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To determine how binuclear giardia swim, we used video microscopy to observe trophozoites of Giardia intestinalis, which were labeled with an amino-specific Alexa Fluor dye that highlighted the flagella and adherence disc. Giardia swam forward by means of the synchronous beating of anterior, posterolateral, and ventral flagella in the plane of the ventral disc, while caudal flagella swam in a plane perpendicular to the disc. Giardia turned in the plane of the disc by means of a rudder-like motion of its tail, which was constant rather than beating. To determine how giardia divide, we used three-dimensional confocal microscopy, the same surface label, nuclear stains, and antitubulin antibodies. Giardia divided with mirror-image symmetry in the plane of the adherence disc, so that the right nucleus of the mother became the left nucleus of the daughter. Pairs of nuclei were tethered together by microtubules which surrounded nuclei and prevented mother or daughter giardia from receiving two copies of the same nucleus. New adherence discs formed upon a spiral backbone of microtubules, which had a clockwise rotation when viewed from the ventral surface. These dynamic observations of the parasite begin to reveal how giardia swim and divide.

Entamoeba histolytica causes amebic colitis and liver abscess in developing countries such as Mexico and India. Entamoeba dispar is morphologically identical but is not associated with disease. Here we determined the ploidy of E. histolytica and developed PCR-based methods for distinguishing field isolates of E. histolytica or E. dispar. Fluorescence in situ hybridization showed that E. histolytica trophozoites are diploid for five "single-copy" probes tested. Intergenic sequences between superoxide dismutase and actin 3 genes of clinical isolates of E. histolytica from the New and Old Worlds were identical, as were those of E. dispar. These results suggest a bottleneck or demographic sweep in entamoebae which infect humans. In contrast, E. histolytica and E. dispar genes encoding repeat antigens on the surface of trophozoites (Ser-rich protein) or encysting parasites (chitinase) were highly polymorphic. chitinase alleles suggested that the early axenized strains of E. histolytica, HM-1 from Mexico City, Mexico, and NIH-200 from Calcutta, India, are still present and that similar E. dispar parasites can be identified in both the New and Old Worlds. Ser-rich protein alleles, which suggested the presence of the HM-1 strain in Mexico City, included some E. histolytica genes that predicted Ser-rich proteins with very few repeats. These results, which suggest diversifying selection at chitinase and Ser-rich protein loci, demonstrate the usefulness of these alleles for distinguishing clinical isolates of E. histolytica and E. dispar.

The infectious stage of amebae is the chitin-walled cyst, which is resistant to stomach acids. In this study an extraordinarily abundant, encystation-specific glycoprotein (Jacob) was identified on two-dimensional protein gels of cyst walls purified from Entamoeba invadens. Jacob, which was acidic and had an apparent molecular mass of approximately 100 kDa, contained sugars that bound to concanavalin A and ricin. The jacob gene encoded a 45-kDa protein with a ladder-like series of five Cys-rich domains. These Cys-rich domains were reminiscent of but not homologous to the Cys-rich chitin-binding domains of insect chitinases and peritrophic matrix proteins that surround the food bolus in the insect gut. Jacob bound purified chitin and chitin remaining in sodium dodecyl sulfate-treated cyst walls. Conversely, the E. histolytica plasma membrane Gal/GalNAc lectin bound sugars of intact cyst walls and purified Jacob. In the presence of galactose, E. invadens formed wall-less cysts, which were quadranucleate and contained Jacob and chitinase (another encystation-specific protein) in secretory vesicles. A galactose lectin was found to be present on the surface of wall-less cysts, which phagocytosed bacteria and mucin-coated beads. These results suggest that the E. invadens cyst wall forms when the plasma membrane galactose lectin binds sugars on Jacob, which in turn binds chitin via its five chitin-binding domains.

Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall. Here, anti-chitinase antibodies identified hundreds of small secretory vesicles in encysting E. invadens parasites and in E. histolytica trophozoites overexpressing chitinase under an actin gene promoter. Abundant small secretory vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol. Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few vesicles of mixed sizes. A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear vesicles, was identified with antibodies to ADP-ribosylating factor and to epsilon-COP. We conclude that the amebic secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.