Characterization of cytotoxic heat-stable enterotoxin (ST-L) produced by Escherichia coli isolated from drinking water / Caracterização da enterotoxina citotoxica termoestavel (ST-L) produzida por Escherichia coli isoladas de agua de consumo

AUTOR(ES)
DATA DE PUBLICAÇÃO

2009

RESUMO

Studies on the virulence properties of Escherichia coli associated with intestinal infections are important for the understanding of diarrheic diseases mechanisms. In a previous work we have described a factor presenting enterotoxic and cytotoxic activities in the culture supernatants of E. coli isolated from drinking water in the city of Ouro Preto, MG. This factor was called as thermo stable enterotoxin - like (ST-L) and it was purified from cultured supernatants produced with modified CAYE medium, fractionated with ammonium sulfate, being subsequently ultrafiltrated, subjected to molecular exclusion chromatography and then to reversed-phase chromatography in HPLC-system. The ST-L showed to be pure by the electrophoretic analysis in the bidimensionl electrophoresis (2D), being the isoelectric point (pI) of 6.19 and the molecular mass of approximately 2kDa. The toxin can not be inactivated by enzimes like trypsin, proteinase K, ribonuclease or pepsin. The cytotoxic activity of ST-L does not change whith pH variations (3 – 9), does not change after treatment with organic solvents and resists heating treatments of 100°C showing it to be heat-stable. Biological characterization of the toxin showed, through the tests of cell viability with the ST-L, that several cell lines were sensitive to the cytotoxin action, with major cellular and nuclear changes observed within 24 hour assays, presenting cytoplasmic vacuoles and cell death, what was verified by tests of cell viability of lysosomes, mitochondria, cell membranes and quantification of the cell population on cell lines like HEp-2, Vero, CaCo-2 and HT-29. The rabbits ileal intestinal loop tests, performed with the ST-L, did not show positive results, but the toxin acts by inducing intestinal fluid accumulation into the newborn mice test (Dean). Through molecular characterization tests, we found that the gene enconding for the toxin ST-L is in a plasmid and has a conserved region with high similarity to estA2, what justifies the presence of the positive enterotoxic result in the Dean test. However, it was not possible to determine the fragment responsible for cytotoxic activity of ST-L. According to the characteristics presented we suggest that the toxin-ST-L is a variant group of the heat-stable enterotoxin (ST) with cytotoxic activity produced by the diarrheagenic E. coli.

ASSUNTO(S)

toxinas termoestaveis enterotoxinas enterotoxins heat-stable toxins escherichia coli citotoxicidade cytotoxicity escherichia coli

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