Avaliação de moléculas bioativas como inibidores do sistema antioxidante de peroxirredoxinas 2-Cys típicas (AhpC) em bactérias
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Data
2021-03-03
Autores
Santos, Melina Cardoso dos
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Universidade Estadual Paulista (Unesp)
Resumo
O aumento da resistência aos antibióticos por microrganismos tem crescido vertiginosamente e especialistas têm alertado quanto à rápida ineficácia dos medicamentos frente a linhagens de bactérias super-resistentes. A resistência de bactérias aos antibióticos pode estar diretamente relacionada ao estresse oxidativo, pois antibióticos distintos possuem a capacidade convergente de produzir espécies reativas de oxigênio, que causam danos às macromoléculas, em especial ao DNA, acentuando as taxas de mutação o que pode contribuir para o aparecimento de linhagens super-resistentes. Espécies reativas de oxigênio e nitrogênio também são produzidas pelo sistema imunológico de hospedeiros para combater patógenos e estudos indicam que a inibição da expressão de enzimas antioxidantes de patógenos é capaz de gerar um aumento intracelular significativo dessas espécies, o que interfere de forma substancial na sua sobrevivência. Recentemente foi demonstrado que um composto natural denominado adenantina (Adn) é capaz de inibir a atividade peroxidásica de peroxirredoxinas (Prx) 2-Cys típicas de humanos (PrxI e PrxII), resultando na mortalidade de células tumorais, mas apresentando baixa toxicidade para células normais. As Prx 2-Cys são capazes de decompor hidroperóxidos utilizando um resíduo de cisteína (CysP) e sua a alta reatividade sobre diferentes tipos de hidroperóxido é alcançada por interações com os resíduos de Thr/Ser e Arg, absolutamente conservados entre as Prx 2-Cys. Em conjunto, CysP, Thr/Ser e Arg, recebem o nome de tríade catalítica (TC). As Prx 2-Cys típicas também estão presentes em bactérias e são denominadas de AhpCs, sendo consideradas como uma das frentes de defesa oxidativas fundamentais para o patógeno. Em projeto anterior, avaliamos os efeitos da presença do resíduo de Thr ou Ser na TC de duas Prx-2 Cys típicas de levedura sobre a atividade inibitória de Adn. Demonstramos então que Adn possui maior afinidade por enzimas contendo Ser na TC e que a presença da Ser nesta posição existe essencialmente em procariotos, sobretudo em bactérias. No presente estudo, dando continuidade a este trabalho, verificamos que AhpCs contendo uma Ser na TC são mais resistentes à superoxidação e apresentariam ainda uma atividade de holdase em situações de estresse térmico mais acentuada do que AhpCs contendo uma Thr nessa posição. Uma vez que o estresse oxidativo e térmico são barreiras impostas à infecção por bactérias pelo organismo hospedeiro, a inibição desse sistema de defesa poderia constituir uma estratégia de importância no combate a esses patógenos. Nesse sentido, avaliamos a capacidade inibitória de Adn sobre AhpCs e verificamos que Adn apresenta maior afinidade por AhpC de Escherichia coli (EcAhpC), independente da presença de Thr ou Ser na TC (EcAhpCWT e EcAhpCT44S) do que para as isoformas de humanos, apresentando um valor de IC50 ≈ 5 e 50 vezes inferior do que para PrxI e PrxII, respectivamente. Adicionalmente, observamos que Adn também inibiu a atividade de Trx de E. coli (EcTrx1) com maior eficiência que a Trx de humanos. Verificamos ainda que Adn mostrou-se tóxica para bactérias Gram-positivas, apresentando valores de IC50 na faixa de 110-140µM, e para Gram-negativa (E. coli) quando tratadas com polimixina B nonapeptídeo (PMBN). Uma vez que PMBN também apresenta atividade antibiótica, sua utilização em associação com Adn pode representar uma potencial nova terapia antimicrobiana. Adicionalmente, realizamos uma triagem de compostos naturais oriundos da biota florística do Estado de São Paulo e identificamos uma molécula capaz de inibir a atividade peroxidásica de EcAhpC com eficiência semelhante à Adn, além de inibir também EcTrx1 com menor eficiência. Este composto representa o primeiro inibidor de Prx da classe das lactonas sesquiterpênicas, além de ser também o primeiro inibidor proveniente da biodiversidade brasileira. Por fim caracterizamos uma nova tiól proteína de X. fastidiosa e demonstramos que ela é capaz de proteger as células bacterianas contra o cianeto e o estresse oxidativo gerado por H2O2. Coletivamente os resultados obtidos neste trabalho contribuem para a descoberta de novas abordagens de combate a bactérias patogênicas.
The increase in resistance to antibiotics by microorganisms has grown dramatically and experts have warned of the rapid ineffectiveness of drugs against super-resistant bactéria strains. The bacterial resistance to antibiotics can be directly related to oxidative stress, since different antibiotics have the convergent capacity to produce reactive oxygen species, which cause damage to macromolecules, especially to DNA, increasing mutation rates, which can contribute to appearance of super-resistant strains. Reactive oxygen and nitrogen species are also produced by the host immune system to combat pathogens and studies indicate that inhibition of the expression of pathogen antioxidant enzymes is able to generate a significant intracellular increase in these species, which significantly interferes in their survival. It has recently been demonstrated that a natural compound named adenantin (Adn) is able to inhibit the peroxidase activity of typical 2-Cys peroxyredoxins (Prx) from humans (PrxI and PrxII), resulting in the mortality of tumor cells, but presenting low toxicity to normal cells . 2-Cys Prx are capable of decomposing hydroperoxides using a cysteine residue (CysP) and their high reactivity over different types of hydroperoxide is achieved by interactions with Thr/Ser and Arg residues, absolutely conserved between 2-Cys Prx. Together, CysP, Thr / Ser and Arg, are called catalytic triad (CT). Typical 2-Cys Prx are also present in bacteria and are named AhpC, being considered one of the fundamental oxidative defense fronts for the pathogen. In a previous project, we have evaluated the effects of the presence of the Thr or Ser residue on the TC of two yeast typical 2-Cys Prx on the inhibitory activity of Adn. We then demonstrated that Adn has higher affinity for enzymes containing Ser on TC and that the presence of Ser in this position exists essentially in prokaryotes, especially in bacteria. In the present study, continuing this work, we have found that AhpCs containing a Ser on TC are more resistant to overoxidation and would have higher holdase activity during thermal stress than AhpCs containing a Thr in that position. Since oxidative and thermal stress are barriers imposed to bacteria infection by the host organism, the inhibition of this defense system could be an important strategy in the fight against these pathogens. So, we have evaluated the inhibitory capacity of Adn over AhpCs and found that Adn has a higher affinity for AhpC of Escherichia coli (EcAhpC), regardless of the presence of Thr or Ser on TC (EcAhpCWT and EcAhpCT44S), than for human isoforms, presenting an IC50 value ≈ 5 and 50 times lower than for PrxI and PrxII, respectively. Additionally, we observed that Adn also inhibited E. coli Trx activity (EcTrx1) with higher efficiency than human Trx. We also found that Adn was toxic to Gram-positive bacteria, with IC50 values in the range of 110-140µM, and to Gram-negative (E. coli) when treated with polymyxin B nonapeptide (PMBN). Since PMBN also has antibiotic activity, its use in association with Adn may represent a potential new antimicrobial therapy. Additionally, we carry out a screening of natural compounds from the floristic biota of the State of São Paulo and identified a molecule capable of inhibiting the peroxidase activity of EcAhpC with similar efficiency to Adn, besides inhibiting EcTrx1 with less efficiency. This compound represents the first Prx inhibitor in the sesquiterpene lactone class, in addition to being the first inhibitor derived from Brazilian biodiversity. Finally, we have characterized a new thiol protein from Xylella fastidiosa and demonstrated that it is able to protect bacterial cells against cyanide and oxidative stress generated by H2O2. Collectively, the results obtained in this work contribute to the discovery of new approaches to combat pathogenic bacteria.
The increase in resistance to antibiotics by microorganisms has grown dramatically and experts have warned of the rapid ineffectiveness of drugs against super-resistant bactéria strains. The bacterial resistance to antibiotics can be directly related to oxidative stress, since different antibiotics have the convergent capacity to produce reactive oxygen species, which cause damage to macromolecules, especially to DNA, increasing mutation rates, which can contribute to appearance of super-resistant strains. Reactive oxygen and nitrogen species are also produced by the host immune system to combat pathogens and studies indicate that inhibition of the expression of pathogen antioxidant enzymes is able to generate a significant intracellular increase in these species, which significantly interferes in their survival. It has recently been demonstrated that a natural compound named adenantin (Adn) is able to inhibit the peroxidase activity of typical 2-Cys peroxyredoxins (Prx) from humans (PrxI and PrxII), resulting in the mortality of tumor cells, but presenting low toxicity to normal cells . 2-Cys Prx are capable of decomposing hydroperoxides using a cysteine residue (CysP) and their high reactivity over different types of hydroperoxide is achieved by interactions with Thr/Ser and Arg residues, absolutely conserved between 2-Cys Prx. Together, CysP, Thr / Ser and Arg, are called catalytic triad (CT). Typical 2-Cys Prx are also present in bacteria and are named AhpC, being considered one of the fundamental oxidative defense fronts for the pathogen. In a previous project, we have evaluated the effects of the presence of the Thr or Ser residue on the TC of two yeast typical 2-Cys Prx on the inhibitory activity of Adn. We then demonstrated that Adn has higher affinity for enzymes containing Ser on TC and that the presence of Ser in this position exists essentially in prokaryotes, especially in bacteria. In the present study, continuing this work, we have found that AhpCs containing a Ser on TC are more resistant to overoxidation and would have higher holdase activity during thermal stress than AhpCs containing a Thr in that position. Since oxidative and thermal stress are barriers imposed to bacteria infection by the host organism, the inhibition of this defense system could be an important strategy in the fight against these pathogens. So, we have evaluated the inhibitory capacity of Adn over AhpCs and found that Adn has a higher affinity for AhpC of Escherichia coli (EcAhpC), regardless of the presence of Thr or Ser on TC (EcAhpCWT and EcAhpCT44S), than for human isoforms, presenting an IC50 value ≈ 5 and 50 times lower than for PrxI and PrxII, respectively. Additionally, we observed that Adn also inhibited E. coli Trx activity (EcTrx1) with higher efficiency than human Trx. We also found that Adn was toxic to Gram-positive bacteria, with IC50 values in the range of 110-140µM, and to Gram-negative (E. coli) when treated with polymyxin B nonapeptide (PMBN). Since PMBN also has antibiotic activity, its use in association with Adn may represent a potential new antimicrobial therapy. Additionally, we carry out a screening of natural compounds from the floristic biota of the State of São Paulo and identified a molecule capable of inhibiting the peroxidase activity of EcAhpC with similar efficiency to Adn, besides inhibiting EcTrx1 with less efficiency. This compound represents the first Prx inhibitor in the sesquiterpene lactone class, in addition to being the first inhibitor derived from Brazilian biodiversity. Finally, we have characterized a new thiol protein from Xylella fastidiosa and demonstrated that it is able to protect bacterial cells against cyanide and oxidative stress generated by H2O2. Collectively, the results obtained in this work contribute to the discovery of new approaches to combat pathogenic bacteria.
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Palavras-chave
Biologia molecular, Antibióticos, Peroxirredoxinas, Inibição enzimática