Earl Weidner

Overcoating of Toxoplasma parasitophorous vacuoles with host cell vimentin type intermediate filaments.

The interaction between the Toxoplasma parasitophorous vacuole and vimentin‐type intermediate filaments in Vero cells was investigated via immunofluorescence microscopy. A significant rearrangement of host cell vimentin around the Toxoplasma parasitophorous vacuoles occurs throughout the course of infection. Host cell vimentin associates with the parasitophorous vacuoles within an hour after invasion. This vimentin overcoating of the vacuole is initiated at the host cell nuclear surface. During parasite multiplication, vimentin retains a closely defined association with the cytosolic surface of the parasitophorous vacuole. In addition, the vimentin intermediate filaments originating from the host cell nuclear surface are progressively rearranged around the enlarging parasitophorous compartment. During infections, the order of vimentin cytoskeleton is normal throughout the cell and appears redefined only at the vicinity of the parasitophorous vacuole. Depolymerization of the intermediate filaments was achieved with the phosphatase inhibitors okadaic acid and calyculin A. Disruption of the intermediate filament networks resulted in displacement of the parasitophorous vacuoles from the host cell nuclear surface. The data indicate that host cell vimentin binds to the Toxoplasma parasitophorous vacuoles and that the host intermediate filament network serves to dock the parasite compartment to the host cell nuclear surface.

Interactions between Encephalitozoon cuniculi and macrophages

SummaryEncephalitozoon cuniculi grow within ever-increasing parasitophorous vacuoles (PV) in peritoneal macrophages. The PV boundary membrane conforms to a rich arrangement of blebs; similar, but free vesicles were observed within the PV space. An iron dextran-concanavalin A marker was used to express visually clustered distributions of Con A receptors on the PV boundary blebs and free vesicles; no marker was observed on other membrane surfaces within the PV. These results, combined with the observation that the PV grows while the host cytoplasm decreases in mass, implicate the PV boundary blebs of interiorizing into vesicles by a pinocytic mechanism.Phagocytic vacuoles, secondary lysosomes and pinocytic vesicles were labeled by incubating infected macrophages in minimum essential medium with ferritin. Ferritin readily accumulated in secondary lysosomes and phagocytic vacuoles; however, ferritin was excluded from parasitophorous vacuoles containing E. cuniculi. Acid phosphatase cytochemical reaction product was observed in lysosomes and phagocytic vacuoles; however, parasitophorous vacuoles with vegetative E. cuniculi were always negative.

Differentiation of microsporidian spore-tails in Inodosporus spraguei Gen. et Sp. N.

SummaryThe new genus Inodosporus was erected to accept I. spraguei, a new species having eight sporoblasts per pansporoblast with each subsequent spore possessing three or four basal spore-tails and one branched apical one. It is primarily by the apical tail that the species is separated from the only other recognized species, I. octospora (Henneguy, 1892) comb. n., formerly Thelohania octospora.Spore-tails of I. spraguei are membranous channels which originate within differentiating pansporoblasts during genesis of sporonts into sporoblasts. During the switch from vegetative to spore-forming development, cytoplasmic constituents of I. spraguei segregate into two distinctive domains for which we originate the terms “pansporoblast-determinate area” (PDA) and “sporont-determinate area” (SDA). Membrane channels, which form spore-tails, develop within the PDA.The following observations indicate that the tails of I. spraguei are continuous with the outer pansporoblast envelope: lanthanum marker readily penetrates pansporoblasts and localizes in channels, in spore-tail attachment points, and between extra-sporoblast membrane and sporoblasts; a positive reaction for adenosine triphosphatase product accumulates within spore-tails at their sites of attachment to sporoblasts; and spore-tails occasionally remain attached to pansporoblast envelopes after mechanical disruption.An extensive PAS-positive glycocalyx-like material is found within newly developing pansporoblasts. This observation, plus the presence of an apparent adenosine triphosphatase system on pansporoblast membranes, indicates that the pansporoblast may serve as a molecular or ion transport system during initial phases of sporont differentiation. Inodosporus spraguei infects each muscle fiber completely until filaments are destroyed, and infections are spread throughout the animal until most fibers are infected. Curiously, uninfected muscle cells seldom show serious pathological changes caused by massive infections of neighboring cells.

Superoxide dismutase and catalase in Toxoplasma gondii

Catalase and superoxide dismutase detected in both RH and C strain Toxoplasma gondii tachyzoites were distinctly different in electrophoretic mobility from host cell enzymes. Catalase and superoxide dismutase activity levels were similar in both Toxoplasma strains and showed narrow pH optima around 8.0. Toxoplasma superoxide dismutase was resistant to cyanide but inhibited by azide or peroxide, consistent with an iron-containing enzyme typical of protozoan parasites. These enzymes may play a role in intracellular survival; however, they do not appear to be the basis for differences in virulence to mice.

Polyhedra without virions in a vertically transmitted nuclear polyhedrosis virus

Abstract Polyhedra were observed in larvae, pupae, and adults (f1 generation) of Spodoptera frugiperda whose parents had survived exposure to the S. frugiperda nuclear polyhedrosis virus (NPV). These polyhedra were isolated from the f1 insects and fed to first instars. The polyhedra from f1 larvae and pupae infected the first instars (86–98 and 69–88% infection, respectively), but most of those recovered from adults of the f1 generation did not (0–11% infection). Electron microscopy indicated that the infectious polyhedra from adults contained virions, but the noninfectious polyhedra from adults did not. A monoclonal anti-polyhedrin antibody used in conjunction with fluorescence microscopy reacted positively with the empty, noninfectious polyhedra, indicating that the polyhedrin protein was present. Thus, when the S. frugiperda NPV is vertically transmitted, the infections in the f1 adults are nonlethal and are manifested mainly as noninfectious, empty polyhedra which are composed of the polyhedrin protein.

Microsporidian Spore Envelope Keratins Phosphorylate and Disassemble During Spore Activation

The microsporidian spore stage of the nerve parasite, Spraguea lophii, consists of outer envelope stabilized in part by keratins, including K4 and K13. The nonepidermal K4 and K13 keratins were found only in the spore envelope and were absent in the internal microsporidian sporoplasm. At the time of spore activation, the keratin‐based outer spore envelope assemblage dissociated and became phosphorylated when the spores were placed in the presence of labeled ATP. Verapamil or lanthanum, agents which block S. lophii spore activation, also blocked spore envelope keratin disassembly and phosphorylation when the spores were incubated in activation medium with labeled ATP. However, after the removal of the verapamil or lanthanum, the spores regained the capacity to activate in discharge medium and the keratin analogues appeared to dissociate and phosphorylate.

The plaque matrix (PQM) and tubules at the surface of intramuscular parasite, Trachipleistophora hominis

ABSTRACT. Surface plaque matrix (PQM) and a tubular arrangement of filaments border Trachipleistophora hominis parasites during growth within host muscle. The PQM at the parasite surface forms a network of processes which can be associated with filamentous tubules. Peroxidase tracer delineated the PQM and showed apparent connections with the tubules. The tubules at the interface of T. hominis‐infected cells are structurally similar to the extrasporular tubules of the microsporidian, Ameson michaelis. The extrasporular tubules of A. michaelis and the proteins from T. hominis‐infected muscle reacted to keratin antibodies, K8.13, K4 and K13. Conversely, antibodies produced to T. hominis‐infected muscle, reacted with the extrasporular tubular proteins of A. michaelis. The PQM and tubular elements are thought to play an important role in affecting molecular traffic between the host and parasite.

Evidence for Heterotrimeric GTP‐Binding Proteins in Toxoplasma gondii

Toxoplasma gondii, an intracellular protozoan parasite, resides within a host‐derived vacuole that is rapidly modified by a parasite‐secreted membranous tubular network. In this study we investigated the involvement of heterotrimeric G proteins in the secretory pathway of T. gondii. Aluminum fluoride (AIFn), a specific activator of heterotrimeric G proteins, induced secretion from isolated tachyzoites of T. gondii in vitro, as seen by light optics and electron microscopy. In Western blot analyses, antibodies to G protein α subunits reacted with 39–42 kDa proteins from T. gondii isolates. Antibodies to Goα and Gsα coupled to the fluorescent probe fluorescein isothiocyanate localized to the paranuclear region of T. gondii. Gi3α immunoprobes were confined to the cytoplasmic matrix of T. gondii and also labeled the parasitophorous vesicle. Fluorescein isothiocyanate‐conjugated GA/1, an antipeptide antisera directed toward the GTP binding site common to G protein α subunits, was confined to the lateral cytoplasmic domain of the parasites where secretion is most prominent. In time‐sequence studies using the GA/1 probe, the immunoreactive material shifted position daring invasion of target cell to areas of active secretion.

Sporogenesis of a myxosporidan with motile spores

SummaryThree types of cells comprise each Fabespora vermicola sporoblast: valvogenic (VAV), capsulogenic (CAP), and germinative (GEM). Walls, polar caps, and sutures are the main assemblages produced by the VAV cells. The unique polar cap organelle extends over the aperture region of the polar capsule component of the CAP cell. The VAV cell also assembles a wall located on the cytoplasmic side of the plasma membrane facing the sporoblast exterior. Bundles of 7 nm microfilaments develop within the extracellular space between the VAV and interior cells of the sporoblast. These microfilaments assemble late in sporogenesis when the spore acquires the capacity for locomotion. Polar filament construction takes place exclusively within the polar capsule primordium (PCP) by apparent self-assembly prior to the PCP being enveloped by membranes. The CAP and GEM cells accumulate considerable glycogen during sporogenesis. The first identifiable GEM cell is single, but has two unpaired nuclei. These GEM cell nuclei later form a paired structure which is sustained into the spore stage.

Cytoskeletal Proteins Expressed by Microsporidian Parasites

Microsporidians are intracellular parasites characterized by a small missile cell spore that when activated, fires a discharge tube through which infective sporo-plasm is injected into a target cell (Weidner, 1972). Microsporidians develop directly in the cytoplasm of host cells and therefore are not confined to a parasitophorous vacuole.