CHARACTERIZATION OF INVASION PLASMID SPONTANEUS MUTANTS OF Shigella spp.

FERNÁNDEZ MARCELO ADRIÁN; CORTÉS LEANDRO; ROBLES LUNA GABRIEL; ROJAS FLORENCIA; SERGIO GIUGNO; PATRICIO DE URRAZA

Rosario, Santa Fe, Argentina

Congreso; XLII Reunión anual; 2006

Sociedad Argentina de investigación Bioquímica y Biología Molecular

It is known that the ability of Shigella spp. to invade and escape from enterocytes cells is conferred by loci products encoded on a 220 kb virulence plasmid (megaplasmid pINV). In a previous work we described clinical isolates of Shigella which had the ability to invade Hep-2 cells monolayers even thought the absence of the plasmid pINV. In the present work we characterized different spontaneous mutants with deletions in the ipaBCDA and replication regions of the plasmid pINV, and analyzed then the ability to invade Hep-2 cells monolayers. A total of 100 clinical isolates of Shigella spp. were employed. All isolates were assayed for the presence of genes encoding in the ipaBCDA and replication region, by PCR using specific primers for each gene with boiled culture as template. Isolates that give negative amplification for one o more genes were analyzed for the ability of invade Hep-2 cells. Twenty mutants were characterized as negative by one or more of the genes. Seven were negative only for the replication region, three for the ipaBCDA region and ten for both regions. Four mutants in the replication region were invasive in Hep-2 cells assay, as well as one for the ipaBCDA region and six for both regions. Invasion of Shigella was also confirmed by fluorescent microscopy using green fluorescent protein (gfp) labeled mutants.