Principal

CROSSTALK AMONG COMMENSAL E. coli AND ENTEROPATHOGENIC Escherichia coli (EPEC)



AC39


Paiva, FPT; Sircili, MP


Laboratório de bacteriologia, Instituto Butantan


franpaiva@butantan.gov.br

Introduction: Quorum sensing is used to designate a mechanism of gene regulation depending on cellular density. The bacteria produce substances that accumulate in growth media, and when these substances reached a threshold concentration, a variety of responses can occur. These molecules are called autoinducers and the phenomena called quorum sensing. Quorum sensing is widespread in gram negative and gram positive bacteria and is involved in various cellular mechanisms, including virulence regulation. At least, three autoinducers were described in literature, and were designated AI-1 (autoinducer 1), AI-2 (autoinducer 2) and AI-3 (autoinducer 3). Objectives: This study was developed to verify the possibility of crosstalk among EPEC and other enterobacteria, in intestinal infections. Methods: Pre conditioned medium : Bacterial strains were grown to 37ºC with agitation of 250 rpm in LB 0.4M NaCl until OD600 of 1.0. The growth was centrifuged, and supernatant were filtered in 0.22 µm membrane. βgalactosidase assay: A reporter TEVS232 strain containing the LEE1:lacZ fusion  was grown in pre conditioned medium until reach an OD600 ≤ 0.2, and  βgalactosidase activity was measured in Miller units. Adherence assays: Bacterial strains were grown for 18h in LB medium at 37ºC. From the uninduced overnight cultures, 105 CFU was added to HEp-2 cells, incubated for 3h at 37ºC with 5% CO2 , washed with PBS, fixed with methanol and stained with Giemsa stain. For quantification of adherence, after 3h incubation, the nonadherent bacteria were removed by PBS washing and HEp-2 cells were lysed with 1% Triton. Serial dilutions of the bacterial cells were plated in LB agar, and CFU were counted. Results: It was possible to verify the production of autoinducer AI-3 in the strains tested, and also quantify their ability of induction. Apparently, the inoculation of cell cultures with enterobacteria influences the cellular responses to EPEC strains. Discussion and conclusion: Pre conditioned media of this strains were capable to activate the transcription of  LEE1:lacZ fusion, indicating that the molecule of autoinducer can be produced by a strain and recognized by another, highlighting the possibility of interspecific crosstalk. Using strains of enterobacteria in adherence assays, we observed a different interaction of EPEC with HEp-2 cells.

quorum sensing, enteropathogenic Escherichia coli (EPEC)

FAPESP

Categorias: Parasitologia, Microbiologia, Imunologia e Hematologia