC have been found to be clinically resistant to the antipseudomonal ß-lactam-ß-lactamase inhibitor (BLI) combinations ceftolozane-tazobactam and ceftazidime-avibactam. Here, we compare the enzymatic activity of wild-type (WT) AmpC from PAO1 to those of four of these reported AmpC mutants, bearing mutations E247K (a change of E to K at position 247), G183D, T96I, and ΔG229-E247 (a deletion from position 229 to 247), to gain detailed insights into how these mutations allow the circumvention of these clinically vital antibiotic-inhibitor combinations. We found that these mutations exert a 2-fold effect on the catalytic cycle of AmpC. First, they reduce the stability of the enzyme, thereby increasing its flexibility. This appears to increase the rate of deacylation of the enzyme-bound ß-lactam, resulting in greater catalytic efficiencies toward ceftolozane and ceftazidime. Second, these mutations reduce the affinity of avibactam for AmpC by increasing the apparent activation barrier of the enzyme acylation step. This does not influence the catalytic turnover of ceftolozane and ceftazidime significantly, as deacylation is the rate-limiting step for the breakdown of these antibiotic substrates. It is remarkable that these mutations enhance the catalytic efficiency of AmpC toward ceftolozane and ceftazidime while simultaneously reducing susceptibility to inhibition by avibactam. Knowledge gained from the molecular analysis of these and other AmpC resistance mutants will, we believe, aid in the design of ß-lactams and BLIs with reduced susceptibility to mutational resistance.
Asunto(s)
Farmacorresistencia Bacteriana/genética , Pseudomonas aeruginosa , Antibacterianos/farmacología , Compuestos de Azabiciclo/farmacología , Ceftazidima/farmacología , Cefalosporinas/farmacología , Combinación de Medicamentos , Hidrólisis , Pruebas de Sensibilidad Microbiana , Mutación , Pseudomonas aeruginosa/genética , beta-Lactamasas/genéticaRESUMEN
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
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
The development of a potent mechanism-based inactivator of NagZ, an enzyme critical to the production of inducible AmpC ß-lactamase in Gram-negative bacteria, is presented. This inactivator significantly reduces MIC values for important ß-lactams against a clinically relevant strain of Pseudomonas aeruginosa.
Asunto(s)
Acetilglucosaminidasa/antagonistas & inhibidores , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Compuestos Epoxi/farmacología , Pseudomonas aeruginosa/metabolismo , Resistencia betalactámica/efectos de los fármacos , Acetilglucosaminidasa/metabolismo , Antibacterianos/síntesis química , Antibacterianos/metabolismo , Aztreonam/farmacología , Proteínas Bacterianas/metabolismo , Burkholderia cenocepacia/enzimología , Ceftazidima/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Compuestos Epoxi/síntesis química , Compuestos Epoxi/metabolismo , Humanos , Imipenem/farmacología , Pruebas de Sensibilidad Microbiana , Unión Proteica , Pseudomonas aeruginosa/enzimología , beta-N-Acetilhexosaminidasas/antagonistas & inhibidoresRESUMEN
Inducible AmpC ß-lactamases deactivate a broad-spectrum of ß-lactam antibiotics and afford antibiotic resistance in many Gram-negative bacteria. The disturbance of peptidoglycan recycling caused by ß-lactam antibiotics leads to accumulation of GlcNAc-1,6-anhydroMurNAc-peptides, which are transported by AmpG to the cytoplasm where they are processed into AmpC inducers. AmpG transporters are poorly understood; however, their loss restores susceptibility toward ß-lactam antibiotics, highlighting AmpG as a potential target for resistance-attenuating therapeutics. We prepare a GlcNAc-1,6-anhydroMurNAc-fluorophore conjugate and, using live E. coli spheroplasts, quantitatively analyze its transport by AmpG and inhibition of this process by a competing substrate. Further, we use this transport assay to evaluate the function of two AmpG homologues from Pseudomonas aeruginosa and show that P. aeruginosa AmpG (Pa-AmpG) but not AmpP (Pa-AmpP) transports this probe substrate. We corroborate these results by AmpC induction assays with Pa-AmpG and Pa-AmpP. This fluorescent AmpG probe and spheroplast-based transport assay will enable improved understanding of PG recycling and of permeases from the major facilitator superfamily of transport proteins and may aid in identification of AmpG antagonists that combat AmpC-mediated resistance toward ß-lactam antibiotics.
Asunto(s)
Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana , Colorantes Fluorescentes/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Peptidoglicano/metabolismo , Pseudomonas aeruginosa/enzimología , Transporte Biológico , CinéticaRESUMEN
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.