Differences in cap formation between invasive Entamoeba histolytica and non-invasive Entamoeba dispar.

The rapid redistribution of surface antigen-antibody complexes in trophozoites of the human protozoan parasite Entamoeba histolytica, in a process known as capping, has been considered as a means of the parasite to evade the host immune response. So far, capping has been documented in the invasive E. histolytica, whereas the mobility of surface components in the non-invasive Entamoeba dispar is not known. E. dispar does not induce liver lesions in rodent experimental models, in contrast to the liver abscesses produced by E. histolytica in the same animal model. We have therefore analyzed the mobility of surface receptors to the lectin concanavalin A and of Rab11, a membrane-associated protein, in both species of Entamoebae by confocal fluorescence microscopy and transmission and scanning electron microscopy. The great majority of E. histolytica trophozoites became morphologically polarized through the formation of well-defined caps at the posterior pole of the parasite. Actin colocalized with the lectin caps. Antibodies against the membrane protein Rab 11 also produced capping. In striking contrast, in E. dispar, the mobility of concanavalin A surface receptors was restricted to the formation of irregular surface patches that did no progress to constitute well-defined caps. Also, anti-Rab 11 antibodies did not result in capping in E. dispar. Whether the failure of E. dispar to efficiently mobilize surface molecules in response to lectin or antibodies as shown in the present results is related to its non-invasive character represents an interesting hypothesis requiring further analysis.

Entamoeba histolyticaelectrondense granules secretion in vitro and in vivo: ultrastructural study.

Electron dense granules (EDGs) were identified by transmission electron microscopy in Entamoeba histolytica trophozoites recovered from hamster liver lesions. Abundant granules were present in trophozoites recovered after 15 min of liver inoculation. Variation in the size and morphology of these EDGs was also observed. Numerous granules were present in the plasma membrane when these parasites were incubated for 5 min with MDCK monolayers. Release of these EDGs was suggested by the presence of granules in contact with the surface of the target cell plasma membrane. Parasite phagocytic invaginations were observed after 10 min of parasite-monolayer interaction. In these structures, scarce granules were seen. Granules secretion was corroborated by obtaining of a pellet of these small structures from the incubation of trophozoites with collagen supernatant. Collagenase and gellatinase activity of this pellet was identified in SDS-PAGE gels. EDGs were also present in amebic hamster liver lesions. Our observations corroborate that these granules are secreted and suggest that may participate in the cytopathic effect of E. histolytica both in vitro and in vivo.

Ultrastructural study of the encystation and excystation processes in Naegleria sp.

An important aspect of the biology of Naegleria sp. is the differentiation processes that occur during encystation and excystation. We studied these using both fluorescence and transmission electron microscopy techniques. In the initial stages of encystation, the cisternae of the endoplasmic reticulum became densely filled with a fibrillar material. Vesicles with a similar content that appeared to be derived from the cisternae were also observed in close contact with the plasma membrane. As encystation progressed, the fibrillar material became localized on the surface of the amoeba. An irregular compaction was observed in some areas of the cyst wall, which contained thin extensions of the cyst wall fibrillar material. Completely formed cysts had two to three ostioles, each sealed by an operculum. The operculum contained two areas in which a differential compaction of the fibrillar structure was observed. When excystation was induced, small dense granules (DGs), which were in close contact with fibrillar material were observed in the cyst cytoplasm and in the peritrophic space. During excystation, the more compact component of the operculum moves to enable the pseudopod of the emerging trophozoite to penetrate the ostiole. Vacuoles containing a fibrillar material, probably derived from the cyst wall, were observed in the cytoplasm of the pseudopodia. Our results provide a platform for further studies using biochemical markers to investigate the origin of the cyst wall as well as the role of DGs during excystation in Naegleria.

Acanthamoeba castellanii: identification and distribution of actin cytoskeleton.

The presence of the cytoskeleton of Acanthamoeba castellanii was observed by means of cryo-electronmicroscopy and immunofluorescence techniques. This structure is formed largely by fibers and networks of actin located mainly in cytoplasmic locomotion structures as lamellipodia and as well as in various endocytic structures. In addition, the comparison between total actin content in whole extracts among different amoebae was made. The molecular weight of actin in A. castellanii was 44 kDa, and 45 kDa for Naegleria fowleri and Entamoeba histolytica.

Cell surface differences of Naegleria fowleri and Naegleria lovaniensis exposed with surface markers.

Differences in the distribution of diverse cell surface coat markers were found between Naegleria fowleri and Naegleria lovaniensis. The presence of carbohydrate-containing components in the cell coat of the two species was detected by selective staining with ruthenium red and alcian blue. Using both markers, N. fowleri presented a thicker deposit than N. lovaniensis. The existence of exposed mannose or glucose residues was revealed by discriminatory agglutination with the plant lectin Concanavalin A. These sugar residues were also visualized at the cell surface of these parasites either by transmission electron microscopy or by fluorescein-tagged Concanavalin A. Using this lectin cap formation was induced only in N. fowleri. The anionic sites on the cell surface detected by means of cationized ferritin were more apparent in N. fowleri. Biotinylation assays confirmed that even though the two amoebae species have some analogous plasma membrane proteins, there is a clear difference in their composition.

Acanthamoeba castellanii: structural basis of the cytopathic mechanisms.

In this study we report observations on the structural mechanisms of the cytopathic effect of Acanthamoeba castellanii trophozoites on cultured MDCK cell monolayers. Co-incubations were carried out for a maximum of 24h. The first evidence of damage to the cell monolayer was detected by measuring the transepithelial resistance of cell monolayers that interacted with the amoebae. At 6h, transepithelial resistance diminished to 51% and amoebae required 5-6h to produce evidence of structural injury at the light microscopy level. Following 12h of incubation, the cell monolayer was severely damaged. After making intimate contact with the surface of target cells, trophozoites detached cells from the substrate, lysed and by means of food-cups ingested the damaged cells. There was no morphological evidence of modifications in MDCK cell membranes, membrane fusion or junction formation between the amoeba and host plasma membrane. The lytic capacity of the amoebas appears to be the result of cytotoxic factors secreted by the amoebae since, when monolayers were incubated with conditioned medium, there was also a decrease in the transepithelial resistance. Besides, mechanical injury produced by the attachment and movement of the trophozoites may contribute to the disruption of the cell monolayer. As in other pathogenic amoebae, the cytopathic action of A. castellanii on the cell monolayers can subjectively be separated into four stages: adhesion, cytolysis, phagocytosis, and intracellular degradation.

Entamoeba histolytica: mechanism of decrease of virulence of axenic cultures maintained for prolonged periods.

Intraportal injection of non-virulent E. histolytica (derived from prolonged axenic culture of virulent E. histolytica) strain HM1-IMSS in normal hamsters results in no liver lesions and disappearance of the parasites 48-72 h after injection. Viability of non-virulent E. histolytica after 2 h of in vitro incubation in either fresh or decomplemented hamster serum is the same as control virulent E. histolytica (50-90%). In hamsters made leukopenic, or both leukopenic+hypocomplementemic, or hypocomplementemic+sephadex microspheres (to produce focal liver ischemia) intraportally injected non-virulent E. histolytica cause no lesions and disappear after 24 h. In addition, neither hypocomplementemia nor immunosuppression with cyclosporin A prolonged the survival of non-virulent E. histolytica. Methyl prednisolone treatment of hamsters resulted in survival of large numbers of non-virulent E. histolytica in the liver, with little inflammation and minimal tissue damage, for up to 7 days. Inflammatory cells (macrophages) would appear to be chiefly responsible for elimination of non-virulent E. histolytica. Parallel experiments with E. dispar suggest a different mechanism for its non-pathogenicity.