RESUMO
Adherence of the enteric protozoan parasite Entamoeba histolytica is mediated by an N-acetyl D-galactosamine (GalNAc)-specific lectin, a heterodimer of heavy (170 kDa) and light (35/31 kDa) subunits. The gene families encoding the lectin subunits were characterized using clamped homogeneous electric field (CHEF) gel electrophoresis in the strain HM1:IMSS. The heavy subunit was shown to be encoded by a family of five hgl genes, which were physically mapped to five distinct HindIII restriction fragments. The light subunit was shown to be encoded by a family of lgl genes located at six loci in the genome. Heavy and light subunit genes did not appear to be linked. Partial sequences of new members of the hgl and lgl gene families were obtained. Several different strains of E. histolytica were found to contain multiple hgl loci in their genomes. Expression of hgl and lgl genes in HM1:IMSS trophozoites was examined under different growth conditions using the reverse transcription-polymerase chain reaction (RT-PCR). mRNA transcripts were detected from three hgl genes and three lgl genes, with no significant differences between cultured amoebae and amoebae from liver abscesses. The complexity of GalNAc lectin gene expression observed suggests distinct biological functions for the products of the individual genes during pathogenesis.
Assuntos
Acetilgalactosamina/metabolismo , Adesão Celular/genética , Entamoeba histolytica/metabolismo , Lectinas/química , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Primers do DNA/química , Eletroforese em Gel de Ágar , Expressão Gênica/genética , Genótipo , Lectinas/genética , Dados de Sequência Molecular , Mapeamento por Restrição , Alinhamento de Sequência , Análise de SequênciaRESUMO
Amebic destruction of neutrophils and macrophages is contact-dependent. Adherence is mediated by a galactose-specific surface lectin on the amebic membrane. The pathway by which contact-dependent cytolysis of the target cell occurs is unknown. We hypothesized that target cell death is due to the triggering of apoptosis (programmed cell death) by the amebae. The purpose of this study was to determine whether target cell DNA is fragmented into a ladder pattern characteristic of apoptosis and to test whether overexpression of Bcl-2, a protein that confers resistance to apoptotic death from some stimuli, blocks target cell killing. The murine myeloid cell line FDC-P1 transfected with a retrovirus construct expressing the Bcl-2 protein was shown to be resistant to the apoptotic death that the parental line undergoes upon growth factor deprivation. 51Cr-labeled FDC-P1 control or bcl-2-transfected cells were incubated with Entamoeba histolytica (4:1 cell/ameba ratio) and killing of the cells was assessed by 51Cr release. Both cell lines were susceptible to contact-dependent killing. Death induced by the amebae in the bcl-2-transfected cells resulted in a DNA ladder fragmentation pattern (using [125I]iododeoxyuridine-labeled target cell DNA) identical to that seen in the control cells undergoing apoptosis upon growth factor withdrawal. Target cell DNA fragmentation was inhibited by blocking adherence with galactose. Our data suggest that target cell killing by E. histolytica can occur via Bcl-2-independent apoptotic mechanism.
