Predição de proteínas parceiras de interação da trealase, enzima relacionada com a patogenicidade de Xanthomonas citri subsp. citri
Resumo
Known as one of the main phytopathologies that affect the citrus industry worldwide, citrus canker is caused by the bacterium Xanthomonas citri subsp. citri (XAC) and is responsible for causing premature drop and reduction in fruit quality, directly affecting productivity and causing a relevant economic impact. Through previous proteomic analysis of our research group, it was possible to identify the periplasmic trehalase enzyme (encoded by the XAC 0604 gene) as a protein expressed in XAC when cultivated in pathogenicity-inducing medium (XAM-M). For the purpose of functional studies, a mutant strain deleted in the gene encoding the trehalase enzyme (XACΔ0604) was previously obtained by our group, as well as the construction of the vector to obtain the recombinant trehalase protein by heterologous expression in E. coli. Considering the scarcity of works in the literature about the functionality of this enzyme, especially in XAC, the present work aimed to verify if there is an interaction between trehalase and other cellular proteins and if there is a change in the interaction profile when using the mutant strain lysate (XACΔ0604), compared to the wild strain (XAC). Thus, the recombinant trehalase was immobilized in a column for a pull-down experiment (in vitro), followed by the addition of cell lysate from wild (XAC306) or mutant (XACΔ0604) strains, cultivated in duplicate (A and B) in pathogenicity-inducing medium (XAM-M). After successive washing steps, the complex was eluted from the column with 250 mM and 2 M imidazole. Analysis of the profiles of the constituent proteins of the eluate was performed by SDS-PAGE, the most evident bands were isolated and digested using trypsin for further analysis by mass spectrometry. For the wild strain, bands that co-eluted with the expected size band for trehalase (~61kDa) for replica A were observed, with approximate molecular mass values of 51, 46, 27 and 19 kDa, while for the replica B, the bands that stood out were those with values of 51, 46 and 27 kDa. The eluate obtained with the mutant strain lysate showed bands of 51 and 27 kDa by SDS-PAGE, similar for both cultures A and B (replicas), in addition to the band predicted for trehalase. Thus, the 51 and 27 kDa bands were recurrent between the two strains. The identification step by mass spectrometry of the proteins present in the isolated bands was made impossible due to the interruption of LNBio-Campinas activities during the COVID-19 pandemic, which was an important setback for the completion of the present work. Thus, we performed the protein-protein interaction prediction by the STRING bioinformatics tool, where it could be foreseen that a likely interaction partner of trehase in XAC is the protein trehalose-6-phosphate phosphatase (OtsB), involved in trehalose biosynthesis, which has a molecular mass of 27.0 kDa, which corresponds to the mass of one of the recurrent bands identified by the pull-down both in the wild and in the mutant. The pull-down results, after further identification of the XAC trehalase partner proteins by mass spectrometry, may complement the differential proteomics analysis previously performed in our group, between the trehalase deletion mutant and XAC, and may contribute to greater understanding the functionality of trehalase in XAC and its relationship to pathogenicity.
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