RESUMO
Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Previously, we showed strong huntingtin reduction and prevention of neuronal dysfunction in HD rodents using an engineered microRNA targeting human huntingtin, delivered via adeno-associated virus (AAV) serotype 5 vector with a transgene encoding an engineered miRNA against HTT mRNA (AAV5-miHTT). One of the challenges of rodents as a model of neurodegenerative diseases is their relatively small brain, making successful translation to the HD patient difficult. This is particularly relevant for gene therapy approaches, where distribution achieved upon local administration into the parenchyma is likely dependent on brain size and structure. Here, we aimed to demonstrate the translation of huntingtin-lowering gene therapy to a large-animal brain. We investigated the feasibility, efficacy, and tolerability of one-time intracranial administration of AAV5-miHTT in the transgenic HD (tgHD) minipig model. We detected widespread dose-dependent distribution of AAV5-miHTT throughout the tgHD minipig brain that correlated with the engineered microRNA expression. Both human mutant huntingtin mRNA and protein were significantly reduced in all brain regions transduced by AAV5-miHTT. The combination of widespread vector distribution and extensive huntingtin lowering observed with AAV5-miHTT supports the translation of a huntingtin-lowering gene therapy for HD from preclinical studies into the clinic.
Assuntos
Terapia Genética/métodos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Doença de Huntington/terapia , Animais , Animais Geneticamente Modificados , Dependovirus/genética , Modelos Animais de Doenças , Vetores Genéticos/genética , Humanos , Doença de Huntington/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Suínos , Porco Miniatura , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Recombinant adeno-associated virus (rAAV) has become the vector of choice for the development of novel human gene therapies. High-yield manufacturing of high-quality vectors can be achieved using the baculovirus expression vector system. However, efficient production of rAAV in this insect cell-based system requires a genetic redesign of the viral protein 1 (VP1) operon. In this study, we generated a library of rationally designed rAAV serotype 5 variants with modulations in the translation-initiation region of VP1 and investigated the potency of the resulting vectors. We found that the initiation strength at the VP1 translational start had downstream effects on the VP2/VP3 ratio. Excessive incorporation of VP3 into a vector type decreased potency, even when the VP1/VP2 ratio was in balance. Finally, we successfully generated a potent rAAV vector based on serotype 5 with a balanced VP1/VP2/VP3 stoichiometry.
Assuntos
Terapia Genética , Vetores Genéticos/genética , Parvovirinae/genética , Proteínas Virais/genética , Baculoviridae/genética , Proteínas do Capsídeo/genética , Dependovirus , Vetores Genéticos/uso terapêutico , Humanos , Óperon/genética , Sorogrupo , Proteínas Virais/uso terapêuticoRESUMO
Overexpression of short hairpin RNA (shRNA) often causes cytotoxicity and using microRNA (miRNA) scaffolds can circumvent this problem. In this study, identically predicted small interfering RNA (siRNA) sequences targeting apolipoprotein B100 (siApoB) were embedded in shRNA (shApoB) or miRNA (miApoB) scaffolds and a direct comparison of the processing and long-term in vivo efficacy was performed. Next generation sequencing of small RNAs originating from shApoB- or miApoB-transfected cells revealed substantial differences in processing, resulting in different siApoB length, 5' and 3' cleavage sites and abundance of the guide or passenger strands. Murine liver transduction with adeno-associated virus (AAV) vectors expressing shApoB or miApoB resulted in high levels of siApoB expression associated with strong decrease of plasma ApoB protein and cholesterol. Expression of miApoB from the liver-specific LP1 promoter was restricted to the liver, while the H1 promoter-expressed shApoB was ectopically present. Delivery of 1 × 10(11) genome copies AAV-shApoB or AAV-miApoB led to a gradual loss of ApoB and plasma cholesterol inhibition, which was circumvented by delivering a 20-fold lower vector dose. In conclusion, incorporating identical siRNA sequences in shRNA or miRNA scaffolds results in differential processing patterns and in vivo efficacy that may have serious consequences for future RNAi-based therapeutics.
Assuntos
Apolipoproteína B-100/genética , MicroRNAs/genética , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/genética , Animais , Sequência de Bases , Western Blotting , Colesterol/sangue , Técnicas de Silenciamento de Genes , Vetores Genéticos , Células HEK293 , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/química , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/químicaRESUMO
Although nisin is a model lantibiotic, our knowledge of the specific interactions of prenisin with its modification enzymes remains fragmentary. Here, we demonstrate that the nisin modification enzymes NisB and NisC can be pulled down in vitro from Lactococcus lactis by an engineered His-tagged prenisin. This approach enables us to determine important intermolecular interactions of prenisin with its modification machinery within L. lactis. We demonstrate that (i) NisB has stronger interactions with precursor nisin than NisC has, (ii) deletion of the propeptide part keeping the nisin leader intact leads to a lack of binding, (iii) NisB point mutants of highly conserved residues W616, F342A, Y346F and P639A are still able to dehydrate prenisin, (iv) NisB Δ(77-79)Y80F mutant decreased the levels of NisB-prenisin interactions and resulted in unmodified prenisin, (v) substitution of an active site residue H331A in NisC leads to higher amounts of the co-purified complex, (vi) NisB is present in the form of a dimer, and (vii) the region FNLD (-18 to -15) of the leader is an important site for binding not only to NisB, but also to NisC.
Assuntos
Proteínas de Bactérias/metabolismo , Lactococcus lactis/metabolismo , Proteínas de Membrana/metabolismo , Nisina/metabolismo , Mapeamento de Interação de Proteínas , Precursores de Proteínas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nisina/genética , Ligação Proteica , Precursores de Proteínas/genética , Estados UnidosRESUMO
Membrane proteins responsible for the active efflux of structurally and functionally unrelated drugs were first characterized in higher eukaryotes. To date, a vast number of transporters contributing to multidrug resistance (MDR transporters) have been reported for a large variety of organisms. Predictions about the functions of genes in the growing number of sequenced genomes indicate that MDR transporters are ubiquitous in nature. The majority of described MDR transporters in bacteria use ion motive force, while only a few systems have been shown to rely on ATP hydrolysis. However, recent reports on MDR proteins from gram-positive organisms, as well as genome analysis, indicate that the role of ABC-type MDR transporters in bacterial drug resistance might be underestimated. Detailed structural and mechanistic analyses of these proteins can help to understand their molecular mode of action and may eventually lead to the development of new strategies to counteract their actions, thereby increasing the effectiveness of drug-based therapies. This review focuses on recent advances in the analysis of ABC-type MDR transporters in bacteria.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Farmacorresistência Bacteriana Múltipla , Transportadores de Cassetes de Ligação de ATP/química , Animais , Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Humanos , Modelos Biológicos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The lantibiotic nisin is a potent antimicrobial substance, which contains unusual lanthionine rings and dehydrated amino acid residues and is produced by Lactococcus lactis. Recently, the nisin biosynthetic machinery has been applied to introduce lanthionine rings in peptides other than nisin with potential therapeutic use. Due to difficulties in the isolation of the proposed synthetase complex (NisBTC), mechanistic information concerning the enzymatic biosynthesis of nisin is scarce. Here, we present the molecular characterization of a number of nisin mutants that affect ring formation. We have investigated in a systematic manner how these mutations influence dehydration events, which are performed enzymatically by the dehydratase NisB. Specific mutations that hampered ring formation allowed for the dehydration of serine residues that directly follow the rings and are normally unmodified. The combined information leads to the conclusion that 1) nisin biosynthesis is an organized directional process that starts at the N terminus of the molecule and continues toward the C terminus, and 2) NisB and NisC are alternating enzymes, whose activities follow one after another in a repetitive way. Thus, the dehydration and cyclization processes are not separated in time and space. On the basis of these results and previous knowledge, a working model for the sequence of events in the maturation of nisin is proposed.
Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Lactococcus lactis/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multienzimáticos/metabolismo , Nisina/biossíntese , Biossíntese de Peptídeos Independentes de Ácido Nucleico/fisiologia , Alanina/análogos & derivados , Alanina/química , Alanina/genética , Alanina/metabolismo , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Lactococcus lactis/química , Lactococcus lactis/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Nisina/química , Nisina/genética , Sulfetos/química , Sulfetos/metabolismoRESUMO
Recent studies showed that the nisin modification machinery can successfully dehydrate serines and threonines and introduce lanthionine rings in small peptides that are fused to the nisin leader sequence. This opens up exciting possibilities to produce and engineer larger antimicrobial peptides in vivo. Here we demonstrate the exploitation of the class I nisin production machinery to generate, modify, and secrete biologically active, previously not-yet-isolated and -characterized class II two-component lantibiotics that have no sequence homology to nisin. The nisin synthesis machinery, composed of the modification enzymes NisB and NisC and the transporter NisT, was used to modify and secrete a putative two-component lantibiotic of Streptococcus pneumoniae. This was achieved by genetically fusing the propeptide-encoding sequences of the spr1765 (pneA1) and spr1766 (pneA2) genes to the nisin leader-encoding sequence. The chimeric prepeptides were secreted out of Lactococcus lactis, purified by cation exchange fast protein liquid chromatography, and further characterized. Mass spectrometry analyses demonstrated the presence and partial localization of multiple dehydrated serines and/or threonines and (methyl)lanthionines in both peptides. Moreover, after cleavage of the leader peptide from the prepeptides, both modified propeptides displayed antimicrobial activity against Micrococcus flavus. These results demonstrate that the nisin synthetase machinery can be successfully used to modify and produce otherwise difficult to obtain antimicrobially active lantibiotics.
Assuntos
Bacteriocinas/biossíntese , Streptococcus pneumoniae/metabolismo , Sequência de Aminoácidos , Antibacterianos/biossíntese , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/química , Bacteriocinas/genética , Bacteriocinas/farmacologia , Genes Bacterianos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Família Multigênica , Nisina/biossíntese , Engenharia de Proteínas/métodos , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência de Aminoácidos , Streptococcus pneumoniae/genéticaRESUMO
Gene therapy is being developed for the treatment of inherited diseases, whereby a therapeutic gene is continuously expressed in patients after delivery via viral vectors such as adeno-associated virus (AAV). Depending on the transgene, there could be a limited therapeutic window, and regulating timing and levels of transgene expression is advantageous. To control transgene transcription, the regulatory system GeneSwitch (GS) was evaluated in detail both in vitro and in vivo. The classical two-plasmid mifepristone (MFP)-inducible GS system was put into one plasmid or a single AAV5 vector. Our data demonstrate the inducibility of multiple transgenes and the importance of promoter and regulatory elements within the GS system. Mice injected with AAV5 containing the GS system transiently expressed mRNA and protein after MFP induction. The inducer MFP could be measured in plasma and liver tissue, and assessment of MFP and its metabolites showed rapid clearance from murine plasma. In a head-to-head comparison, our single vector outclassed the classical two-vector GS system. Finally, we show repeated inducibility of the transgene that also translated into a dynamic phenotypic change in mice. Taken together, this in-depth analysis of the GS system shows its applicability for regulated gene therapy.
RESUMO
Gene therapy for severe hemophilia B is advancing and offers sustained disease amelioration with a single treatment. We have reported the efficacy and safety of AMT-060, an investigational gene therapy comprising an adeno-associated virus serotype 5 capsid encapsidating the codon-optimized wild-type human factor IX (WT hFIX) gene with a liver-specific promoter, in patients with severe hemophilia B. Treatment with 2 × 1013 gc/kg AMT-060 showed sustained and durable FIX activity of 3%-13% and a substantial reduction in spontaneous bleeds without T cell-mediated hepatoxicity. To achieve higher FIX activity, we modified AMT-060 to encode the R338L "Padua" FIX variant that has increased specific activity (AMT-061). We report the safety and increased FIX activity of AMT-061 in non-human primates. Animals (n = 3/group) received intravenous AMT-060 (5 × 1012 gc/kg), AMT-061 (ranging from 5 × 1011 to 9 × 1013 gc/kg), or vehicle. Human FIX protein expression, FIX activity, and coagulation markers including D-dimer and thrombin-antithrombin complexes were measured. At equal doses, AMT-060 and AMT-061 resulted in similar human FIX protein expression, but FIX activity was 6.5-fold enhanced using AMT-061. Both vectors show similar safety and transduction profiles. Thus, AMT-061 holds great promise as a more potent FIX replacement gene therapy with a favorable safety profile.
RESUMO
Upon prolonged exposure to cholate and other toxic compounds, Lactococcus lactis develops a multidrug resistance phenotype that has been attributed to an elevated expression of the heterodimeric ABC-type multidrug transporter LmrCD. To investigate the molecular basis of bile acid resistance in L. lactis and to evaluate the contribution of efflux-based mechanisms in this process, the drug-sensitive L. lactis NZ9000 DeltalmrCD strain was challenged with cholate. A resistant strain was obtained that, compared to the parental strain, showed (i) significantly improved resistance toward several bile acids but not to drugs, (ii) morphological changes, and (iii) an altered susceptibility to antimicrobial peptides. Transcriptome and transport analyses suggest that the acquired resistance is unrelated to elevated transport activity but, instead, results from a multitude of stress responses, changes to the cell envelope, and metabolic changes. In contrast, wild-type cells induce the expression of lmrCD upon exposure to cholate, whereupon the cholate is actively extruded from the cells. Together, these data suggest a central role for an efflux-based mechanism in bile acid resistance and implicate LmrCD as the main system responsible in L. lactis.
Assuntos
Proteínas de Bactérias/genética , Ácidos e Sais Biliares/farmacologia , Lactococcus lactis/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Transporte Biológico Ativo/efeitos dos fármacos , Colatos/farmacologia , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana Múltipla/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Lactococcus lactis/metabolismo , Lactococcus lactis/ultraestrutura , Microscopia Eletrônica de Varredura , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
LmrCD is an ABC-type multidrug transporter in Lactococcus lactis. LmrR encodes a putative transcriptional regulator. In a DeltalmrR strain, lmrCD is up-regulated. LmrR binds the promoter region of lmrCD and interacts with drugs that cause lmrCD up-regulation. This suggests that LmrR is a drug-dependent transcriptional regulator of lmrCD expression.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Regulação Bacteriana da Expressão Gênica/fisiologia , Lactococcus lactis/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Proteínas Repressoras/fisiologia , DNA Bacteriano/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/genética , Lactococcus lactis/genética , Ligação Proteica , Proteínas Repressoras/genéticaRESUMO
Since the recent discovery that the nisin modification and transport machinery can be used to produce and modify peptides unrelated to nisin, specific questions arose concerning the specificity of the modification enzymes involved and the limits of their promiscuity with respect to the dehydration and cyclization processes. The nisin leader peptide has been postulated to fulfill a recognition and binding function required for these modifications. Here, we investigated whether the relative positions of the modifiable residues in the nisin prepeptide, with respect to the leader peptide, could influence the efficiency of their modification. We conducted a systematic study on the insertion of one to four alanines in front of either ring A or ring D to change the "reading frame" of modifiable residues, resulting in altered distance and topology of the modifiable residues relative to the leader. The insertion of N-terminal and hinge-located Ala residues had only a modest influence on the modification efficiency, demonstrating that the "phasing" of these residues relative to the leader peptide is not a critical factor in determining modification. However, in all cases, but especially with the N-terminal insertions, the antimicrobial activities of the fully modified nisin species were decreased.
Assuntos
Antibacterianos/farmacologia , Cisteína/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Nisina/química , Nisina/metabolismo , Peptídeos/farmacologia , Serina/metabolismo , Treonina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/crescimento & desenvolvimento , Dados de Sequência Molecular , Plasmídeos , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologiaRESUMO
Nisin is a posttranslationally-modified antimicrobial peptide that has the ability to induce its own biosynthesis. Serines and threonines in the modifiable core peptide part of precursor nisin are dehydrated to dehydroalanines and dehydrobutyrines by the dehydratase NisB, and subsequently cysteines are coupled to the dehydroamino acids by the cyclase NisC. In this study, we applied extensive site-directed mutagenesis, together with direct binding studies, to investigate the molecular mechanism of the dehydratase NisB. We use a natural nisin-producing strain as a host to probe mutant-NisB functionality. Importantly, we are able to differentiate between intracellular and secreted fully dehydrated precursor nisin, enabling investigation of the NisB properties needed for the release of dehydrated precursor nisin to its devoted secretion system NisT. We report that single amino acid substitutions of conserved residues, i.e., R83A, R83M, and R87A result in incomplete dehydration of precursor nisin and prevention of secretion. Single point NisB mutants Y80F and H961A, result in a complete lack of dehydration of precursor nisin, but do not abrogate precursor nisin binding. The data indicate that residues Y80 and H961 are directly involved in catalysis, fitting well with their position in the recently published 3D-structure of NisB. We confirm, by in vivo studies, results that were previously obtained from in vitro experiments and NisB structure elucidation and show that previous findings translate well to effects seen in the original production host.
RESUMO
LmrCD is a lactococcal, heterodimeric multidrug transporter, which belongs to the ABC superfamily. It consists of two half-transporters, LmrC and LmrD, that are necessary and sufficient for drug extrusion and ATP hydrolysis. LmrCD is asymmetric in terms of the conservation of the functional motifs of the nucleotide-binding domains (NBDs). Important residues of the nucleotide-binding site of LmrC and the C loop of LmrD are not conserved. To investigate the functional importance of the LmrC and LmrD subunits, the putative catalytic base residue adjacent to the Walker B motif of both NBDs were substituted for the respective carboxamides. Our data demonstrate that Glu587 of LmrD is essential for both drug transport and ATPase activity of the LmrCD heterodimer, whereas mutation of Asp495 of LmrC has a less severe effect on the activity of the complex. Structural and/or functional asymmetry is further demonstrated by differential labeling of both subunits by 8-azido-[alpha-32P]ATP, which, at 4 degrees C, occurs predominantly at LmrC, while aluminiumfluoride (AlF(x))-induced trapping of the hydrolyzed nucleotide at 30 degrees C results in an almost exclusive labeling of LmrD. It is concluded that the LmrCD heterodimer contains two structurally and functionally distinct NBDs.
Assuntos
Proteínas de Bactérias/metabolismo , Lactococcus lactis/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Nucleotídeos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Compostos de Alumínio/metabolismo , Sequência de Aminoácidos , Azidas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Transporte Biológico Ativo , Domínio Catalítico , Dimerização , Resistência a Múltiplos Medicamentos/genética , Fluoretos/metabolismo , Lactococcus lactis/enzimologia , Lactococcus lactis/genética , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutagênese Sítio-Dirigida , Nucleotídeos/química , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
When Lactococcus lactis is challenged with drugs it displays a multidrug resistance (MDR) phenotype. In silico analysis of the genome of L. lactis indicates the presence of at least 40 putative MDR transporters, of which only four, i.e. the ABC transporters LmrA, LmrC and LmrD, and the major facilitator LmrP, have been experimentally associated with the MDR. To understand the molecular basis of the MDR phenotype in L. lactis, we have performed a global transcriptome analysis comparing four independently isolated drug-resistant strains of L. lactis with the wild-type strain. The results show a strong and consistent upregulation of the lmrC and lmrD genes in all four strains, while the mRNA levels of other putative MDR transporters were not significantly altered. Deletion of lmrCD renders L. lactis sensitive to several toxic compounds, and this phenotype is associated with a reduced ability to secrete these compounds. Another gene, which is strongly upregulated in all mutant strains, specifies LmrR (YdaF), a local transcriptional repressor of lmrCD that belongs to the PadR family of transcriptional regulators and that binds to the promoter region of lmrCD. These results demonstrate that the heterodimeric MDR ABC transporter LmrCD is a major determinant of both acquired and intrinsic drug resistance of L. lactis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Farmacorresistência Bacteriana Múltipla/fisiologia , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras GenéticasRESUMO
The fluorescent compound Hoechst 33342 is a substrate for many multidrug resistance (MDR) transporters and is widely used to characterize their transport activity. We have constructed mutants of the adenosine triphosphate (ATP) binding cassette (ABC)-type MDR transporter LmrA of Lactococcus lactis that are defective in ATP hydrolysis. These mutants and wild-type LmrA exhibited an atypical behavior in the Hoechst 33342 transport assay. In membrane vesicles, Hoechst 33342 transport was shown to be independent of the ATPase activity of LmrA, and it was not inhibited by orthovanadate but sensitive to uncouplers that collapse the proton gradient and to N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. In contrast, transport of Hoechst 33342 by the homologous, heterodimeric MDR transporter LmrCD showed a normal ATP dependence and was insensitive to uncouplers of the proton gradient. With intact cells, expression of LmrA resulted in an increased rate of Hoechst 33342 influx while LmrCD caused a decrease in the rate of Hoechst 33342 influx. Cellular toxicity assays using a triple knockout strain, i.e., L. lactis delta lmrA delta lmrCD, demonstrate that expression of LmrCD protects cells against the growth inhibitory effects of Hoechst 33342, while in the presence of LmrA, cells are more susceptible to Hoechst 33342. Our data demonstrate that the LmrA-mediated Hoechst 33342 transport in membrane vesicles is influenced by the transmembrane pH gradient due to a pH-dependent partitioning of Hoechst 33342 into the membrane.
Assuntos
Proteínas de Bactérias/fisiologia , Benzimidazóis/metabolismo , Lactococcus lactis/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Força Próton-Motriz/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/fisiologia , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzimidazóis/farmacologia , Transporte Biológico/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dicicloexilcarbodi-Imida/farmacologia , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacologia , Concentração de Íons de Hidrogênio , Ionóforos/farmacologia , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/genética , Lipossomos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Metilaminas/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutagênese Sítio-Dirigida , Nigericina/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Valinomicina/farmacologia , Vanadatos/farmacologiaRESUMO
Multidrug resistance (MDR)-type transporters mediate the active extrusion of structurally and functionally dissimilar compounds from the cells, thereby rendering cells resistant to a range of drugs. The ydaG and ydbA genes of Lactococcus lactis encode two ATP-binding cassette half-transporters, which both share homology with MDR proteins such as LmrA from L. lactis or the mammalian P-glycoprotein. The ydaG/ydbA genes were cloned and expressed separately and jointly in L. lactis using the nisin-inducible system. When both proteins are co-expressed, several structurally dissimilar drugs such as ethidium, daunomycin, and BCECF-AM are extruded from the cell. YdaG and YdbA could be co-purified as a stable heterodimer. ATPase activity was found to be associated with the YdaG/YdbA heterodimer only and not with the individual subunits. Both the ydaG and ydbA genes are up-regulated in multidrug-resistant L. lactis strains selected for growth in the presence of a variety of toxic compounds. This is the first demonstration of a functional heterodimeric ATP-binding cassette-type MDR transporter.