Investigando possíveis interações físicas entre componentes do sistema de detecção de fosfato e do sistema de secreção tipo III em Xanthomonas citri subsp. citri
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Data
2019-03-12
Autores
Mello, Jéssica Alcimari Ferreira
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Xanthomonas citri subsp. citri (Xac) é um fitopatógeno responsável por causar o Cancro Cítrico, relacionado a grandes perdas econômicas para a citricultura. Esta bactéria utiliza diversos mecanismos moleculares para infectar o hospedeiro, dentre eles, o Sistema de Secreção Tipo III (T3SS), considerado o principal fator de virulência expresso in planta pela Xac. Sabe-se que a ativação do T3SS é controlada por dois reguladores: HrpG e HrpX. Porém, os mecanismos que controlam estes reguladores e posteriormente a expressão do T3SS in planta ainda são pouco compreendidos. Estudos realizados por nosso grupo de pesquisa sugerem que a concentração de fosfato inorgânico (Pi) presente em meio de cultura ou em espaço apoplástico, onde a Xac causa a infecção em plantas, pode estar relacionado com a ativação do T3SS, sugerindo uma possível relação entre o Pi com os reguladores HrpG e HrpX. Em Xac, a detecção e transportorte do Pi é realizada pelo sistema de dois componentes (TCS) PhoR/PhoB, codificado pelo operon pho. Afim de averiguar a possível relação entre PhoR/PhoB com os reguladores do T3SS HrpG e HrpX, ensaios de modelagem molecular foram realizados, indicando que a proteína PhoR poderia interagir fisicamente com HrpG, fosforilando-a, e sugerindo um possível mecanismo de interação entre o TCS PhoR/PhoB e os genes reguladores do T3SS. Para testar a hipótese de interação física entre HrpG e PhoR, as proteínas recombinantes PhoR, PhoB e HrpG de Xac foram expressas em células de Escherichia coli BL21 com a finalidade de realização de ensaios de pull-down. Através da purificação por cromatografia de afinidade em coluna de Ni2+-Sepharose, as proteínas HrpG e PhoB recombinantes foram obtidas com alto grau de pureza e na forma solúvel. Entretanto a proteína PhoR se mostrou insolúvel em todos os testes de purificação e de renaturação realizados, impossibilitando a realização dos ensaios de interação física até o momento. De qualquer modo, este trabalho representa importantes avanços sobre a regulação do T3SS em Xac, tendo em vista que até o atual momento configura-se como o primeiro estudo relacionando o TCS PhoR/PhoB com HrpG.
Xanthomonas citri subsp. citri (Xac) is a plant pathogen that causes citrus canker, responsible for significant economic losses in citriculture. This bacteria uses several molecular mechanisms to infect the host, such as the Type III Secretion System (T3SS), which is considered the main factor of virulence expressed in plant by Xac. Activation of T3SS is controlled by two regulators, HrpG and HrpX, however, the mechanisms that control these regulators and subsequently the expression of T3SS in planta are poorly understood. Previosuly, our research group identified that the concentration of inorganic phosphate (Pi) present in culture media or in the apoplastic space, where Xac causes infection in plants, may be related to the activation of T3SS, suggesting a possible relationship between Pi and the HrpG and HrpX regulators. In Xac, the detection and transport of Pi is performed by the two-component system (TCS) PhoR/PhoB, encoded by the operon pho. In order to investigate the possible relationship between PhoR/PhoB and the regulators of T3SS HrpG and HrpX, molecular modeling analyses were performed and indicated that the PhoR protein could physically interact with HrpG and phosphorylate it, suggesting a possible mechanism of interaction between TCS PhoR / PhoB and the T3SS regulatory genes with subsequent activation of T3SS. To test the hypothesis, recombinant forms of PhoR, PhoB and HrpG were expressed in Escherichia coli BL21 cells for the purpose of performing pull-down assays. By using Ni2+-Sepharose affinity chromatography, recombinant HrpG and PhoB proteins were obtained with high purity and in their soluble forms. However, the PhoR protein was insoluble in all the purification and renaturation tests performed, making the subsequent physical tests impossible. Nevertheless, this work represents important towards the understanding of T3SS regulation in Xac, since this is the first study correlating the TSC PhoR /PhoB proteins and HrpG.
Xanthomonas citri subsp. citri (Xac) is a plant pathogen that causes citrus canker, responsible for significant economic losses in citriculture. This bacteria uses several molecular mechanisms to infect the host, such as the Type III Secretion System (T3SS), which is considered the main factor of virulence expressed in plant by Xac. Activation of T3SS is controlled by two regulators, HrpG and HrpX, however, the mechanisms that control these regulators and subsequently the expression of T3SS in planta are poorly understood. Previosuly, our research group identified that the concentration of inorganic phosphate (Pi) present in culture media or in the apoplastic space, where Xac causes infection in plants, may be related to the activation of T3SS, suggesting a possible relationship between Pi and the HrpG and HrpX regulators. In Xac, the detection and transport of Pi is performed by the two-component system (TCS) PhoR/PhoB, encoded by the operon pho. In order to investigate the possible relationship between PhoR/PhoB and the regulators of T3SS HrpG and HrpX, molecular modeling analyses were performed and indicated that the PhoR protein could physically interact with HrpG and phosphorylate it, suggesting a possible mechanism of interaction between TCS PhoR / PhoB and the T3SS regulatory genes with subsequent activation of T3SS. To test the hypothesis, recombinant forms of PhoR, PhoB and HrpG were expressed in Escherichia coli BL21 cells for the purpose of performing pull-down assays. By using Ni2+-Sepharose affinity chromatography, recombinant HrpG and PhoB proteins were obtained with high purity and in their soluble forms. However, the PhoR protein was insoluble in all the purification and renaturation tests performed, making the subsequent physical tests impossible. Nevertheless, this work represents important towards the understanding of T3SS regulation in Xac, since this is the first study correlating the TSC PhoR /PhoB proteins and HrpG.
Descrição
Palavras-chave
T3SS, TCS PhoR/PhoB, Expressão heteróloga, Purificação de proteínas