Host tissue destruction by Entamoeba histolytica: molecules mediating adhesion, cytolysis, and proteolysis

Entamoeba histolytica, the protozoan parasite causing human amoebisis, has recently been found to comprise two genetically distinct forms, potentially pathogenic and constitutively nonpathogenic ones. Host tissue destruction by pathogenic forms is belived to result from cell functions mediaed by a lectin-type adherence receptor, a pore-forming peptide involved in host cell lysis, and abundant expression of cysteine proteinase(s). Isolation and molecular cloning of these amoeba products have provided the tools for structural analyses and manipulations of cell functions including comparisons between pathogenic and nonpathogenic forms

Diagnóstico microscópico de protozoarios intestinales / Microscopic diagnosis of intestinal protozoan

Se realizó una experiencia para evaluar la efectividad de las preparaciones permanentes de muestras obtenidas previa purga en fijador de Brooke-Goldman (PVA), con muestras espontáneas de los mismos pacientes, en fijador de Junod (SAF). También fueron comparados los porcentajes obtenidos para E. histolytica y D. fragilis, con los modelos de recolección y procesamiento de heces más usados en nuestro país. Fueron estudiados 248 pacientes. Se tomaron tres muestras de cada uno de ellos. La primera (M1) y segunda muestra (M2) fueron recolectados con heces espontáneas, de días no consecutivos, en fijador de Junod (SAF) y, una tercera (M3), previa purga, en fijador de Brooke-Goldman (PVA). Las muestras recolectadas en SAF (M1 y M2) fueron analizadas por microscopia de preparaciones húmedas directas y previo enriquecimiento por los métodos Ritchie y Faust. Las muestras fijadas en PVA se colorearon con la técnica tricrómica modificada. El análisis de los datos obtenidos mostró diferencias significativas entre las muestras espontáneas de días no consecutivos (M1 y M2) para: Entamoeba histolytica, Giordia lamblia y Endolimax nana. También el examen estadístico reveló diferencias significativas entre las muestras uno y dos, tomadas juntamente, con respecto a los resultados obtenidos en PVA (M3), para: Entamoeba histolytica, Giardia lambia, Dientamoeba fragilis y Endolimax nana. Se compararon los porcentajes obtenidos por coloraciones permanentes para E. hitolytica y D. fragilis, con los correspondientes a otras formas de diagnóstico: 9.088 muestras procesadas por los métodos directo y Ritchie, y 4.228 estudiados por los métodos directo, Ritchie, Faust y coloraciones sólo en casos sospechosos. Estos datos indicaron una mayor eficiencia de la recolección en PVA, previa purga y posterior coloración para el diagnóstico de los dos protozoarios. (AU)

Diagnóstico microscópico de protozoarios intestinales / Microscopic diagnosis of intestinal protozoan

Se realizó una experiencia para evaluar la efectividad de las preparaciones permanentes de muestras obtenidas previa purga en fijador de Brooke-Goldman (PVA), con muestras espontáneas de los mismos pacientes, en fijador de Junod (SAF). También fueron comparados los porcentajes obtenidos para E. histolytica y D. fragilis, con los modelos de recolección y procesamiento de heces más usados en nuestro país. Fueron estudiados 248 pacientes. Se tomaron tres muestras de cada uno de ellos. La primera (M1) y segunda muestra (M2) fueron recolectados con heces espontáneas, de días no consecutivos, en fijador de Junod (SAF) y, una tercera (M3), previa purga, en fijador de Brooke-Goldman (PVA). Las muestras recolectadas en SAF (M1 y M2) fueron analizadas por microscopia de preparaciones húmedas directas y previo enriquecimiento por los métodos Ritchie y Faust. Las muestras fijadas en PVA se colorearon con la técnica tricrómica modificada. El análisis de los datos obtenidos mostró diferencias significativas entre las muestras espontáneas de días no consecutivos (M1 y M2) para: Entamoeba histolytica, Giordia lamblia y Endolimax nana. También el examen estadístico reveló diferencias significativas entre las muestras uno y dos, tomadas juntamente, con respecto a los resultados obtenidos en PVA (M3), para: Entamoeba histolytica, Giardia lambia, Dientamoeba fragilis y Endolimax nana. Se compararon los porcentajes obtenidos por coloraciones permanentes para E. hitolytica y D. fragilis, con los correspondientes a otras formas de diagnóstico: 9.088 muestras procesadas por los métodos directo y Ritchie, y 4.228 estudiados por los métodos directo, Ritchie, Faust y coloraciones sólo en casos sospechosos. Estos datos indicaron una mayor eficiencia de la recolección en PVA, previa purga y posterior coloración para el diagnóstico de los dos protozoarios.

Entamoeba histolytica: molecules involved in the target cell-parasite relationship.

To detect molecules of Entamoeba histolytica involved in the trophozoite-target cell interaction, three different antisera were generated: (a) two rabbit antisera, one against total amebic proteins and another directed specifically to the 112-kDa adhesin; and (b) a mouse antiserum against amebic molecules adhering to the red blood cell (RBC) surface after incubation of RBCs with total soluble protein from trophozoites (anti-adhesion serum). All three antisera recognized the 112-kDa adhesion. Adhesion of this molecule to the RBC surface was temperature-dependent. More of the 112-kDa adhesion was found on the surface of RBCs incubated with trophozoites at 37 degrees C than on RBCs incubated at room temperature or at 0 degree C. Experiments using both anti-adhesin and anti-total ambebic protein sera revealed the presence of 210, 160, 112, 90, 70, 50, and 24-kDa proteins on RBC incubated with trophozoites. Surface proteins obtained from iodinated MDCK cells recognized amebic proteins of 112, 90, and 48-50 kDa. Virulence-deficient mutants presented a similar amount of the 112-kDa adhesin to the wild-type strain. However, in mutants, the adhesion was not functional, since they did not adhere to RBCs. 90- and 24-kDa proteins were also found to be altered in mutants.

Isolation, characterization and partial purification of a transferable membrane channel (amoebapore) produced by Entamoeba histolytica.

Entamoeba histolytica kills cells by contact dependent cytolysis. The mechanism underlying this process must be of rapid onset because target cells round up and show marked zeiosis within 15 min of contact. In earlier work, we identified a remarkable ion-channel forming protein which we named amoebapore, that may contribute to the amoeba-induced target cell killing. Within the amoeba it exists as part of a supramolecular aggregate together with other proteins of unknown function. In this work we report the purification of a solubilized form of the amoebapore. Amoebapore was found to exist as an apparent dimer of the previously reported protein whose molecular weight had been determined under denaturing conditions. Two isoforms of this dimer, with pI values of 6.8 and 5.3 present at a ratio of 7 to 1, were identified and purified. Both isoforms demonstrate ion-channel forming activity in planar lipid membranes. These channels show a unit conductance of 5-20 pS and remain open for less than 1 s. Upon lateral aggregation, opening becomes concerted to a greater degree with channel conductance are observed. The isolated particulate form of amoebapore depolarizes cells.

Subunit structure of the galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica.

The galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica is a cell surface protein which mediates parasite adherence to human colonic mucus, colonic epithelial cells, and other target cells. The amebic lectin was purified in 100-micrograms quantities from detergent-solubilized trophozoites by monoclonal antibody affinity chromatography. The adherence lectin was purified 500-fold as judged by radioimmunoassay. The nonreduced lectin had a molecular mass of 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of pH 6.2. The amebic lectin reduced with beta-mercaptoethanol consisted of 170- and 35-kDa subunits. Both subunits could be labeled on the cell surface with 125I, and both were metabolically labeled with [3H]glucosamine. The amino termini of the subunits had unique amino acid sequences, and polyclonal antisera to the heavy subunit did not cross-react with the light subunit. The yield of phenylthiohydantoin derivatives from the second and third positions in the sequence of the heavy and light subunits gave a molar ratio of one 170- to one 35-kDa subunit. Antibodies directed to the heavy subunit inhibited amebic adherence to Chinese hamster ovary cells by 100%, suggesting that the heavy subunit is predominantly responsible for mediating amebic adherence.

Demonstration of histamine receptors on the surface of Entamoeba histolytica.

Histamine receptors on the surface of E. histolytica could be demonstrated by histochemical method using three isolates of the protozoa grown and maintained in modified Boeck and Drbohlav's medium. Prior treatment of E. histolytica with cimetidine a H2 blocker, blocked the histamine uptake. Similar treatment with mepyramine maleate, a H1 blocker, did not prevent histamine uptake by the protozoa. It is postulated that E. histolytica has H2 receptors on its surface.

Appearance of sialoglycoproteins in encysting cells of Entamoeba histolytica.

Amoeba-bacterium cultures of Entamoeba histolytica transferred to a hypoosmotic medium depleted of nutrients changed morphologically and biochemically. The cells ejected grains of rice starch, rounded up, and formed a distinct cell wall that was resistant to detergent, bound the sialic acid-specific lectin from Limulus polyphemus, and became fluorescent with Calcofluor M2R. A subpopulation of these cells displayed more than one nucleus. All these signs are characteristic of encysting cells and were also observed in cysts obtained from a human patient. The morphological changes were accompanied by the appearance of two new glycoproteins with apparent molecular sizes of 100 and 150 kilodaltons which contained sialic acid. Sialic acid has been reported to be absent from trophozoites of Entamoeba species. The presence of this sugar residue on cyst-specific proteins parallels recently reported findings during the encystation of the related reptilian parasite Entamoeba invadens. This may indicate a basic role for sialic acid in the encystation of Entamoeba parasites.