Full text
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| Author(s): |
Raquel Caserta
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2008-01-09 |
| Examining board members: |
Alessandra Alves de Souza;
Anete Pereira de Souza;
Wellington Luiz Araujo
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| Advisor: | Alessandra Alves de Souza |
| Abstract | |
The importance of citriculture in Brazilian's economy is unquestionable. Brazil is the major exporter of concentrate juice in the world. In 2007, Brazilian exportations of orange almost reached 400 million boxes, reflecting the importance of a product that generates an enormous diversity of direct and indirect jobs, even helping the insums industry. In Brazil, São Paulo state has a great prominence in orange production. In the 80's, the citrus fields were severely harmed by Citrus Variegated Chlorosis (CVC), a disease that causes losses of million dollars since it affects the fruit size and, consequently, juice production. It was proved that the Xylella fastidiosa, a phytopathogen that grows limited to the xylem vessels, is the causal agent of CVC. Due to the importance of citriculture in the São Paulo state, the genome of X. fastidiosa was sequenced and there were found many genes related to adhesion, some of them similar to human and animal pathogens. This fact suggested that adhesion and biofilm formation were essential for the survey of the bacteria within the plant. This hypothesis is supported by the fact that symptomatic plants have colonies of bacteria adhered to the walls in xylem vessels. Subsequent works demonstrated that the colonization occurs through the formation of a biofilm that blocks the vessels, reducing the efficiency of water and sap transportation. In this context, the study of the role of the fimbrial or afimbrial adhesion proteins is fundamental to the elucidation of the biofilm structure and formation. These adhesins show different functions, and because of that they are not expressed at the same time during biofilm formation. In face of what was shown above, the aim of this work was to monitor the expression of two fimbrial and two non fimbrial adhesins involved in biofilm formation in vitro. Western blot and fluorescence microscopy using polyclonal antibodies developed against the target proteins were performed, and the results revealed that fimbrial adhesins are expressed preferentially at the initial phases of biofilm formation, while non fimbrial adhesins are expressed at the late phases, when the biofilm already presents cellular aggregation traits. This expression pattern suggests that the initial adhesion of the bacteria to the substrate is mediated by fimbrial adhesins and that the function of cell to cell adhesion is performed by non fimbrial adhesins. Besides, each protein studied is found more or less to be expressed during all stages of biofilm development, suggesting the existence of a regulatory mechanism that results in a biological interaction between these proteins, in order to keep the stability and structure of the biofilm. In order to accompany the expression of these adhesins in vivo, ultra thin sections of petioles of periwinkle, hibiscus and citrus infected by X. fastidiosa and presenting symptoms were prepared. All the sections showed the presence of the target proteins, suggesting that they are necessary to the infectious process of the plants. The results demonstrated that fimbrial and non fimbrial proteins, as well as in human pathogens, are necessary for the biofilm formation by X. fastidiosa. This is the only phytopathogen which requires the formation of a biofilm to cause the disease. In this way, the results obtained in this work may contribute with the development of possible approaches to control diseases caused by biofilm formation, once the expression profile of each adhesin was determined. In a possible attempt to prevent the symptoms, the interaction between these adhesins could be blocked, breaking the stability of biofilm (AU) | |