Estudo da interação da proteína repressora de arginina (ArgR) de Clostridium botulinum, Porphyromonas gingivalis, Nocardia farcinica e Corynebacterium pseudotuberculosis com possíveis ligantes
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Data
2021-07-08
Autores
Jeronymo, Giovanna Tavares
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Universidade Estadual Paulista (Unesp)
Resumo
Clostridium botulinum é o agente etiológico do botulismo, que ocorre principalmente após o consumo de alimentos contaminados com a toxina botulínica. Essa toxina impede a contração muscular e provoca paralisia, podendo afetar os nervos autônomos. Já a bactéria Porphyromonas gingivalis está envolvida na patogênese da periodontite, sendo considerada também um fator de risco para outras complicações. A espécie Nocardia farcinica é um actinomiceto responsável por infecções pulmonares, abscessos subcutâneos e cerebrais e bacteremia. A bactéria Corynebacterium pseudotuberculosis é o agente etiológico da linfadenite caseosa (LC), doença infectocontagiosa crônica e de relevância econômica, que causa perdas econômicas significativas na produção de ovinos e caprinos. Todas essas espécies codificam a proteína repressora de arginina (ArgR), de forma que após atingir uma determinada concentração de arginina no citoplasma, esse ligante interage com a ArgR e o complexo formado (ArgR/Arg) se acopla ao promotor do DNA interrompendo a síntese. A sequência de aminoácidos da ArgR dos patógenos citados apresenta particularidades em relação ao sítio de ligação da arginina de forma que a ArgR de C. botulinum e C. pseudotuberculosis apresenta um resíduo de prolina na posição 115 (Pro115), enquanto P. gingivalis apresenta alanina (Ala115) e N. farcinica glicina (Gly115), diferentemente da maioria das ArgRs, as quais possuem aminoácidos Gln ou His na posição 115. O conhecimento dessas particularidades pode ser utilizado para o desenvolvimento de ligantes que possam inibir a via de síntese da arginina desses patógenos. Levando isso em conta, o objetivo do presente trabalho foi expressar a proteína ArgR de C. botulinum, P. gingivalis, N. farcinica e ArgR mutante P115H de C. pseudotuberculosis em Escherichia coli, purificá-las em condições nativas e desnaturantes e realizar ensaios de cristalização para a obtenção das estruturas tridimensionais cristalográficas dessas proteínas. Além disso, também foram realizados Ensaios de Espalhamento Dinâmico de Luz (DLS), Dicroísmo Circular (CD), modelagem por homologia e simulações de Dinâmica Molecular. Ensaios de Espalhamento Dinâmico de Luz (DLS) sugerem que o ligante Tirosina pode induzir a hexamerização das ArgRs de P. gingivalis e N. farcinica. Já resultados obtidos por meio de modelagem e dinâmica molecular indicam que outros fatores além do volume do pocket das ArgRs podem influenciar na especificidade pelo ligante. Essas constatações podem ser usadas para o desenvolvimento de ligantes que possam ocupar o sítio de ligação da arginina da ArgR dos patógenos citados, de forma que sua via de síntese seja interrompida e resulte na inibição da proliferação desse patógenos.
Clostridium botulinum is the etiological agent of botulism, which occurs mainly after consumption of foods contaminated with botulinum toxin. This toxin prevents muscle contraction and causes paralysis, which can affect the autonomic nerves. The bacterium Porphyromonas gingivalis is involved in the pathogenesis of periodontitis, and is also considered a risk factor for other complications. The species Nocardia farcinica is an actinomycete responsible for lung infections, subcutaneous and cerebral abscesses and bacteremia. The bacterium Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis (CL), a chronic infectious disease of economic relevance, which causes significant economic losses in the production of sheep and goats. All these species encode the arginine repressor protein (ArgR), so that after reaching a certain concentration of arginine in the cytoplasm, this ligand interacts with the ArgR and the complex formed (ArgR/Arg) couples to the DNA promoter, interrupting the synthesis. The amino acid sequence of the ArgR of the aforementioned pathogens has particularities in relation to the arginine binding site, such that the ArgR of C. botulinum and C. pseudotuberculosis has a proline residue at position 115 (Pro115), while P. gingivalis has alanine (Ala115) and N. farcinica glycine (Gly115), unlike most ArgRs, which have amino acids Gln or His at position 115. The knowledge of these particularities can be used for the development of ligands that can inhibit the arginine synthesis pathway of these pathogens. Taking this into account, the aim of the present work was to express the ArgR protein from C. botulinum, P. gingivalis, N. farcinica and P115H mutant ArgR from C. pseudotuberculosis in Escherichia coli, purify them under native and denaturing conditions and carry out assays of crystallization to obtain the three-dimensional crystallographic structures of these proteins. In addition, Dynamic Light Scattering (DLS), Circular Dichroism (CD), homology modeling and Molecular Dynamics simulations were also performed. Dynamic Light Scattering Assays (DLS) suggest that the Tyrosine ligand can induce the hexamerization of P. gingivalis and N. farcinica ArgRs. On the other hand, results obtained through modeling and molecular dynamics indicate that factors other than the pocket volume of ArgRs can influence the specificity for the ligand. These findings can be used for the development of ligands that can occupy the arginine binding site of the ArgR of the aforementioned pathogens, so that its synthesis pathway is interrupted and results in the inhibition of the proliferation of these pathogens.
Clostridium botulinum is the etiological agent of botulism, which occurs mainly after consumption of foods contaminated with botulinum toxin. This toxin prevents muscle contraction and causes paralysis, which can affect the autonomic nerves. The bacterium Porphyromonas gingivalis is involved in the pathogenesis of periodontitis, and is also considered a risk factor for other complications. The species Nocardia farcinica is an actinomycete responsible for lung infections, subcutaneous and cerebral abscesses and bacteremia. The bacterium Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis (CL), a chronic infectious disease of economic relevance, which causes significant economic losses in the production of sheep and goats. All these species encode the arginine repressor protein (ArgR), so that after reaching a certain concentration of arginine in the cytoplasm, this ligand interacts with the ArgR and the complex formed (ArgR/Arg) couples to the DNA promoter, interrupting the synthesis. The amino acid sequence of the ArgR of the aforementioned pathogens has particularities in relation to the arginine binding site, such that the ArgR of C. botulinum and C. pseudotuberculosis has a proline residue at position 115 (Pro115), while P. gingivalis has alanine (Ala115) and N. farcinica glycine (Gly115), unlike most ArgRs, which have amino acids Gln or His at position 115. The knowledge of these particularities can be used for the development of ligands that can inhibit the arginine synthesis pathway of these pathogens. Taking this into account, the aim of the present work was to express the ArgR protein from C. botulinum, P. gingivalis, N. farcinica and P115H mutant ArgR from C. pseudotuberculosis in Escherichia coli, purify them under native and denaturing conditions and carry out assays of crystallization to obtain the three-dimensional crystallographic structures of these proteins. In addition, Dynamic Light Scattering (DLS), Circular Dichroism (CD), homology modeling and Molecular Dynamics simulations were also performed. Dynamic Light Scattering Assays (DLS) suggest that the Tyrosine ligand can induce the hexamerization of P. gingivalis and N. farcinica ArgRs. On the other hand, results obtained through modeling and molecular dynamics indicate that factors other than the pocket volume of ArgRs can influence the specificity for the ligand. These findings can be used for the development of ligands that can occupy the arginine binding site of the ArgR of the aforementioned pathogens, so that its synthesis pathway is interrupted and results in the inhibition of the proliferation of these pathogens.
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Palavras-chave
Proteína repressora de arginina, Tirosina, Ligantes, Modelagem, Arginine repressor protein, Tyrosine, Ligands, Modeling