Characterization of in vitro invasion of Shigella spp. ipaBCDA negative mutants

ROJAS, FLORENCIA; CORTÉS, LEANDRO E.; FERNÁNDEZ, MARCELO A.; SERRADELL, MARÍA; DE URRAZA, PATRICIO J.

Mar del Plata

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Bioquímica y Biología Molecular (SAIB); 2007

Sociedad Argentina de Bioquímica y Biología Molecular (SAIB)

It is known that genes encoded in the ipaBCDA operon of Shigella spp are necessary to invade and escape from infected enterocytes. In previous works we described clinical isolates of Shigella that invade Hep-2 cells monolayers even thought the absence of amplification of the genes of the ipaBCDA operon by PCR assays. The aims of this work are analyze ipaBCDA mutants for Ipa proteins production by immunoblotting and in vitro invasion by Confocal Microscopy. Four isolates of ipaBCDA negative mutants were analyzed by dot-blot and Western-blot with anti-IpaB monoclonal antibodies. In these assays whole cell extract of bacteria with or without incubation with Hep-2 cells were used. Three of these mutants were tagged with GFP reporter plasmids and used for invasion assays in Hep-2 cells. For this assay WT Shigella ipaBCDA positive isolate were labeled with carboxyfluorescein. Hep-2 infected cells with WT and mutants of Shigella were analyzed by Confocal Microscopy to locate intracellular bacteria. One of the ipa- mutants was located in the cytoplasm of the Hep-2 infected cells, as well as WT. Dot and Western-blot analysis of the mutant confirm the absence of IpaB protein in detectable limits of all four ipaBCDA negative mutants. According with these observations it is possible that Shigella ipa- mutant posses a different in vitro invasion mechanism than WT bacteria.