RESUMEN
Recent investigations into quinoline and phenanthrene methanol resistance in Plasmodium falciparum have described a linkage between amplification of the mdr homologue pfmdr1 and decreased susceptibility to mefloquine (MQ) and halofantrine (HF). We have examined the current theories on cross-resistance patterns and pfmdr1 gene expression by comparing the chloroquine (CQ) resistant isolate K1 with K1Hf, developed from the K1 isolate by intermittent exposure to halofantrine. Reduced halofantrine susceptibility in K1Hf was accompanied by reduced sensitivity to mefloquine and increased sensitivity to chloroquine. These sensitivity changes were reflected by changes in parasite drug accumulation. The loss of high level chloroquine resistance in K1Hf was associated with an inability of verapamil to enhance chloroquine sensitivity or accumulation. In contrast verapamil retained the chemosensitising potential against quinine in this isolate. The changes in phenotype were achieved without any amplification or increased expression of pfmdr1 or reversion of the Tyr86 mutation in the gene. Our data indicates that acquisition of halofantrine and mefloquine resistance and the loss of high level chloroquine resistance can be achieved without enhanced expression of the pfmdr1 gene product.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Antimaláricos/farmacología , Resistencia a Múltiples Medicamentos/genética , Fenantrenos/farmacología , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/biosíntesis , Selección Genética , Animales , Transporte Biológico , Cloroquina/metabolismo , Cloroquina/farmacología , Dermatoglifia del ADN , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Dosificación de Gen , Expresión Génica , Immunoblotting , Mefloquina/farmacología , Proteínas Protozoarias/genética , Quinina/metabolismo , Quinina/farmacología , Análisis de Secuencia de ADN , Verapamilo/farmacologíaAsunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Cloroquina/farmacología , Genes Protozoarios/genética , Mutación/genética , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Animales , Resistencia a Medicamentos/genética , Técnicas In Vitro , Plasmodium falciparum/clasificaciónRESUMEN
KefC is a glutathione-gated K(+)-efflux system that is widespread in Gram-negative bacteria and which plays a role in the protection of cells from the toxic effects of electrophilic reagents, such as N-ethylmaleimide (NEM). The KefC gene from Escherichia coli has been cloned and the DNA sequenced. A number of kefC mutants that affect K+ retention by the KefC system have been isolated and all retain activation by NEM. Cloned kefC was found to suppress the phenotype of two such mutants kefC121 and kefC103. Analysis of this phenomenon has shown that suppression is specific to the KefC system, but that cloned kefC from Klebsiella and Erwinia can also mediate suppression of the mutant phenotype. Plasmid constructs of the E. coli gene in which expression of the cloned gene was diminished showed induced ability to suppress the mutant phenotype. KefC'-'LacZ hybrid proteins were inserted in the membrane but did not suppress the mutant phenotype. Cloned kefC did not suppress a mutant kefB allele that exhibited a similar phenotype to the kefC121 allele. These data suggest that suppression is unlikely to arise from exclusion of the mutant form of the protein from the membrane. Furthermore, NEM-activated K+ efflux from a strain carrying both the mutant and cloned wild-type alleles was faster than when either allele was present in cells alone, suggesting that both forms of the protein are inserted into the membrane. These data are discussed in terms of a model for the KefC protein in which the protein is composed of one or more identical subunits that interact in the membrane.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Canales de Potasio , Potasio/metabolismo , Proteínas Bacterianas/química , Clonación Molecular , Erwinia/genética , Expresión Génica , Genes Bacterianos , Transporte Iónico/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Fenotipo , Conformación Proteica , Supresión GenéticaRESUMEN
Control of falciparum malaria has become almost impossible in many areas due to the development of resistance to chloroquine and other antimalarial drugs. Verapamil and a number of unrelated compounds which chemosensitise multi-drug resistant cancer cells also enhance chloroquine susceptibility in Plasmodium falciparum. Chloroquine is accumulated to lower levels in resistant plasmodia, hence the reversal of chloroquine resistance has been attributed to the ability of chemosensitising agents to increase the amount of chloroquine accumulated by the resistant parasite. We have conducted a detailed examination of the effect of verapamil on chloroquine sensitivity and its relationship to chloroquine accumulation. The ability of verapamil to increase steady-state chloroquine accumulation was found to be totally insufficient to explain the increase in chloroquine sensitivity caused by the drug. In contrast, when chloroquine accumulation was increased by raising the pH gradient, the corresponding shifts in sensitivity to chloroquine could be accurately predicted. These results were confirmed with other classes of chemosensitisers and we conclude that an alternative mechanistic explanation is required to completely explain the reversal of chloroquine resistance in P. falciparum.
Asunto(s)
Cloroquina/farmacología , Cloroquina/farmacocinética , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , Desipramina/farmacología , Resistencia a Medicamentos , Humanos , Concentración de Iones de Hidrógeno , Cinética , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Verapamilo/farmacologíaRESUMEN
Recent reports suggest that lower levels of chloroquine accumulation in chloroquine-resistant isolates of Plasmodium falciparum are achieved by energy-dependent chloroquine efflux from resistant parasites. In support of this argument, a rapid chloroquine efflux phenotype has been observed in some chloroquine-resistant isolates of P. falciparum. In this study, no relationship was found between chloroquine sensitivity and the rate of [3H]chloroquine efflux from four isolates of P. falciparum with a greater than 10-fold range in sensitivity to chloroquine. All the isolates tested displayed the rapid efflux phenotype, irrespective of sensitivity. However, chloroquine sensitivity of these isolates was correlated with energy-dependent rate of drug accumulation into these parasites. Verapamil and a variety of other compounds reverse chloroquine resistance. The reversal mechanism is assumed to result from competition between verapamil and chloroquine for efflux protein translocation sites, thus causing an increase in steady-state accumulation of chloroquine and hence a return to sensitivity. Verapamil accumulation at a steady-state is increased by chloroquine, possibly indicating competition for efflux of the two substrates. Increases in steady-state verapamil concentrations caused by chloroquine were identical in sensitive and resistant strains, suggesting that similar capacity efflux pumps may exist in these isolates. These data suggest that differences in steady-state chloroquine accumulation seen in these isolates can be attributed to changes in the chloroquine concentrating mechanism rather than the efflux pump. It seems likely that chloroquine resistance generally in P. falciparum, results at least in part from a change in the drug concentrating mechanism and that changes in efflux rates per se are insufficient to explain chloroquine resistance.
Asunto(s)
Cloroquina/farmacología , Plasmodium falciparum/efectos de los fármacos , Animales , Cloroquina/metabolismo , Resistencia a Medicamentos , Concentración de Iones de Hidrógeno , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Factores de Tiempo , Verapamilo/metabolismoRESUMEN
Using a variety of techniques the distribution of the glutathione-regulated KefC K(+)-transport system among bacterial species was investigated. The presence of similar systems in a number of Gram-negative bacteria was demonstrated. In contrast, the system appeared to be absent from most Gram-positive bacteria tested with the exception of Staphylococcus aureus. Using the cloned Escherichia coli kefC gene as a probe for Southern hybridization it was shown that only limited DNA sequence homology exists with other bacteria, even when closely related members of the enteric group were examined.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Canales de Potasio , Potasio/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico Activo/efectos de los fármacos , Southern Blotting , Clonación Molecular , Sondas de ADN/genética , Etilmaleimida/farmacología , Glutatión/análogos & derivados , Glutatión/análisis , Glutatión/metabolismo , Bacterias Gramnegativas/metabolismo , Bacterias Grampositivas/metabolismo , Cinética , Homología de Secuencia de Ácido Nucleico , Staphylococcus aureus/metabolismo , Succinimidas/análisisRESUMEN
The kefC gene of Escherichia coli encodes a potassium-efflux system that is regulated by glutathione metabolites. The close proximity of the E. coli kefC gene to the folA gene, encoding dihydrofolate reductase, has been utilized to clone the structural gene for the system from a Clarke-Carbon plasmid. The cloned gene has been refined to a region of DNA approximately 2.1 kb in length using exonuclease III-generated deletions and random MudII1734 (lacZ) insertions. The direction of transcription has been deduced from the orientation of the Mu insertions in the cloned DNA. A hybrid protein consisting of approximately two thirds of the KefC protein fused to beta-galactosidase has been shown to be membrane-located. The DNA sequence of the gene has been determined and an open reading frame of 1.86 kb has been located which could encode a protein of 620 amino acids (79010 Da). Using the T7 expression system a membrane protein, of apparent molecular mass 55-60 kDa, has been shown to be encoded by the kefC gene. The predicted protein sequence shows a highly hydrophobic amino-terminus and a strongly hydrophilic carboxy-terminus. Comparison of the amino acid sequence of the kefC gene product with those of two glutathione-utilizing enzymes, glyoxalase and dehalogenase, has revealed some similarities.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Canales de Potasio , Potasio/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/análisis , Secuencia de Bases , Transporte Biológico , Membrana Celular/química , Clonación Molecular , Escherichia coli/análisis , Escherichia coli/metabolismo , Glutatión/análogos & derivados , Glutatión/farmacología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas Recombinantes de Fusión/análisis , Mapeo Restrictivo , Succinimidas/farmacologíaRESUMEN
The mechanism by which N-ethylmaleimide (NEM) elicits potassium efflux from Escherichia coli has been investigated. The critical factor is the formation of specific glutathione metabolites that activate transport systems encoded by the kefB and kefC gene products. Formation of N-ethyl-succinimido-S-glutathione (ESG) leads to the activation of potassium efflux via these transport systems. The addition of dithiothreitol and other reducing agents to cells reverses this process by causing the breakdown of ESG and thus removing the activator of the systems. Chlorodinitrobenzene, p-chloromercuribenzoate and phenylmaleimide provoke similar effects to NEM. lodoacetate, which leads to the formation of S-carboxymethyl-glutathione, does not activate the systems but does prevent the action of NEM. It is concluded that the KefB and KefC systems are gated by glutathione metabolites and that the degree to which they are activated is dependent upon the nature of the substituent on the sulphydryl group.