INVESTIGADORES
SERRADELL Maria De Los Angeles
congresos y reuniones científicas
Título:
Caracterización de la invasión in vitro de mutantes negativos para ipaBCDA de Shigella spp
Autor/es:
ROJAS, F; CORTÉS, L; FERNÁNDEZ, M; SERRADELL, M; DE URRAZA, P
Lugar:
Mar del Plata, Argentina
Reunión:
Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2007
Institución organizadora:
Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)
Resumen:
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.