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Biochemical and functional characterization of Xylella fastidiosa XadA3 adhesin

Grant number: 14/03617-1
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): May 01, 2014
Effective date (End): July 31, 2016
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Aline Maria da Silva
Grantee:Ana Paula Silva de Souza
Home Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Xylella fastidiosa causes serious diseases in citrus, grapevines, almond, coffee among other plants. This phytopathogen presents on its surface several fimbrial and afimbrial adhesins that are important for efficient colonization of the insect vector as well as the host plant. Among the encoded adhesins in the X. fastidiosa genome (strain 9a5c isolated from citrus or Temecula strain, isolated from vines) there are three adhesins classified as trimeric autotransporters (XadA1, Hsf/XadA2 and Hsf/XadA3). Involvement XadA1 and XadA2 in biofilm formation and some aspects of their expression regulation have been partially studied in previous work. However XadA3 function and biochemical properties are still unknown. In this scenario we started the project aiming the biochemical and functional characterization of Xylella fastidiosa XadA3 adhesin (FAPESP Masters Scholarship/Process 2011/15217-0). The results we obtained show that the XadA3 adhesin is not secreted and shows higher expression in biofilm than in planktonic cells, as expected for trimeric autotransporter proteins, which are located in the outer membrane of Gram-negative bacteria. The XadA3 mutant (delta-xadA3) showed decreased cell aggregation and biofilm formation when compared to the wild type strain Temecula. Assays performed in collaboration with the group of Prof. Steve Lindow showed that delta-xadA3 mutant is deficient in transmission by the insect vector and displays a hypervirulent phenotype in grapevines. The results we have accumulated so far reveals the importance of this adhesin to biofilm formation and cell-cell adhesion in X. fastidiosa in vitro, in the insect vector and the in xylem vessels. The project presented here will deepen the biochemical and functional characterization of X. fastidiosa XadA3 protein through additional analysis of the phenotype of the delta-xadA3 mutant as their ability to substrate adhesion, biofilm formation and motility under flow conditions. The effect of the xadA3 deletion on the expression level of other adhesins and other genes will be evaluated and compared to the effect of deletion of the gene XadA1 and XadA2 adhesin. The xadA3 expression regulation will be investigated by analyzing the expression level of its transcript in strains defective in signaling pathways. We will also initiate studies aiming at unraveling the XadA3 adhesion mechanisms through the utilization of anti-XadA3 antibody as a potential interferer in intercellular and surface adhesion. Another approach to investigate the XadA3adhesion mechanism will consist in expression of this protein in the outer membrane of E. coli, which may result in agglutination of these cells and/or increased adhesion of these cells to the surface (glass or plastic). The success of this gain of function experiment will allow further analysis of the adhesion properties of XadA3 polymorphic variants already detected in the genome of 18 strains isolated from different hosts such as citrus, vine, plum, hibiscus and coffee. (AU)