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Comparative proteomic analysis between wild-type Xanthomonas citri subsp citri and its deletion mutant treA, and the in vitro interaction of the respective proteomes with the recombinant trehalase

Grant number: 17/23196-9
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): February 01, 2018
Effective date (End): January 31, 2019
Field of knowledge:Biological Sciences - Microbiology - Applied Microbiology
Principal Investigator:Maria Teresa Marques Novo Mansur
Grantee:Solange Cristina Antão
Home Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil

Abstract

Citrus canker provides a drop in citrus fruit yield and quality as a result of a lack of efficient control and eradication measures. It is caused by the bacterium Xanthomonas citri subsp citri (Xcc), of fast dispersion and high degree of virulence. In a work previously performed by our research group, a mutant of Xcc was produced by deletion of the treA gene, present in a single copy in the genome of Xcc strain 306 and encoding the enzyme trehalase. This enzyme catalyzes the hydrolysis of the disaccharide trehalose in two glucose monomers. There are studies that relate the performance of trehalose with mechanisms of protection of the bacterium, and in this sense, the deletion of the treA gene led to a greater virulence of Xcc. The objective of this work is to compare the wild-type proteome with that of the mutant strain (Xcc”treA) for a better understanding of the role of trehalase in the infectious process of Xcc, since the identification of other proteins affected by gene deletion may reveal possible pathways of interaction. Growth curves of the two strains will be performed in XAM-M pathogenicity inducer broth to determine the logarithmic phase of bacterial growth, at which point the collection of cells will be performed for the differential proteomic analysis. Total proteins will be extracted, quantified and used in two-dimensional polyacrylamide gel electrophoresis (2D-PAGE and / or 2D-DIGE) assays. Spots identified as being differential between the two strains will be excised, trypsin digested and subjected to identification by mass spectrometry. A Pull Down assay will also be conducted to investigate possible interactions of Xcc trehalase, produced in recombinant form in our research group, with other Xcc proteins in vitro. The results may give indications of the mechanisms involved in the metabolism of trehalose and its relation with the pathogenicity in Xcc. (AU)