Analise comparativa da expressão de genes de Xylella fastidiosa associados a patogenicidade e formação de biofilme

AUTOR(ES)
DATA DE PUBLICAÇÃO

2004

RESUMO

The main goal of this study is to survey the expression of genes possibly involved in the pathogenicity of strain 9a5c of X. fastidiosa. Freshly-isolated bacteria from symptomatic plants (first passage condition or FP) as well as bacteria obtained after 46 transfers to axenic culture (several passage condition or SP) were utilized in this work. A possible lost of virulence of the X. fastidiosa in the SP condition was verified after inoculation into sweet orange and periwinkle plants. Using real-time quantitative PCR, we verified that the colonization of SP cells was less efficient in both hosts. The DNA microarray technology was used to investigate the global changes in gene expression associated with the pathogenic FP condition. Most of the differentially expressed genes encode hypothetical proteins. Genes potentially involved with pathogenicity, virulence and adaptation were induced in the FP condition. Three of these genes (fimA, hsf and uspA1) are associated with adhesion to the host surfaces and four (msrA, acrA, cvaC e xpsE) with adaptation in the host environment. The induction of these genes in the FP condition was confirmed by RT-PCR both in vitro and in planta 15 days after inoculation, period in which the initial colonization of the vessels was taking place. Ninety days after inoculation, when the colonization had reached to more advanced stages and the first symptoms were developed, the expression levels of the adhesion genes were similar although a higher expression was observed for genes related to adaptation in the pathogenic condition. These results suggest that the adhesion is important at the beginning of the biofilm formation, whereas the genes related to adaptation are essential for the maintenance of this biofilm in planta. We also evaluated the expression of these genes in vitro during biofilm formation by semiquantitative RT-PCR after 3, 5, 10, 20 and 30 days of growth at the medium-air interface in a glass flask. The gene expression observed under in vitro condition was similar to that observed in planta for the adhesion genes whose expression occurred mainly at the initial step of biofilm formation. These results indicate that these genes can be involved in adhesion to different attachment surfaces. In relation to the adaptation-related genes, xpsE and acrA showed and expression pattern similar to that observed in planta, with over-expression in the mature biofilm. On the other hand, the expression of cvaC and msrA did not show the same pattern as found in the in planta. These genes showed little differences in relation to the stages of biofilm development. This difference could have resulted from the different experimental designs utilized since the genetic reprogramming of gene expression of the bacterial biofilm depends on the changes in multiple environmental conditions to which the bacterium is exposed. Light transmission microscopy analysis of different phases of the biofilm formed in glass covers revealed that the X. fastidiosa biofilm development presented five distinct stages. At the 20th day, the biofilm showed high cell density. A considerable increase in the gene expression number studies involving biofilm formation has been observed, mainly for bacteria causing human diseases, since the biofilm formation has been associated with serious diseases. However, limited information is available concerning gene expression involved in biofilm formation in plant-pathogen interactions. For this reason and because biofilm formation seems to be the main pathogenicity mechanism in X. fastidiosa, we utilized the microarray technology to access changes in gene expression in mature biofilm cells when compared with planktonic cells. A total of 202 genes (9.18%) were significantly up-regulated, while 32 genes (1.45%) were downregulated in the mature biofilm. The majority of the differentially expressed genes encodes hypothetical proteins. Under the biofilm condition we observed an increase in the expression of some housekeeping genes responsible for metabolic functions. We also found a large number of genes from the pXF51 plasmid being differentially expressed. This could possibly be associated with lateral gene transfer in the X. fastidiosa biofilm. Moreover most of the pathogenicity-related genes over-expressed in the biofilm condition were associated with toxin production, detoxification and adaptation to atypical conditions. The expression of genes associated with adaptation and competitiveness in the habitat to be colonized gives a clear indication of the importance of such factors in the established biofilm of X. fastidiosa. This demonstrates that the physiological properties of this biofilm are similar to the ones observed in human pathogens, indicating that the pathogenicity mechanisms of bacteria of different host may show common characteristics.

ASSUNTO(S)

genomas bacterias dna

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