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Estudo dos antiporters Na+/H+ NhaC e NhaE de Neisseria meningitidis
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Os antiporters Na+/H+ são proteínas membranares, encontradas em todos os reinos e envolvidas na homeostasia do Na+ e do pH. Dois antiporters Na+/H+, denominados NmNhaC e NmNhaE foram identificados no genoma da bactéria patogénica, Neisseria meningitidis, e estudados através da sua expressão heteróloga numa estirpe de E. coli deficiente em antiporters Na+/H+, a estirpe E. coli KNabc. Os antiporters Na+/H+ de N. meningitidis, foram capazes de conferir resistência ao Na+, a esta estirpe, sugerindo que os dois possam contribuir para a homeostasia do Na+ e pH. O NmNhaE é também capaz de transportar Li+, e parece ser o principal antiporter Na+/H+ a contribuir para este papel em N. meningitidis. O NmNhaE tem atividade de antiporter Na+/H+ entre valores de pH 6.5 a 9, e a atividade Na+(Li+)/H+ ótima é observada a pH 7. Além disso, verificou-se que o NmNhaE tem maior afinidade para o Na+. Com o objetivo de encontrar resíduos importantes na função e estrutura do NmNhaE, dez resíduos de aminoácidos foram selecionados apartir de comparações de alinhamentos entre sequências de aminoácidos, e também através de estudos anteriores de mutagénese dirigida noutros antiporters Na+/H+, tendo as mutações pontuais sido intruzidas no gene nhaE, resultando nas mutações E91Q, H161L, G174S, D182N, H201L, V258T, H272L, H356L, H361L e G413S. Quando a expressão dos mutantes foi induzida em E. coli KNabc, todos os mutantes, com exceção dos mutantes G174S e H201L, foram capazes de complementar a estirpe KNabc, permitindo que esta crescesse em meio LBK líquido com NaCl 500 mM. Este facto sugere que as mutações G174S e H201L podem impedir a atividade de antiporter Na+/H+. Nos mutantes E91Q, V258T e H361L o Km determinado a pH 7, para o Na+, é semelhante ao determinado no NhaE selvagem, contudo o seu Vmax sofreu uma descida de quase duas vezes. Além disso, a atividade medida a pH 7 para os mutantes V258T e E91Q, e a atividade medida a 7.5 para o mutante V258T, diminuiu relativamente ao NhaE selvagem. Este resultado juntamente com as atividades medidas a pH 7 e 7.5, sugerem que os resíduos E91, V258, H361 estejam envolvidos na resposta ao pH. No mutante H272L, verificou-se um aumento no Km determinado para o Na+ a pH 7 para quase o dobro, enquanto o Vmax se manteve inalterado em relação ao NhaE selvagem. Os parâmetros cinéticos e as atividades medidas a pH 7 e 7.5 sugerem que o resíduo H272 pode estar envolvido no transporte de Na+ e também na resposta ao pH. Nos restantes residuos não se verificou atividade de antiporter Na+/H+. Este trabalho contribuiu para uma melhor compreensão do transporte de Na+ nos antiporters Nha de N. menigitidis, nomeadamente no funcionamento e estrutura do NhaE. No futuro, este trabalho pode fornecer informação relevante quanto ao papel do Na+ na infeção e sobrevivência desta bactéria no hospedeiro.
Na+/H+ antiporters are membrane proteins present in all kingdoms involved in Na+ and pH homeostasis. Two Na+/H+ antiporters, NmNhaC and NmNhaE, from the pathogenic bacterium, Neisseria meningitis, were identified in the genome and studied through heterologous expression in the E. coli KNabc, a strain lacking the major Na+/H+ antiporters. N. meningitidis Na+/H+ antiporters, were able to restore Na+ resistance to E. coli KNabc, suggesting that they may contribute to the Na+ and pH homeostasis. NmNhaE is also able to transport Li+ ions and appears to be the main Na+/H+ antiporter contributing to this role in N. meningitidis. The Na+/H+ antiporter activity of NmNhaE is active between pH 6.5 to 9, the optimal Na+(Li+)/H+ antiporter activity being observed at pH 7. Furthermore, NmNhaE displayed higher affinity towars Na+. To address important functional and structural residues in NmNhaE, ten amino acid residues were selected from sequence alignments comparisions and previous studies of site directed mutagenesis in other Na+/H+ antiporters and point mutations were introduced in the nhaE gene, resulting in E91Q, H161L, G174S, D182N, H201L, V258T, H272L, H356L, H361L and G413S mutations. When their expression was induced, all mutants but G174S and H201L, allowed E. coli KNabc strain it to grow in LBK with NaCl 500 mM. This suggests that mutations G174S and H201L may block the antiporter activity. No alteration was seen regarding the Km measured for Na+ at pH 7 of mutants E91Q, V258T and H361L when compared to the NhaE wild type, however their Vmax suffered a decrease of almost two times. In addition, the activities measured at pH 7 and 7.5 showed a decrease in V258T, while E91Q showed a decrease in activity at pH 7, when compared with the NhaE wild type. The determined kinetic parameters and the measured activities suggests that residues E91, V258, H361 may be involved in the pH response. The comparision between the NhaE kinetic parameters with those of the recombinant H272L showed almost one fold increase in the Km for Na+ at pH 7, while the Vmax showed no difference. The kinetic parameters and the activities measured at pH 7 and pH 7.5 suggests that residue H272 may be involved in the translocation of Na+ and also in pH response. No Na+/H+ antiporter activity was observed in the remaining residues. This work contributed for a better understanding of Na+ transport in N. meningitidis Nha Na+/H+ antiporters namely the function of Na+/H+ antiporters and important residues to their function. In the future, this work can provide useful information about the role of Na+ in the infection and survival of this bacterium in the host.
Na+/H+ antiporters are membrane proteins present in all kingdoms involved in Na+ and pH homeostasis. Two Na+/H+ antiporters, NmNhaC and NmNhaE, from the pathogenic bacterium, Neisseria meningitis, were identified in the genome and studied through heterologous expression in the E. coli KNabc, a strain lacking the major Na+/H+ antiporters. N. meningitidis Na+/H+ antiporters, were able to restore Na+ resistance to E. coli KNabc, suggesting that they may contribute to the Na+ and pH homeostasis. NmNhaE is also able to transport Li+ ions and appears to be the main Na+/H+ antiporter contributing to this role in N. meningitidis. The Na+/H+ antiporter activity of NmNhaE is active between pH 6.5 to 9, the optimal Na+(Li+)/H+ antiporter activity being observed at pH 7. Furthermore, NmNhaE displayed higher affinity towars Na+. To address important functional and structural residues in NmNhaE, ten amino acid residues were selected from sequence alignments comparisions and previous studies of site directed mutagenesis in other Na+/H+ antiporters and point mutations were introduced in the nhaE gene, resulting in E91Q, H161L, G174S, D182N, H201L, V258T, H272L, H356L, H361L and G413S mutations. When their expression was induced, all mutants but G174S and H201L, allowed E. coli KNabc strain it to grow in LBK with NaCl 500 mM. This suggests that mutations G174S and H201L may block the antiporter activity. No alteration was seen regarding the Km measured for Na+ at pH 7 of mutants E91Q, V258T and H361L when compared to the NhaE wild type, however their Vmax suffered a decrease of almost two times. In addition, the activities measured at pH 7 and 7.5 showed a decrease in V258T, while E91Q showed a decrease in activity at pH 7, when compared with the NhaE wild type. The determined kinetic parameters and the measured activities suggests that residues E91, V258, H361 may be involved in the pH response. The comparision between the NhaE kinetic parameters with those of the recombinant H272L showed almost one fold increase in the Km for Na+ at pH 7, while the Vmax showed no difference. The kinetic parameters and the activities measured at pH 7 and pH 7.5 suggests that residue H272 may be involved in the translocation of Na+ and also in pH response. No Na+/H+ antiporter activity was observed in the remaining residues. This work contributed for a better understanding of Na+ transport in N. meningitidis Nha Na+/H+ antiporters namely the function of Na+/H+ antiporters and important residues to their function. In the future, this work can provide useful information about the role of Na+ in the infection and survival of this bacterium in the host.
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Parasitologia médica Biologia molecular Medicina tropical
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Instituto de Higiene e Medicina Tropical