Morphology and Ultrastructure of Cryptobia eilatica n. sp. (Bodonidae: Kinetoplastida), an Ectoparasite from the Gills of Marine Fish

ABSTRACT. A marine kinetoplastid flagellate, Cryptobia eilatica n. sp., is described from the gills of cultured gilt-head sea bream Sparus aurata L. and wild black-spot sea bream Diplodus noct (Valenciennes) in the Red Sea. The trophozoite is elongated and lacks a contractile vacuole and undulating membrane. The body averages 13.5 × 4.1 μm, anterior flagellum 9.7 μm, and free portion of recurrent flagellum 15.2 μm. The ultrastructural features of the species exhibit great similarity to various previously studied Cryptobiids. Cryptobia eilatica trophozoites feed on bacteria, show a preference for the branc hial interlamellar crypts, and attach to the host epithelium by means of the recurrent flagellum. Neither penetration into the epithelial cells, nor any direct damage to host tissue was observed. Cryptobia eilatica inhabits a purely marine habitat, but its trophozoite tolerates salinities as low as 10 ppt.     

Toxoplasma gondii: purification of trophozoites propagated in cell culture.

A new procedure is described for the purification of trophozoites from the virulent RH strain of Toxoplasma gondii propagated in baby hamster kidney (BHK-21) cell cultures. The culture medium containing host cell debris and trophozoites was filtered through glass-wool filtering fiber, which removed most host cell material. The filtrate containing trophozoites was centrifuged, and the trophozoite pellet was resuspended and washed in phosphate-buffered saline. An average of about 75% of the original number of trophozoites was recovered. No loss of trophozoite viability was observed as determined by the rate of host cell culture monolayer destruction. The amount of host cell material contamination in the final trophozoite fraction was negligible as determined by measuring radioactivity in the trophozoite fraction after cofiltration with noninfected host cell material which had been prelabeled with radioactive precursors.     

Ultrastructure of Cryptosporidium wrairi from the Guinea Pig

SYNOPSIS. The ultrastructure of the known tissue stages of Cryptosporidium wrairi Vetterling, Jervis, Merrill, and Sprinz, 1971 parasitizing the ileum of guinea pigs is described. Young trophozoites are surrounded by 4 unit membranes, the outer 2 of host origin, the inner 2 the pellicle of the parasite. Each trophozoite contains a vesicular nucleus with a large nucleolus. Its cytoplasm contains ribosomes, but eventually fills with cisternae of the rough endoplasmic reticulum. As the trophozoite matures the area of attachment of the parasite to the host cell becomes vacuolated, with vertical membranous folds. It is apparent that the parasite acquires nourishment from the host cell thru this area of attachment. As schizonts develop, (a) multiple nuclei appear, (b) the endoplasmic reticulum enlarges, (c) the attachment zone increases in area, (d) large vacuoles, which develop as endocytotic vesicles in the attachment area, are found in the cytoplasm and (e) the inner unit membrane of the parasite pellicle is resorbed around the sides of the developing schizont. Following nuclear division, merozoites develop from the schizont by budding. Merozoites have an ultrastructure similar to that described for other coccidia except that no mitochondria, micropores, or subpellicular tubules were observed. Merozoites penetrate the epithelial cell causing invagination of the microvillar membrane and lysing it. No unit membrane is formed between the parasite and the host cell. However, the cell produces one or 2 dense bands adjacent to the parasite attachment area. The macrogamete contains a nucleus, endoplasmic reticulum, attachment zone, and large vacuoles. It also contains a variety of granules, some of which are polysaccharide. The immature microgametocyte contains multiple compact nuclei. No mature microgametocytes or zygotes were found.     

A cytological study of a chonotrichous ciliate protozoan,Lobochona prorates,from the gribble

The Ciliophora are unique in that they are heterokaryote. In certain ciliates, namely the Chlamydodontidae and Dysteriidae of the Order Holotrichida and Order Chonotrichida, the macronucleus has a chromophilic portion and a chromophobic portion. The similarities in structure and behavior of the macronucleus of Lobochona prorates to that of some lower holotrichs as well as the genesis of the ciliary mechanism suggest the close affinities of the Order Chonotrichida to the lower holotrichs — the cyrtophorine gymnostomes. Budding, a somewhat modified binary fission preserving the fundamental structure of the parent individual, appears to be an adaption to the sessile life of the Chonotrichida. Throughout the order the process is basically similar, but there are noticeable differences from genus to genus. There appear to be two distinct patterns of budding in the Chonotrichida: (1) the parent retains its fundamental structure, and (2) the parental cytosome diminishes. Both patterns may produce one or more (multiple) buds. Diminutive budding appears to be a mechanism of these sessile organisms which enables them to abandon the exuvium of the crustacean host and to reinfest the original host or a new host. Sexual reproduction is by conjugation. In L. prorates it occurs between adjacent individuals on the same pleopod of Limnoria tripunctata and not between a stationary individual and a “swarming” larva.     

Ultrastructure of Infection, Development and Gametocyst Formation of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) in Its Mosquito Host, Aedes albopictus (Diptera: Culicidae)

ABSTRACT. The life history of the protozoan parasite Ascogregarina taiwanensis in mosquito larvae ( Aedes albopictus , collected in southern Taiwan) was shown to consist of two consecutive stages—intracellular and extracellular. Light microscopy showed that most trophozoites moved into the Malpighian tubules and developed into giant trophozoites during the first day pupa. The locomotion may be associated with bristle-like ridges of the trophozoite. The stage for sexual reproduction, i.e. the gamete, was then formed by segmentation of the giant trophozoite and twisting off the anucleate extremities of the body. Sexual reproduction occurred via fertilization by fusion of two resulting gametes, presumably two opposed sexes. The fused gametes finally generate the formation of the gametocyst, within which oocysts develop by budding from the cytoplasmic mass. This type of sexual reproduction has not been reported previously in any gregarine protozoa. We here proposed it as a new hypothesis for further elucidation of the protozoan reproduction.     

A comparison of the Giardia lamblia trophozoite and cyst transcriptome using microarrays

Background  

Compared with many protists, Giardia lamblia has a simple life cycle alternating between cyst and trophozoite. Most research on the molecular biology of Giardia parasites has focused on trophozoites and the processes of excystation and encystation, whereas cysts have attracted less interest. The striking morphological differences between the dormant cyst and the rapidly dividing and motile trophozoite implies profound changes in the metabolism as the parasite encysts in the host's intestine and excysts upon ingestion by a new host.     

Immunoelectron microscopic localization of circumsporozoite antigen in the differentiating exoerythrocytic trophozoite of Plasmodium berghei

The distribution of the circumsporozoite (CS) antigens in the 24 hour exoerythrocytic trophozoite of P. berghei was studied using Lowicryl immunogold electron microscopy. These antigens were present on the plasmalemma of the parasite, in disrupted areas of the host cell cytoplasm adjacent to the trophozoite and around inclusions of the host cell cytoplasm. There was evidence of a redistribution of the CS antigens away from the pellicular region of the sporozoite.     

Late domain-dependent inhibition of equine infectious anemia virus budding

The Gag proteins of a number of different retroviruses contain late or L domains that promote the release of virions from the plasma membrane. Three types of L domains have been identified to date: Pro-Thr-Ala-Pro (PTAP), Pro-Pro-X-Tyr, and Tyr-Pro-Asp-Leu. It has previously been demonstrated that overexpression of the N-terminal, E2-like domain of the endosomal sorting factor TSG101 (TSG-5') inhibits human immunodeficiency virus type 1 (HIV-1) release but does not affect the release of the PPPY-containing retrovirus murine leukemia virus (MLV), whereas overexpression of the C-terminal portion of TSG101 (TSG-3') potently disrupts both HIV-1 and MLV budding. In addition, it has been reported that, while the release of a number of retroviruses is disrupted by proteasome inhibitors, equine infectious anemia virus (EIAV) budding is not affected by these agents. In this study, we tested the ability of TSG-5', TSG-3', and full-length TSG101 (TSG-F) overexpression, a dominant negative form of the AAA ATPase Vps4, and proteasome inhibitors to disrupt the budding of EIAV particles bearing each of the three types of L domain. The results indicate that (i) inhibition by TSG-5' correlates with dependence on PTAP; (ii) the release of wild-type EIAV (EIAV/WT) is insensitive to TSG-3', whereas this C-terminal TSG101 fragment potently impairs the budding of EIAV when it is rendered PTAP or PPPY dependent; (iii) budding of all EIAV clones is blocked by dominant negative Vps4; and (iv) EIAV/WT release is not impaired by proteasome inhibitors, while EIAV/PTAP and EIAV/PPPY release is strongly disrupted by these compounds. These findings highlight intriguing similarities and differences in host factor utilization by retroviral L domains and suggest that the insensitivity of EIAV to proteasome inhibitors is conferred by the L domain itself and not by determinants in Gag outside the L domain.     

Eugregarine trophozoite detachment from the host epithelium via epimerite retraction: Fiction or fact?

Eugregarines represent a diverse group of Apicomplexa parasitising numerous invertebrates. Their sporozoites generally develop into epicellular trophozoites attached to the host epithelium by a specialised attachment organelle known as an epimerite. They are considered peculiar protists due to their unique cell architecture and dimensions as well as their attachment strategy which is similar to that of cryptosporidia. Using electron and fluorescence microscopy, the fine structure of the epimerite with associated structures and the mechanism of trophozoite detachment from the host epithelium were studied in Gregarina polymorpha parasitising the intestine of Tenebrio molitor larvae. The epimerite appears to be a very dynamic structure whose shape dramatically changes depending on whether or not it is embedded into the host epithelium. The trophozoite’s most fragile zone is the area below the membrane fusion site at the epimerite base. The epimerite plasma membrane forms basal radial ribs which are involved in increasing its surface and strengthening the epimerite-host cell junction. FITC-phalloidin labelling demonstrated the presence of filamentous actin in trophozoites along with its accumulation at the epimerite base and in the apical end of the protomerite, as well as a patch accumulation of filamentous actin in the protomerite of maturing and mature trophozoites. Indirect immunofluorescence revealed the presence of myosin in the cortical zone of the epimerite and in the membrane fusion site area. The data obtained strongly suggest that these structures could facilitate the detachment of a mature trophozoite from the host epithelium. Supported by data presented herein and our previous observations, we propose a new hypothesis on the mechanism of trophozoite detachment from the host epithelium based on epimerite retraction into the protomerite. This is contrary to the commonly accepted hypothesis describing gradual epimerite constriction and subsequent separation facilitated by contractility of the membrane fusion site (osmiophilic ring).     

More than one door - Budding of enveloped viruses through cellular membranes

Enveloped viruses exit their host cell by budding from a cellular membrane and thereby spread from one cell to another. Virus budding in general involves the distortion of a cellular membrane away from the cytoplasm, envelopment of the viral capsid by one or more lipid bilayers that are enriched in viral membrane glycoproteins, and a fission event that separates the enveloped virion from the cellular membrane. While it was initially thought that virus budding is always driven by viral transmembrane proteins interacting with the inner structural proteins, it is now clear that the driving force may be different depending on the virus. Research over the past years has shown that viral components specifically interact with host cell lipids and proteins, thereby adopting cellular functions and pathways to facilitate virus release. This review summarizes the current knowledge of the cellular membrane systems that serve as viral budding sites and of the viral and cellular factors involved in budding. One of the best studied cellular machineries required for virus egress is the ESCRT complex, which will be described in more detail.     

Sporogony in Pneumocystis carinii: Synaptonemal Complexes and Meiotic Nuclear Divisions Observed in Precysts1

Evidence for meiosis was demonstrated electron microscopically for the first time in Pneumocystis carinii in rat alveoli by the observation of synaptonemal complexes followed by nuclear divisions. Synaptonemal complexes indicating meiotic nuclear divisions were observed in uninuclear precysts. Additionally, owing to the use of tannic acid as a fixative, spindle microtubules were also observed for the first time in the precyst. Based on these facts, a new life cycle of the organism is proposed. The precyst has generally been considered an intermediate form between the trophozoite and the cyst. The present paper proposes that the precyst is additionally defined as the cell in which eight intracystic bodies are produced through meiotic reduction. The most characteristic feature of the precyst is a clump of mitochondria in the cytoplasm. We divide the precyst phase into three forms, which are named early, intermediate, and late. Synaptonemal complexes were only observed in the early precyst, which is a uninuclear cell with a thin pellicle. In the intermediate precyst, nuclear divisions are observed as follows: meiosis I produces two haploid nuclei and each of these divides at meiosis II producing four nuclei. After that, another postmeiotic mitosis takes place, resulting in eight haploid nuclei. In the late precyst, a delimiting membrane originates from the mother plasmalemma and surrounds the daughter nuclei and a small portion of the adjacent cytoplasm. Finally, when the eight intracystic bodies are complete, the precyst changes to a cyst. Thus, we deduce that intracystic bodies resulting from meiotic nuclear division are haploid and, after excystation, they are haploid trophozoites. We consider that this process can be called sporogony. Although we could not distinguish between the haploid and the diploid trophozoite, it is quite plausible that copulation occurs, probably in host alveoli.     

An ultrastructural comparison of the attachment sites between Gregarina steini and Cryptosporidium muris

Early developmental stages of Gregarina steini Berndt, 1902 from the intestine of Tenebrio molitor larvae were studied by transmission electron microscopy. The formation and structure of the eugregarine attachment site were compared with comparable features found on the feeder organelle of Cryptosporidium muris Tyzzer, 1907, from the stomach of experimentally infected rodents. The similarity of the attachment strategy between both organisms was revealed. The membrane fusion site in G. steini, formed by the trophozoite plasma membrane, host cell plasma membrane and a membrane-like structure limiting the cortical zone of the epimerite, resembles the Y-shaped membrane junction between the host cell plasma membrane, the trophozoite plasma membrane and membrane surrounding the anterior vacuole in C. muris. The anterior vacuole of C. muris appears to be the precursor of the feeder organelle and its structure is very similar to the epimeritic bud and the cortical zone of G. steini trophozoites. In both investigated organisms, the apical complex disappears early during cell invasion. The possibility of the epicellular location of Cryptosporidium on the surface of host cells is discussed.     

Soft X-ray microscopy analysis of cell volume and hemoglobin content in erythrocytes infected with asexual and sexual stages of Plasmodium falciparum

Plasmodium falciparum, the most virulent agent of human malaria, undergoes both asexual cycling and sexual differentiation inside erythrocytes. As the intraerythrocytic parasite develops it increases in size and alters the permeability of the host cell plasma membrane. An intriguing question is: how is the integrity of the host erythrocyte maintained during the intraerythrocytic cycle? We have used water window cryo X-ray tomography to determine cell morphology and hemoglobin content at different stages of asexual and sexual differentiation. The cryo stabilization preserves native structure permitting accurate analyses of parasite and host cell volumes. Absorption of soft X-rays by protein adheres to Beer–Lambert’s law permitting quantitation of the concentration of hemoglobin in the host cell compartment. During asexual development the volume of the parasite reaches about 50% of the uninfected erythrocyte volume but the infected erythrocyte volume remains relatively constant. The total hemoglobin content gradually decreases during the 48 h cycle but its concentration remains constant until early trophozoite stage, decreases by 25%, then remains constant again until just prior to rupture. During early sexual development the gametocyte has a similar morphology to a trophozoite but then undergoes a dramatic shape change. Our cryo X-ray tomography analysis reveals that about 70% of the host cell hemoglobin is taken up and digested during gametocyte development and the parasite eventually occupies about 50% of the uninfected erythrocyte volume. The total volume of the infected erythrocyte remains constant, apart from some reversible shrinkage at stage IV, while the concentration of hemoglobin decreases to about 70% of that in an uninfected erythrocyte.     

Plasmodium falciparum-infected erythrocytes: qualitative and quantitative analyses of parasite-induced knobs by atomic force microscopy

We used the combination of an atomic force microscope and a light microscope equipped with epifluorescence to serially image Plasmodium falciparum-infected erythrocytes. This procedure allowed us to determine unambiguously the presence and developmental stage of the malaria parasite as well as the number and size of knobs in singly, doubly, and triply infected erythrocytes. Knobs are not present during the ring stage of a malaria infection but a lesion resulting from invasion by a merozoite is clearly visible on the erythrocyte surface. This lesion is visible into the late trophozoite stage of infection. Knobs begin to form during the early trophozoite stage of infection and have a single-unit structure. Our data suggest the possibility that a two-unit structure of knobs, which was reported by Aikawa et al. (1996, Exp. Parasitol. 84, 339-343) using atomic force microscopy, appears to be a double-tipped image. The number of knobs per unit of host cell surface area is directly proportional to parasite number in both early and late trophozoite stages. These results indicate that knob formation by one parasite does not influence knob formation by other parasites in a multiply infected erythrocyte. In addition, knob volume is not influenced by either parasite stage or number at the late trophozoite stage, indicating that the number of component molecules per knob is constant throughout the parasite maturation process.     

Toxoplasma gondii exploits the host ESCRT machinery for parasite uptake of host cytosolic proteins

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole (PV), the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the host Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV-1 virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV-1 Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of TgGRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PV membrane (PVM). These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission.     

UDP-N-acetylglucosamine 4'-epimerase from the intestinal protozoan Giardia intestinalis lacks UDP-glucose 4'-epimerase activity

The protozoan parasite Giardia intestinalis has a simple life cycle consisting of an intestinal trophozoite stage and an environmentally resistant cyst stage. The cyst is formed when a trophozoite encases itself within an external filamentous covering, the cyst wall, which is crucial to the cyst's survival outside of the host. The filaments in the cyst wall consist mainly of a beta (1-3) polymer of N-acetylgalactosamine. Its precursor, UDP-N-acetylgalactosamine, is synthesized from fructose 6-phosphate by a pathway of five inducible enzymes. The fifth, UDP-N-acetylglucosamine 4'-epimerase, epimerizes UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine reversibly. The epimerase of G. intestinalis lacks UDP-glucose/UDP-galactose 4'-epimerase activity and shows characteristic amino acyl residues to allow binding of only the larger UDP-N-acetylhexosamines. While the Giardia epimerase catalyzes the reversible epimerization of UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine, the reverse reaction apparently is favored. The enzyme has a higher Vmax and a smaller Km in this direction. Therefore, an excess of UDP-N-acetylglucosamine is required to drive the reaction towards the synthesis of UDP-N-acetylgalactosamine, when it is needed for cyst wall formation. This forms the ultimate regulatory step in cyst wall biosynthesis.     

Compartment analysis of ATP in malaria-infected erythrocytes

The ATP concentration of malaria-infected erythrocytes changes substantially with parasite development. These alterations have been attributed to a decline in host cell [ATP], but have not been tested critically hitherto. A method for the compartmental analysis of ATP in malaria (Plasmodium falciparum)-infected human red blood cells has been developed using Sendai virus to permeabilize the host erythrocyte membrane. Permeabilization and release of host cytosol was complete within 6 to 8 minutes and ATP was measured by the luciferin-luciferase bioluminescence assay in the lysate and in the pellet. Equal ATP concentrations were found in host and parasite compartments at the trophozoite and schizont stages. Both were lower than those detected in uninfected cells. Other methods for compartment analysis of ATP are presented and discussed.     

Directed migration of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) in its natural host Aedes albopictus (Diptera: Culicidae)

Directed migration of trophozoites from the midgut toward the Malpighian tubules is essential for Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) to complete its developmental cycle within the natural host Aedes albopictus. We have obtained a 275-bp actin cDNA fragment amplified from extracted mRNAs of migrating trophozoites, suggesting the involvement of actin in trophozoite motility. Down-regulation on the migration of the trophozoite was seen in mosquito larvae fed with cytochalasin D, ML-7, and BDM, indicating that myosin, in the form of an actomyosin system, may also be involved in driving motility of the trophozoite. The "protruding apparatus" (PA) formed at the anterior end of trophozoites during the migrating stage had significant deposits of actin by immunofluorescent microscopy. Moreover, PA formation was enhanced in response to elevated levels of 20-hydroxyecdysone (20-HE) in cultures of alimentary canals in which the trophozite was contained. Thus, 20-HE may also promote expression of actin and perhaps myosin simultaneously.     

Ditrypanocystis sp. (Apicomplexa, Gregarinia, Selenidiidae): the mode of survival in the gut of Enchytraeus albidus (Annelida, Oligochaeta, Enchytraeidae) is close to that of the coccidian genus Cryptosporidium

A selenid gregarine Ditrypanocystis sp. (Apicomplexa, Gregarinia, Selenidiidae), harboring the gut lumen of the oligochaete Enchytraeus albidus, was studied by light and electron microscopy. The trophozoite of Ditrypanocystis sp. is attached to the gut wall with its apical end to be anchored eventually between enterocytes in the crypts. Simultaneously, between the surfaces of the parasite and the host cell a peculiar contact is formed made of membranous channels and vesicles of unknown origin, the host cell surface in the contact area lacking cilia. The trophozoite becomes progressively enclosed within a parasitophorous vacuole made of layers of fused ciliar membranes of enterocytes. The fused cilia may be a source of membranes lining channels and vesicles of the contact area. Such a mode of parasitophorous arrangements has never been described before for gregarines, however, it bears a some likeness with that of the coccidian genus Cryptosporidium (similarity and differences being discussed). With regard to some molecular phylogeny constructions, claiming the "sister" relationship between gregarines and the coccidian genus Cryptosporidium (Carreno et al., 1999; Leander et al., 2003), this common feature in host-parasite relationships enabled us to put forward an idea of a possible evolutionary route from extracellularity of gregarines to intracellularity of coccidia, as exemplified by species of Cryptosporidium.     

Pathogenesis of infection byEntamoeba histolytica

Entamoeba histolytica, a protozoan parasite, is the etiologic agent of amoebiasis in humans. It exists in two forms—the trophozoite which is the active, dividing form, and the cyst which is dormant and can survive for prolonged periods outside the host. In most infected individuals the trophozoites exist as commensals. In a small percentage of infections, the trophozoites become invasive and penetrate the intestinal mucosa, causing ulcers. The trophozoites may reach other parts of the body—mainly liver, where they cause tissue necrosis, leading to lifethreatening abscesses. It is thought that pathogenesis of infection byEntamoeba histolytica is governed at several levels, chief among them are (i) adherence of trophozoite to the target cell, (ii) lysis of target cell, and (iii) phagocytosis of target cell. Several molecules which may be involved in these processes have been identified. A lectin inhibitable by galactose and N-acetyl-D-galactosamine is present on the trophozoite surface. This is implicated in adherence of trophozoite to the target cell. Various amoebic poreforming proteins are known, of which 5kDa protein (amoebapore) has been extensively studied. These can insert into the lipid bilayers of target cells, forming ion-channels. The phagocytic potential of trophozoites is directly linked to virulence as measured in animal models. Factors like association of bacteria with trophozoites also influence virulence. Thus, pathogenesis is determined by multiple factors and a unifying picture taking into account the relative contributions of each factor is sought. Recent technical advances, which includes the development of a transfection system to introduce genes into trophozoites, should help to understand the mechanism of pathogenesis in amoebiasis.