Peter M. Takvorian

A Cell Culture System for Study of the Development of Toxoplasma gondii Bradyzoites

ABSTRACT. Toxoplasma gondii is a ubiquitous apicomplexan parasite and a major opportunistic pathogen under AIDS‐induced conditions, where it causes encephalitis when the bradyzoite (cyst) stage is reactivated. A bradyzoite‐specific Mab, 74.1.8, reacting with a 28 kDa antigen, was used to study bradyzoite development in vitro by immuno‐electron microscopy and immunofluorescence in human fibroblasts infected with ME49 strain T. gondii. Bradyzoites were detected in tissue culture within 3 days of infection. Free floating cyst‐like structures were also identified. Western blotting demonstrated the expression of bradyzoite antigens in these free‐floating cysts as well as in the monolayer. Bradyzoite development was increased by using media adjusted to pH 6.8 or 8.2. The addition of γ‐interferon at day 3 of culture while decreasing the total number of cysts formed prevented tachyzoite overgrowth and enabled study of in vitro bradyzoites for up to 25 days. The addition of IL‐6 increased the number of cysts released into the medium and increased the number of cysts formed at pH 7.2. Confirmation of bradyzoite development in vitro was provided by electron microscopy. It is possible that the induction of an acute phase response in the host cell may be important for bradyzoite differentiation. This system should allow further studies on the effect of various agents on the development of bradyzoites.

Phosphatidylserine Vesicles Enable Efficient En Bloc Transmission of Enteroviruses

A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication.

Brachiola vesicularum, n. g., n. sp., a new microsporidium associated with AIDS and myositis

Brachiola vesicularum, n. g., n. sp., is a new microsporidium associated with AIDS and myositis. Biopsied muscle tissue, examined by light and electron microscopy, revealed the presence of organisms developing in direct contact with muscle cell cytoplasm and fibers. No other tissue types were infected. All parasite stages contain diplokaryotic nuclei and all cell division is by binary fission. Sporogony is disporoblastic, producing 2.9 times 2 μm diplokaryotic spores containing 8‐10 coils of the polar filament arranged in one to three rows, usually two. Additionally, this microsporidium produces electron‐dense extracellular secretions and vesiculotubular appendages similar to Nosema algerae. However, the production of protoplasmic extensions which may branch and terminate in extensive vesiculotubular structures is unique to this parasite. Additionally, unlike Nosema algerae, its development occurred at warm blooded host temperature (37‐38° C) and unlike Nosema connori, which disseminates to all tissue types, B. vesicularum infected only muscle cells. Thus, a new genus and species is proposed. Because of the similarities with the genus Nosema, this new genus is placed in the family Nosematidae. Successful clearing of this infection (both clinically and histologically) resulted from treatment with albendazole and itraconozole.

The molecular characterization of the major polar tube protein gene from Encephalitozoon hellem, a microsporidian parasite of humans

The microsporidia are obligate intracellular protozoan parasites of increasing importance as human pathogens, which are characterized by a small resistant spore with a single polar filament that coils around the sporoplasm. When stimulated, the polar filament rapidly everts out of the spore to form a hollow polar tube through which the sporoplasm passes, thus serving as a unique mechanism of transmission. A genomic library of the human microsporidium Encephalitozoon hellem was screened using a polyclonal rabbit antibody (anti-PTP Eh55) produced to the major HPLC purified polar tube protein (PTP) of E. hellem. This antibody localized to intrasporal polar filaments and extrasporal polar tubes of E. hellem by immunogold electron microscopy confirming the polar tube specificity of the antibody. A total of 14 anti-PTP Eh55 reactive genomic clones were identified and purified. A PTP gene was identified consisting of 1362 bp coding for 453 amino acids. The N-terminus of the translated protein consists of aputative N-terminal signal sequence of 22 amino acids, which when cleaved results in a mature protein of 431 amino acids with a predicted molecular mass of 43 kDa. The protein has a high proline content (14.6%) and contains a central domain of six alternating tandem repeats of 20 amino acids. After ligation of the gene into a glutathione S-transferase (GST) expression vector, a fusion protein was produced that reacted by immunoblotting with the polar tube specific anti-PTP Eh55. The gene was present as a single copy in the genome and there was no homology with other known genes. As the polar tube is a critical structure for the transmission of this organism to a new host cell, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.

Identification of a New Spore Wall Protein from Encephalitozoon cuniculi

ABSTRACT Microsporidia form environmentally resistant spores that are critical for their host-to-host transmission and persistence in the environment. The spore walls of these organisms are composed of two layers, the exospore and the endospore. Two spore wall proteins (SWP1 and SWP2) have been previously identified in members of the Encephalitozoonidae family. These proteins localize to the exospore. The endospore is known to contain chitin, and a putative glycosylphosphatidylinositol (GPI)-anchored chitin deacetylase has been localized to the plasmalemma-endospore interface. Using proteomic techniques, we have identified a new spore wall protein (SWP3) that is located in the endospore. The gene for this protein is located on chromosome 1 and corresponds to the open reading frame ECU01_1270. SWP3 is predicted to have a signal peptide and to be GPI anchored. Consistent with these modifications, two-dimensional electrophoresis demonstrated that SWP3 has an acidic pI and a molecular mass of <20 kDa. By immunoelectron microscopy, this protein was found on the cell surface during sporogony and in the endospore in mature spores. SWP3 has several potential O-glycosylation sites, and it is possible that it is a mannosylated protein like the major polar tube protein (PTP1).

The Effects of Elevated Temperatures and Various Time‐Temperature Combinations on the Development of Brachiola (Nosema) algerae N. Comb. in Mammalian Cell Culture

Abstract Nosema algerae Vávra and Undeen 1970, a microsporidian known to cause infection in mosquitoes, develops in mammalian cell cultures at 24–35 °C and in the tails and footpads of athymic mice. More recently it has been reported to grow at 38 °C in human cell culture. The present study is a two-part temperature/development examination. The first part examines the development of N. algerae in rabbit kidney cell culture at 29 °C, which permits the formation of functional spores within 72 h, and compares the effect of elevated temperatures (36.0, 36.5, 37 °C) on parasite development. At these elevated temperatures, N. algerae infects but undergoes only one or two proliferative divisions, with no evidence of sporogony by 72 h post-inoculation. During this time, however, the host cells continue to divide resulting in fewer infected cells over time and giving the appearance of a diminished parasitemia. Additionally, at 37 °C some organisms degenerate/hibernate by 72 h while others remain viable/active. It is not until 96 h that the parasites appear in large clusters of proliferative stages in the few host cells that are infected. By 120 h post-inoculation, proliferative cells, sporoblasts, and early spores are observed. These results suggest that elevated temperatures impede proliferation rates and the onset of sporogony. The second part of this study evaluates developmental changes in N. algerae when incubation temperatures and times are varied during parasite growth, resulting in abnormal parasite morphology. These abnormalities were not present when parasites were grown at constant temperature (29–37 °C). This report demonstrates that N. algerae can successfully develop at high temperatures (37 °C), justifying its taxonomic relocation to the genus Brachiola.

Ultrastructure and development of Pleistophora ronneafiei n. sp., a microsporidium (Protista) in the skeletal muscle of an immune-compromised individual.

Abstract This report provides a detailed ultrastructural study of the life cycle, including proliferative and sporogonic developmental stages, of the first Pleistophora species (microsporidium) obtained from an immune-incompetent patient. In 1985, the organism obtained from a muscle biopsy was initially identified as belonging to the genus Pleistophora, based on spore morphology and its location in a sporophorous vesicle. Since that initial report, at least two new microsporidial genera, Trachipleistophora and Brachiola, have been reported to infect the muscle tissue of immunologically compromised patients. Because Trachipleistophora development is similar to Pleistophora, and as Pleistophora was only known to occur in cold-blooded hosts, the question of the proper classification of this microsporidium arose. The information acquired in this study makes it possible to compare Pleistophora sp. (Ledford et al. 1985) to the known human infections and properly determine its correct taxonomic position. Our ultrastructural data have revealed the formation of multinucleate sporogonial plasmodia, a developmental characteristic of the genus Pleistophora and not Trachipleistophora. A comparison with other species of the genus supports the establishment of a new species. This parasite is given the name Pleistophora ronneafiei n. sp.

Glycosylation of the Major Polar Tube Protein of Encephalitozoon hellem, a Microsporidian Parasite That Infects Humans

ABSTRACT The microsporidia are ubiquitous, obligate intracellular eukaryotic spore-forming parasites infecting a wide range of invertebrates and vertebrates, including humans. The defining structure of microsporidia is the polar tube, which forms a hollow tube through which the sporoplasm is transferred to the host cell. Research on the molecular and cellular biology of the polar tube has resulted in the identification of three polar tube proteins: PTP1, PTP2, and PTP3. The major polar tube protein, PTP1, accounts for at least 70% of the mass of the polar tube. In the present study, PTP1 was found to be posttranslationally modified. Concanavalin A (ConA) bound to PTP1 and to the polar tube of several different microsporidia species. Analysis of the glycosylation of Encephalitozoon hellem PTP1 suggested that it is modified by O-linked mannosylation, and ConA binds to these O-linked mannose residues. Mannose pretreatment of RK13 host cells decreased their infection by E. hellem, consistent with an interaction between the mannosylation of PTP1 and some unknown host cell mannose-binding molecule. A CHO cell line (Lec1) that is unable to synthesize complex-type N-linked oligosaccharides had an increased susceptibility to E. hellem infection compared to wild-type CHO cells. These data suggest that the O-mannosylation of PTP1 may have functional significance for the ability of microsporidia to invade their host cells.

Identification of a microsporidian polar tube protein reactive monoclonal antibody

ABSTRACT. The microsporidia are characterized by spores containing a single polar tube that coils around the sporoplasm. When triggered by appropriate stimuli, the polar tube rapidly discharges out of the spore forming a hollow tube. The sporoplasm passes out of the spore through this tube serving as a unique vehicle of infection. Due to the unusual functional and solubility properties of the polar tube, the proteins comprising it are likely to be members of a protein family with a highly conserved amino acid composition among the various microsporidia. Polar tube proteins were separated from the majority of other proteins in glass bead disrupted spores of Glugea americanus using sequential 1% sodium dodecyl sulfate (SDS) and 9M urea extractions. The resultant spore pellet demonstrated broken, empty spore coats and numerous polar tubes in straight and twisted formations by negative stain transmission electron microscopy. After subsequent incubation of the pellet with 2% dithiothreitol (DTT), empty spore coats were still observed but the polar tubes were no longer present in the pellet. The DTT supernatant demonstrated four major protein bands by SDS‐PAGE: 23, 27, 34 and 43 kDa. Monoclonal antibodies were produced to these proteins using Hunters Titermax adjuvant. Mab 3C8.23.1 which cross‐reacted with a 43‐kDa antigen by immunoblot analyis, demonstrated strong reactivity with the polar tube of G. americanus spores by immunogold electron microscopy. This antibody will be useful in further characterization of polar tube proteins and may lead to novel diagnostic and therapeutic reagents.

Brachiola algerae Spore Membrane Systems, their Activity During Extrusion, and a New Structural Entity, the Multilayered Interlaced Network, Associated with the Polar Tube and the Sporoplasm

Abstract The microsporidial genus, Brachiola, contains three species: the type species Brachiola vesicularum (identified from an AIDS patient) and two species transferred from the genus Nosema, becoming Brachiola connori and Brachiola algerae. A developmental feature of the genus Brachiola is the “thickened” plasmalemma from sporoplasm through sporoblast stage. The sporoplasm has been reported to have a thick plasmalemma at 1-h postextrusion. The purpose of this investigation was to observe B. algerae spores before, during and after germination to determine if the plasmalemma is thick at the point of extrusion and if not, when and how it forms. New understandings regarding the polar filament position inside the spore, places it outside the sporoplasm proper with the sporoplasm limiting membrane invaginations surrounding it. These invaginations, present a possible location for aquaporins. The multilayered interlaced network (MIN), a new organelle (possibly of Golgi origin from the sporoblast), was observed inside the spore and sporoplasm; it formed an attachment to the end of the extruded polar tube and contributed to the thickening of the sporoplasm plasmalemma. A thin “unit limiting membrane”, present on the sporoplasm at the time of extrusion, is connected to the MIN by many cross-connections forming the “thick blistered” surface by 30 min-postextrusion.