Transcriptional and secretory responses of Entamoeba histolytica to mucins, epithelial cells and bacteria.

Invasive intestinal amebiasis, caused by Entamoeba histolytica, is initiated with attachment of trophozoites to the colonic mucous layer, mucous disruption and/or depletion, and adherence to and cytolysis of host epithelial and inflammatory cells. A current working model of intestinal amebiasis suggests that the microenvironment of the host intestine, particularly intestinal mucins and the bacterial biofilm, may influence the behavior of pathogenic amebae. The invasive phenotype is dependent on expression of a number of virulence factors of which cysteine proteases provide the most convenient experimental probe because their activity is readily monitored. In the present study, we examined the interaction of E. histolytica with GalNAc, mucin, different epithelial cell lines and bacteria both by biochemical assays of protease release and transcriptional profiling using a previously validated genomic microarray. A significant down-regulation of released cysteine protease activity was observed when amebic trophozoites were grown with GalNAc, specific colonic cell lines and bacteria. Transcriptional profiling during GalNAc interaction revealed enhanced expression of the 170-kDa Gal/GalNAc lectin. Decreased protease activity during GalNAc interaction and enhanced expression of the Gal/GalNAc lectin gene are consistent with a program of commensal infection and mucus coat colonization mediated by the lectin. The down-regulation of cysteine protease activity following interaction with a colonic epithelial cell line parallels the presence of secretory mucin having a complex carbohydrate structure rich in Gal and GalNAc. In contrast, interaction of E. histolytica trophozoites with stomach porcine mucin enhanced cysteine protease (EhCP1 and EhCP2) secretion 3-fold. This suggests the specific composition of mucins may affect the Entamoeba phenotype. Transcriptional profiling revealed interaction of Entamoeba with intestinal bacteria induced protein kinase, ABC transporter, Rab family GTPase and hsp 90 gene expression. The enhanced expression of this gene cluster is consistent with enhanced phagocytosis of E. histolytica during interaction with bacteria.

Bcr-Abl-mediated protection from apoptosis downstream of mitochondrial cytochrome c release.

Bcr-Abl, activated in chronic myelogenous leukemias, is a potent cell death inhibitor. Previous reports have shown that Bcr-Abl prevents apoptosis through inhibition of mitochondrial cytochrome c release. We report here that Bcr-Abl also inhibits caspase activation after the release of cytochrome c. Bcr-Abl inhibited caspase activation by cytochrome c added to cell-free lysates and prevented apoptosis when cytochrome c was microinjected into intact cells. Bcr-Abl acted posttranslationally to prevent the cytochrome c-induced binding of Apaf-1 to procaspase 9. Although Bcr-Abl prevented interaction of endogenous Apaf-1 with the recombinant prodomain of caspase 9, it did not affect the association of endogenous caspase 9 with the isolated Apaf-1 caspase recruitment domain (CARD) or Apaf-1 lacking WD-40 repeats. These data suggest that Apaf-1 recruitment of caspase 9 is faulty in the presence of Bcr-Abl and that cytochrome c/dATP-induced exposure of the Apaf-1 CARD is likely defective. These data provide a novel locus of Bcr-Abl antiapoptotic action and suggest a distinct mechanism of apoptosomal inhibition.

Post-cytochrome C protection from apoptosis conferred by a MAPK pathway in Xenopus egg extracts.

In response to many different apoptotic stimuli, cytochrome c is released from the intermembrane space of the mitochondria into the cytoplasm, where it serves as a cofactor in the activation of procaspase 9. Inhibition of this process can occur either by preventing cytochrome c release or by blocking caspase activation or activity. Experiments involving in vitro reconstitution of apoptosis in cell-free extracts of Xenopus laevis eggs have suggested that extracts arrested in interphase are susceptible to an endogenous apoptotic program leading to caspase activation, whereas extracts arrested in meiotic metaphase are not. We report here that Mos/MEK/MAPK pathways active in M phase-arrested eggs are responsible for rendering them refractory to apoptosis. Interestingly, M phase-arrested extracts are competent to release cytochrome c, yet still do not activate caspases. Concomitantly, we have also demonstrated that recombinant Mos, MEK, and ERK are sufficient to block cytochrome c-dependent caspase activation in purified Xenopus cytosol, which lacks both transcription and translation. These data indicate that the MAP kinase pathway can target and inhibit post-cytochrome c release apoptotic events in the absence of new mRNA/protein synthesis and that this biochemical pathway is responsible for the apoptotic inhibition observed in meiotic X. laevis egg extracts.