Monoclonal antibodies to Hammondia hammondi allowing immunological differentiation from Toxoplasma gondii

Five murine monoclonal antibodies (MAbs) were developed against purified sporozoites of Hammondia hammondi. Despite a large antigenic similarity between the 2 closely related coccidia, H. hammondi and Toxoplasma gondii, these MAbs only reacted with H. hammondi. Three MAbs, ID3, 3F2, and 4C9-7, recognized antigens of 38 kDa localized in rhoptries (1D3), in rhoptries and in oocyst and cyst walls (3F2), and in rhoptries and the apical region (4C9-7). Another MAb, 4C9-10, reacted with a 27-kDa antigen in dense granules of sporozoites and tachyzoites, and MAB 11B3 labeled an antigen of >94 kDa located in the pellicular membrane of the 3 stages of the parasite. These MAbs could be used for a rapid discrimination of the 2 coccidia in epidemiological studies or for diagnostic purposes in tissues.     

Antigenic similarity between the coccidian parasites Toxoplasma gondii and Hammondia hammondi

The antigens that are present in the coccidian parasites Toxoplasma gondii and Hammondia hammondi were demonstrated and defined by using SDS-PAGE and immunoenzymatic techniques with 125I-labeled and unlabeled antigens of T. gondii and sera of mice infected orally or intraperitoneally with H. hammondi . All cell surface antigens of T. gondii that were labeled with 125I were recognized by antibodies in the sera of the mice infected with H. hammondi except the antigen of approximate molecular weight of 21.5 Kd. This suggests that this antigen is specific for T. gondii. Various antigens in the T. gondii-lysed antigen preparations were recognized by antibodies to H. hammondi . The number of recognized antigens increased as the infection of the mice with H. hammondi progressed. Oral infection with H. hammondi appeared to induce the formation of antibodies that recognized more T. gondii antigens than infection by intraperitoneal inoculation.     

Redescription of Hammondia hammondi and its differentiation from Toxoplasma gondii

Hammondia hammondi is a protozoan parasite that, until 1975, was misidentified as Toxoplasma gondii. Recently, the validity of H. hammondi has been questioned. In this article, the authors redescribe the parasite and its life cycle, provide accession numbers to its specimens deposited in a museum, and distinguish it structurally and biologically from T. gondii. Hammondia hammondi was found to be structurally, biologically, and molecularly different from T. gondii.     

Isoenzyme analysis of Hammondia hammondi and Toxoplasma gondii sporozoites.

Isoenzyme analysis using isoelectrofocusing in polyacrylamide gels was used to distinguish Hammondia hammondi and Toxoplasma gondii sporozoites. Five enzyme systems were studied: aconitase (EC 4.2.1.3), aspartate aminotransferase (EC 2.6.1.1), glucose phosphate isomerase (EC 5.3.1.9), lactate dehydrogenase (EC 1.1.1.27), and phosphoglucomutase (EC 2.7.5.1). Three stocks of T. gondii belonging to 3 zymodemes were compared to 1 stock of H. hammondi. Hammondia hammondi differed from T. gondii at all 5 loci analyzed. This was observed for all 3 zymodemes of T. gondii. These results indicated clear genetic differences between the 2 species.     

Toxoplasma gondii and Hammondia hammondi: DNA comparison using cloned rRNA gene probes

A mung bean nuclease genomic library of purified DNA from tachyzoites of the RH strain of Toxoplasma gondii was prepared in the bacteriophage lambda gtll and recombinants containing rRNA gene fragments were detected by hybridization with radiolabeled total RNA from the closely related coccidian Eimeria acervulina. Ten recombinants were chosen at random, and five of these were investigated further using probes for the genes of the large and small rRNA of Plasmodium berghei. An insert (called TG4) that hybridized only to the 3' end of the large rRNA coding region of P. berghei and an insert (called TG18) that hybridized only to the small rRNA coding region of P. berghei were purified by electrophoresis in low melting point agarose. Radiolabeled E. acervulina total RNA, TG4, and TG18, were then used to compare the sizes of the large and small rRNA gene fragments after DNA extracted from three strains of T. gondii, and the type strain of the closely related coccidian Hammondia hammondi were cut by one of a series of 10 restriction endonucleases. The patterns obtained for the three T. gondii isolates were identical to those obtained for H. hammondi, for each enzyme tested. In addition, the guanine plus cytosine (G + C) content of H. hammondi DNA was found to be almost identical to that obtained previously for T. gondii DNA.     

Tazonomy of Toxoplasma.

After reviewing reports of the hosts, structure and life cycle of Toxoplasma, the genus is placed in the apicomplexan family Eimeriidae and the folllowing 7 species are recognized: Toxoplasma gondii (Nicolle & Manceaux) (type species) from about 200 species of mammals and birds, with oocysts in felids; Toxoplasma alencari (Da Costa & Pereira) from the frog Leptodactylus ocellatus; Toxoplasma brumpti Coutelen from the iguana Iguana tuberculata; Toxoplasma colubri Tibaldi from the snakes Coluber melanoleucus and Coluber viridiflavus; Toxoplasma hammondi (Frenkel & Dubey) (a new combination for Hammondia hammondi) from the house mouse with oocysts in the domestic cat; Toxoplasma ranae Levine & Nye from the leopard frog Rana Pipiens; and Toxoplasma serpai Scorza, Dagert & Iturriza Arocha from the toad Bufo marinus.     

Identification and disruption of a rhoptry-localized homologue of sodium hydrogen exchangers in Toxoplasma gondii

Na+/H+ exchangers (NHEs) are ubiquitous membrane proteins that catalyze the exchange of Na+ for H+ and are critical in pH and cell volume regulation, as well as osmotolerance. In this study, we identify and characterize a novel NHE, TgNHE2, in Toxoplasma gondii. Immunofluorescence studies show that TgNHE2 is localized to the rhoptries, secretory organelles involved in invasion. TgNHE2 is the first intracellular NHE to be characterized in a protozoan parasite and its localization suggests possible roles for the rhoptries in osmotolerance and/or as secretory lysosomes-like granules.     

Autofluorescence of Toxoplasma gondii and related coccidian oocysts

This is the first report of blue autofluorescence as a useful characteristic in the microscopic detection of Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Neospora caninum, Besnoitia darlingi, and Sarcocystis neurona oocysts or sporocysts. This autofluorescence is of sufficient intensity and duration to allow identification of these oocysts from complex microscopic sample backgrounds. As with the autofluorescence of related coccidia, the oocysts glow pale blue when illuminated with an ultraviolet (UV) light source and viewed with the correct UV excitation and emission filter set.     

A parafusin-related Toxoplasma protein in Ca2+-regulated secretory organelles

We cloned a gene, PRPI, of Toxoplasma gondii encoding a 637-amino-acids protein having a calculated mass of 70 kDa. The sequence showed high homology to parafusin, a protein that in Paramecium tetraurelia participates in Ca2+-regulated exocytosis and is a paralog of phosphoglucomutase. We show that Toxoplasma gondii homogenate and an expressed recombinant PRP1 fusion protein cross-react with a specific peptide-derived antibody to parafusin in Western blots. Antibodies to the recombinant PRP1 showed cross-reaction with parafusin and recognized PRP1, as bands at M, 63 x 10(3) and 68 x 10(3), respectively. PRP1 is labeled when Toxoplasma gondii cells are incubated with inorganic 32P and appears as the major band on autoradiograms of SDS-PAGE gels. The localization of PRP1 was examined in secretory organelles of Toxoplasma gondii by deconvolution light microscopy followed by three dimensional reconstruction using pairwise combinations of specific antibodies. PRP1 localized to the apical third of the cell. It co-localized with micronemes, the only secretory organelle the secretion of which is Ca2+ dependent. Quantification of the co-localized stain suggests that only mature micronemes ready for exocytosis have PRP1. These findings suggest that PRP1, parafusin and other members of the phosphoglucomutase superfamily have a conserved role in Ca2+-regulated exocytic processes.     

Sarcocystis neurona n. sp. (Protozoa: Apicomplexa), the etiologic agent of equine protozoal myeloencephalitis

Sarcocystis neuronan n. sp. is proposed for the apicomplexan taxon associated with myeloencephalitis in horses. Only asexual stages of this parasite presently are known, and they are found within neuronal cells and leukocytes of the brain and spinal cord. The parasite is located in the host cell cytoplasm, does not have a parasitophorous vacuole, and divides by endopolygeny. Schizonts are 5-35 microns x 5-20 microns and contain 4-40 merozoites arranged in a rosette around a prominent residual body. Merozoites are approximately 4 x 1 micron, have a central nucleus, and lack rhoptries. Schizonts and merozoites react with Sarcocystis cruzi antiserum but not with Caryospora bigenetica. Toxoplasma gondii, Hammondia hammondi, or Neospora caninum antisera in an immunohistochemical test.     

Targeting to rhoptry organelles of Toxoplasma gondii involves evolutionarily conserved mechanisms

Intracellular parasites of the phylum Apicomplexa contain specialized rhoptry secretory organelles that have a crucial function in host-cell invasion and establishment of the parasitophorous vacuole. Here we show that localization of the Toxoplasma gondii rhoptry protein ROP2 is dependent on a YEQL sequence in the cytoplasmic tail that binds to micro-chain subunits of T. gondii and mammalian adaptors, and conforms to the YXXstraight phi mammalian sorting motif. Chimaeric reporters, containing the transmembrane domains and cytoplasmic tails of the low-density lipoprotein receptor and of Lamp-1, are sorted to the Golgi or the trans-Golgi network (TGN), and partially to apical microneme organelles of the parasite, respectively. Targeting of these reporters is mediated by YXXstraight phi- and NPXY-type signals. This is the first demonstration of tyrosine-dependent sorting in protozoan parasites, indicating that T. gondii proteins may be targeted to, and involved in biogenesis of, morphologically unique organelles through the use of evolutionarily conserved signals and machinery.     

Membrane topology and transient acylation of Toxoplasma gondii glycosylphosphatidylinositols

Using hypotonically permeabilized Toxoplasma gondii tachyzoites, we investigated the topology of the free glycosylphosphatidylinositols (GPIs) within the endoplasmic reticulum (ER) membrane. The morphology and permeability of parasites were checked by electron microscopy and release of a cytosolic protein. The membrane integrity of organelles (ER and rhoptries) was checked by protease protection assays. In initial experiments, GPI biosynthetic intermediates were labeled with UDP-[6-(3)H]GlcNAc in permeabilized parasites, and the transmembrane distribution of the radiolabeled lipids was probed with phosphatidylinositol-specific phospholipase C (PI-PLC). A new early intermediate with an acyl modification on the inositol was identified, indicating that inositol acylation also occurs in T. gondii. A significant portion of the early GPI intermediates (GlcN-PI and GlcNAc-PI) could be hydrolyzed following PI-PLC treatment, indicating that these glycolipids are predominantly present in the cytoplasmic leaflet of the ER. Permeabilized T. gondii parasites labeled with either GDP-[2-(3)H]mannose or UDP-[6-(3)H]glucose showed that the more mannosylated and side chain (Glc-GalNAc)-modified GPI intermediates are also preferentially localized in the cytoplasmic leaflet of the ER.     

The acidocalcisome Ca2+-ATPase (TgA1) of Toxoplasma gondii is required for polyphosphate storage, intracellular calcium homeostasis and virulence

A large proportion of intracellular Ca2+ in Toxoplasma gondii tachyzoites is stored within acidocalcisomes. These organelles are characterized by their acidic nature and high calcium and polyphosphate (polyP) content. The activity of a Ca2+/H+-ATPase named TgA1 may be important for the accumulation of Ca2+ in these organelles. This enzyme belongs to a group of plasma membrane Ca2+-ATPase (PMCA) that lack a calmodulin-binding domain and have vacuolar localization. To investigate the role of this enzyme, we have generated T. gondii mutants deficient in TgA1 through gene disruption. Proliferation of these mutants decreased dramatically because of deficient cell invasion. In addition, these cells had reduced virulence in a mouse model. Biochemical analysis revealed that the cell polyP content was drastically reduced, and the basal calcium levels were increased and unstable. Microneme secretion under the conditions of stimulation by ionophores was altered. Complementation of null mutants with TgA1 restored most functions. In summary, these results establish a link between TgA1, calcium homeostasis, polyP storage and virulence.     

Proteomics and glycomics analyses of N-glycosylated structures involved in Toxoplasma gondii--host cell interactions

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.     

Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells

Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite.     

Toxoplasma gondii Rab6 mediates a retrograde pathway for sorting of constitutively secreted proteins to the Golgi complex

Toxoplasma gondii relies on protein secretion from specialized organelles for invasion of host cells and establishment of a parasitophorous vacuole. We identify T. gondii Rab6 as a regulator of protein transport between post-Golgi dense granule organelles and the Golgi. Toxoplasma Rab6 was localized to cisternal rims of the late Golgi and trans-Golgi network, associated transport vesicles, and microdomains of dense granule and endosomal membranes. Overexpression of wild-type Rab6 or GTP-activated Rab6(Q70L) rerouted soluble dense granule secretory proteins to the Golgi and endoplasmic reticulum and augmented the effect of brefeldin A on Golgi resorption to the endoplasmic reticulum. Parasites expressing a nucleotide-free (Rab6(N124I)) or a GDP-bound (Rab6(T25N)) mutant accumulated dense granule proteins in the Golgi and associated transport vesicles and displayed reduced secretion of GRA4 and a delay in glycosylation of GRA2. Activated Rab6 on Golgi membranes colocalized with centrin during mitosis, and parasite clones expressing Rab6 mutants displayed a partial shift in cytokinesis from endodyogeny (formation of two daughter cells) to endopolygeny (multiple daughter cells). We propose that Toxoplasma Rab6 regulates retrograde transport from post-Golgi secretory granules to the parasite Golgi.     

Dense-granule organelles of Toxoplasma gondii: their role in the host-parasite relationship

The dense-granule organelles of Toxoplasma gondii are secretory vesicles that play a major role in the structural modifications of the parasitophorous vacuole. In this article, Marie-France Cesbron-Delauw reviews work on a prominent set of molecules recently characterized as components of the dense granules, and discusses various aspects on their post-translational trafficking and delivery within the host cell.     

Hepatic sarcocystosis in a horse

Hepatic sarcocystosis was diagnosed in a horse in association with refractory bacterial osteomyelitis and plasma cell tumor of the maxilla and hepatic salmonellosis. Gross lesions included pleural, pericardial, and peritoneal effusions, hepatomegaly, gastric ulceration, colonic edema, and proliferative tissues filling 2 maxillary dental alveoli. Histologically, liver was characterized by severe suppurative, necrotizing, periportal hepatitis, and severe periacinar necrosis. Hepatocytes frequently contained protozoal schizonts in various stages of development. In mature schizonts, merozoites were often arranged radially around a central residual body, consistent with asexual division by endopolygeny. Ultrastructural features of merozoites included an apical conoid and polar ring, anterior micronemes, central nuclei, and absence of rhoptries. These protozoa did not react to antisera raised against Neospora caninum, Sarcocystis neurona, Toxoplasma gondii, or Hammondia hammondi. The microscopic and ultrastructural characteristics and immunoreactivity of this organism are consistent with a Sarcocystis sp. other than S. neurona. This is the first report of Sarcocystis-associated hepatitis in a horse. The life cycle of this organism and source of infection are unknown.     

Detection and localization of a Ca2+-ATPase activity in Toxoplasma gondii

Toxoplasma gondii, the agent causing toxoplasmosis, is an obligate intracellular protozoan parasite. A calcium signal appears to be essential for intracellular transduction during the active process of host cell invasion. We have looked for a Ca2+-transport ATPase in tachyzoites and found Ca2+-ATPase activity (11-22 nmol Pi liberated/mg protein/min) in the tachyzoite membrane fraction. This ATP-dependent activity was stimulated by Ca2+ and Mg2+ ions and by calmodulin, and was inhibited by pump inhibitors (sodium orthovanadate or thapsigargin). We used cytochemistry and X-ray microanalysis of cerium phosphate precipitates and immunolabelling to find the Ca2+, Mg2+-ATPase. It was located mainly in the membrane complex, the conoid, nucleus, secretory organelles (rhoptries, dense granules) and in vesicles with a high calcium concentration. Thus, Toxoplasma gondii possesses Ca2+-pump ATPase (Ca2+, Mg2+-ATPase) as do eukaryotic cells.