Full text
| |
| Author(s): |
Lígia Segatto Muranaka
Total Authors: 1
|
| Document type: | Master's Dissertation |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2010-06-15 |
| Examining board members: |
Alessandra Alves de Souza;
Anete Pereira de Souza;
Marie Anne Van Sluys
|
| Advisor: | Marco Aurélio Takita; Alessandra Alves de Souza |
| Abstract | |
Xylella fastidiosa is a Gram-negative bacterium that causes several diseases in different plant species, including citrus variegated chlorosis (CVC) in sweet orange (Citrus sinensis L. Osbeck), whose economic damage is of millions of dollars annually. The symptoms development has been associated with the blockage of xylem vessels caused by bacterial biofilm formation. Recently it was found that the biofilm of X. fastidiosa is more resistant to antimicrobial compounds than the planktonic cells. This resistance has been considered as a complex phenomenon that cannot be explained by a single mechanism, but by multi-factors. Accordingly, the intent of this study was to identify through DNA microarray technology, genes possibly involved in adaptation of biofilm cells to the presence of antimicrobial compounds in concentrations that are lethal to cells in planktonic growth, but do not inhibit the cell growth in biofilm. We found 223 (7.87%) genes induced in presence of copper and 150 (5.29%) genes repressed. For tetracycline, there were 450 (15.89%) induced genes and 449 (15.85%) repressed ones. Many genes encoding proteins related to protein synthesis, energy metabolism and cell division were negatively modulated in both, copper and tetracycline treatments, suggesting that these changes could contribute to a state of resistance. When a subinhibitory dose of copper was applied we could also observe the induction of genes related to adhesion and thus biofilm formation and toxin production, suggesting that the bacterial virulence should be increased. The opposite was found for tetracycline. However, we observed the induction of a possible SOS response mechanism in which genes related to a toxin-antitoxin system was overexpressed. This system is probably involved with programmed cell death and formation of persistent cells. We then concluded that subinhibitory doses of antimicrobial compounds could induce bacterial virulence as occurred for copper, or the formation of persistent cells, as observed for tetracycline rather than kill the cells. Another approach of this work was to carry out experiments in vitro and in vivo with an analogue of cysteine, N-acetyl-L-cysteine (NAC), which has been already used in medicine as a drug for disruption of biofilms formed by human pathogenic bacteria. Results of cellular mass quantification, number of viable cells and total exopolysaccharide content revealed that all doses (1.0, 2.0 and 6.0 mg / mL) of NAC tested in in vitro experiments decreased the biofilm formation and inhibited growth of X. fastidiosa, which indicated that this substance could also be toxic for the bacteria. In vivo experiments showed a strong reduction in CVC symptoms in C. sinensis plants infected and treated with different doses of NAC. These studies open a real prospect for the use of this compound in CVC management. (AU) | |