CIDCA   05380
CENTRO DE INVESTIGACION Y DESARROLLO EN CRIOTECNOLOGIA DE ALIMENTOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Adhesion factors and signaling pathways involved in the interaction between Bacillus cereus and cultured human enterocytes.
Autor/es:
MINNAARD, J., ROLNY, I. AND PÉREZ, P. F.
Lugar:
Córdoba
Reunión:
Congreso; VI Congreso Argentino de Microbiología General (SAMIGE); 2009
Institución organizadora:
SAMIGE
Resumen:
Adhesion factors and signaling pathways involved in the interaction between Bacillus cereus and cultured human enterocytes.   Jessica Minnaard1,2, Ivanna S. Rolny1,2, Pablo F. Pérez1,2 *   1. CIDCA (CONICET. La Plata). 2. Cátedra de Microbiología. Facultad de Ciencias Exactas, UNLP. (pfp@biol.unlp.edu.ar)   In the context of intestinal pathologies, Bacillus cereus is involved in two syndromes: emetic and diarrheic. Even though pathogenesis of B. cereus was traditionally ascribed to toxin production, recent studies demonstrated that adhesion-invasion onto enterocytes could also contribute to B. cereus virulence The present study sough to gain further insight on the factors involved in the interaction of B. cereus with cultured human enterocytes as well as on the signaling pathways responsible for the biological effects. Five strains of B cereus were used: ATCC 10876, T1, M2, 2 and B10502. Cultured human enterocytes (Caco-2 cells) were incubated for 2 h (1 x 108 CFU/well ) with bacteria from mid-log cultures in DMEM containing 100 µg/ml cloramphenicol at 37°C in a 5 % CO2 atmosphere. Associated bacteria (adhering + invading) were evaluated by microscopy on stained smears and plate counts. Invasion was evaluated by plate counts after killing of exocellular bacteria by gentamicin. Signaling pathways involved in the interactions were assessed by using specific inhibitors. Cytoskeleton was labeled with FITC-phaloidin. Killing of bacteria by UV irradiation decreases association of B. cereus to enterocytes thus demonstrating that bacterial viability is crucial for the interaction with eukaryotic cells. Treatment of bacteria with LiCl (5 M), proteases (trypsin or chemotrypsin) or exhaustive washing with PBS showed that non-covalent bound structures, proteins and loosely associated material are involved in the interaction in a strain-dependent manner. In contrast when bacteria were treated with metaperiodate (50 mM), association values were not modified. Extracellular factors from strain 2 increased its own association and invasion values and also association of strain B10502. Calcium depletion significantly increased the ability of strains T1 and 2 to gain access to the intracellular domain but did not modify association values. In addition, higher association/invasion values were found for cells cultured for 4 days as compared with. cells cultured for 9, 15 and 21 days. Dantrolene, wortmannin), U73122  and staurosporin were able to abrogate citoskeleton disruption that follows infection of enterocytes by B. cereus. Adhesion of strain T1 decreased in the presence of U73122, wortmannin and when those inhibitors were used together. In contrast, invasion values were diminished by U73122 and not by wortmannin . Results show that different bacterial structures are related to the interaction of B. cereus with cultured human enterocytes. In addition, signalling cascades related to phosphorilated lipids are involved in the profound modifications on the cytoskeleton that follows infection of cells by B. cereus. These changes would in turn facilitate the invasion of the monolayers through both the apical and the basolateral domains.