Do microtubules around the Toxoplasma gondii-containing parasitophorous vacuole in skeletal muscle cells form a barrier for the phagolysosomal fusion?

The intracellular fate of Toxoplasma gondii was studied in primary cultures of skeletal muscle cells (SMC). The labelling of secondary lysosomes with BSA-Au particles showed no phagolysosomal fusion with the vacuole containing the parasite. After internalization of the parasites, the parasitophorous vacuole became involved by closely apposed endoplasmic reticulum (ER) and mitochondria; within 18 h of interaction, microtubules were visualized in association with the parasitophorous vacuole, suggesting that they could form a barrier for the phagolysosomal fusion.     

In vitro light and electron microscope studies on different virulent promastigotes ofLeishmania donovani in hamster peritoneal macrophages

Summary Hamster peritoneal macrophages were infected with arivulent and virulent promastigotes of aL. donovani strain using various ratios (11; 110) of parasites and peritoneal cells. Light microscope studies have shown that there was a significant difference in the number of parasites taken up by phagocytic cells between the macrophage cultures infected with avirulent and virulent promastigotes at 4 h as well as during the following 14 days of infection. In both virulent groups the number of amastigotes were sharply increased. However, the surviving parasites were eliminated continuously when the macrophage cultures were infected with avirulent parasites. Electron microscope examinations of the different infected macrophage cultures did not show any difference in the localization of the surviving parasites. At one and 24 h post-infection, parasites have been observed in typical parasitophorous vacuoles. However, by day 4, 7, and 14 post-infections, the majority of intact parasites were surrounded by a four-laminar membrane without a space between parasite and vacuole membrane. Besides, some amastigotes were seen in large parasitophorous vacuoles. It seemed as if some of these amastigotes were trying to leave the parasitophorous vacuoles. In all cases acid phosphatase could be demonstrated in the parasitophorous vacoules and around the parasites indicating that the lysosomes of the host cell have been fused with the parasitophorous vacuole. It is indicated that the virulentLeishmania parasites are more resistant to the digestive system of the macrophages.     

Exocytosis ofToxoplasma gondii dense granules into the parasitophorous vacuole after host cell invasion

Tachyzoites ofToxoplasma gondii have been shown to exocytose the contents of dense granules into the parasitophorous vacuole after host cell invasion. A monoclonal antibody specific for a 27-kDa protein was used to locate the dense granules by immunoelectron microscopy. The same antibody also reacted with the tubular network found in the parasitophorous vacuole, which confirmed that the dense granules were exocytosed by tachyzoites.     

Ultrastructure of the attachment of Cryptosporidium sporozoites to tissue culture cells

The attachment of Cryptosporidium sporozoites to Madin-Darby canine kidney (MDCK) cells was examined using transmission electron microscopy. As the anterior end of the sporozoite came into close proximity to the MDCK cell, the host cell membrane evaginated around the sporozoite, forming a parasitophorous vacuole. A dense band formed below the host cell membrane at the site nearest to the conoid. Variably electron-dense material was apparently released from the conoid and a large membrane-bound vacuole was formed in the anterior end of the sporozoite, displacing the typical anterior electron-dense organelles (rhoptries and micronemes). The outer membrane of the sporozoite pellicle then fused with the host cell membrane immediately adjacent to the conoid. The membrane surrounding the anterior vacuole was also fused with the common host-parasite membrane, forming Y-shaped membrane junctions where each limb was a unit membrane. A direct link was thereby established between the anterior vacuole of the sporozoite and the host cell cytoplasm. The anterior vacuole membrane separating the sporozoite and the host cell cytoplasm was the precursor of the feeder organelle.     

Localization and activity of various lysosomal proteases in Leishmania amazonensis-infected macrophages.

In mammalian hosts, Leishmania amastigotes are obligatory intracellular parasites of macrophages and multiply within parasitophorous vacuoles of phagolysosomal origin. To understand how they escape the harmful strategies developed by macrophages to kill ingested microorganisms, it is important to obtain information on the functional state of parasitophorous vacuole. For this purpose, we studied the intracellular distribution and activity of host lysosomal proteases in rat bone marrow-derived macrophages infected with Leishmania amazonensis amastigotes. Localization of cathepsins B, H, L, and D was investigated by using specific immunoglobulins. In uninfected macrophages, these enzymes were located in perinuclear granules (most of them were probably secondary lysosomes) which, after infection, disappeared progressively. In infected macrophages, cathepsins were detected mainly in the parasitophorous vacuoles, suggesting that the missing secondary lysosomes had fused with these organelles. Biochemical assays of various proteases (cathepsins B, H, and D and dipeptidyl peptidases I and II) showed that infection was accompanied by a progressive increase of all activities tested, except that of dipeptidyl peptidase II, which remained constant. No more than 1 to 10% of these activities could be attributed to amastigotes. These data indicate that (i) Leishmania infection is followed by an increased synthesis and/or a reduced catabolism of host lysosomal proteases, and (ii) amastigotes grow in a compartment rich in apparently fully active proteases. Unexpectedly, it was found that infected and uninfected macrophages degraded endocytosed proteins similarly. The lack of correlation in infected macrophages between increase of protease activities and catabolism of exogenous proteins could be linked to the huge increase in volume of the lysosomal compartment.     

Depletion of secondary lysosomes in mouse macrophages infected withLeishmania mexicana amazonensis: A cytochemical study

Leishmania amastigotes lodge and multiply within parasitophorous vacuoles, which can fuse with secondary lysosomes of the host macrophages. This study examines the effect of infection with amastigotes ofL. mexicana amazonensis on the secondary lysosomes of mouse macrophage cultures. The cultures were stained for the activities of two lysosomal enzyme markers, acid phosphatase and arylsulfatase, and the light microscopic observations were supplemented by electron microscopy. Nearly all noninfected macrophages contained numerous stained secondary lysosomes. The number of such lysosomes was markedly reduced 24 h postinfection, and the reduction persisted for at least 10 days. Stained secondary lysosomes reppeared after the amastigotes were destroyed by exposure of the cultures to phenazine methosulfate or by placing them at 37.5° C. The depletion of lysosomes shown by cytochemical methods may reflect a high rate of fusion of the lysosomes with the parasitophorous vacuoles, exceeding the rate of formation of new secondary lysosomes. Alternatively, the parasites may inhibit the synthesis of lysosomal hydrolases, or the assembly or formation of primary or secondary lysosomes.     

Ultrastructure of gamonts and gametes and fertilization ofSarcocystis sp. from the roe deer (Capreolus capreolus) in dogs

Gamonts ofSarcocystis sp. from the roe deer were examined in the intestine of dogs 10 h after inoculation. Early macrogamonts were limited by a three-membranous pellicle, and situated in a parasitophorous vacuole. Female sexual stages during fertilization, the macrogametes, were limited by five membranes, and microgametes were observed in the parasitophorous vacuole. The outer membranes of the microgamete and macrogamete fuse, and the nucleoplasm of the microgamete enters the cytoplasm of the macrogamete. No wall-forming bodies were observed in macrogamonts and macrogametes.     

Toxoplasma gondii : an ultrastructural study of host-cell invasion by the bradyzoite stage

The invasion of Toxoplasma gondii tachyzoites and bradyzoites was followed in bovine kidney cells via electron microscopy. The process of invasion differed between bradyzoites and tachyzoites. In the early stages of entry there was evidence of localised formation of membrane projections in the host cell adjacent to the parasite. Parasite reorientation and rhoptry release appeared to be necessary for invasion; however, the tight junction could not be clearly discerned and there was no evidence of constriction or of any membrane shedding from the parasite. The resulting parasitophorous vacuole was smaller than the tachyzoite vacuole and parasites were frequently found to lie immediately under the host cell membrane. The vacuole was rapidly adapted by the release and formation of an intra-phagosomal membrane network, while the parasitophorous vacuole formed a relationship with host-cell endoplasmic reticulum. Received: 28 March 1998 / Accepted: 28 April 1998     

Labeled probes inserted in the macrophage membrane are transferred to the parasite surface and internalized during cell invasion by Toxoplasma gondii

Tachyzoites of Toxoplasma gondii attach to the macrophage surface and are internalized either by a phagocytic process, which can be inhibited by cytochalasin D, or by an active process, independent of host-cell actin. Previous studies have shown that parasite attachment induces the secretion of macromolecules found in the apical organelles (micronemes and rhoptries) and subsequent/concomitant parasite internalization with the formation of a membrane-bound vacuole known as the parasitophorous vacuole. In the present study we labeled the macrophage surface with fluorescent probes that bind to proteins (DiIC16) and lipids (DTAF) and then allowed control or cytochalasin-D-treated cells to interact with untreated or antibody-coated tachyzoites of T. gondii. The interaction was interrupted at different time points by fixation and the distribution of the probes was analyzed by confocal laser scanning microscopy. Following attachment of the parasites to the macrophage surface, intense labeling of the parasite surface was observed, suggesting transfer of components of the macrophage surface to the parasite surface. Nonadherent parasites were not labeled. Immediately after attachment, most of the parasites were internalized and labeling of the internalized parasites as well as of the parasitophorous vacuole, probably of its membrane, was evident, indicating that surface components of the macrophage are involved in the formation of the parasitophorous vacuole. Received: 30 April 1999 / Accepted: 21 July 1999     

Fine structure of sporogonic stages of Goussia clupearum (Apicomplexa: Eimeriidae) in the liver of infected fish (Belone belone L.), using light and electron microscopy

A coccidian species, Goussia clupearum (L.) is reported to parasitize the liver of a new host, Belone belone (Teleostei: Belonidae), caught on the Atlantic coast at the north of Portugal. The parasitophorous vacuole containing oocysts was attached to the host's liver cells. Spherical oocysts (∼ 21.2 μm diameter), each containing four ellipsoidal elongated sporocysts (10.5 × 6.3 μm), were enclosed in the parasitophorous vacuole. Each sporocyst contained two sporozoites. The micropyle was absent, but a polar granule (without Stieda body) was present. Each sporozoite possessed four refractile bodies. During sporoblastogenesis and sporogenesis, one or two dense polar bodies were found within the oocysts. They were composed of a dense homogeneous core, surrounded by a ring of dense granular material. On occasion, we observed some sporocysts in direct contact with host cells. This paper describes the morphology and ultrastructural details of the oocysts, sporocysts and sporozoites of G. clupearum. This species seems to represent the only coccidium described in fish from this Atlantic coast. Received: 5 August 2000 / Accepted: 12 October 2000     

Antigen presentation by Leishmania mexicana-infected macrophages: activation of helper T cells by a model parasite antigen secreted into the parasitophorous vacuole or expressed on the amastigote surface

Leishmania are protozoan parasites which invade mammalian macrophages and multiply as amastigotes in phagolysosomes (parasitophorous vacuoles). Using L. mexicana and bone marrow-derived macrophages (BMM), the question is addressed whether infected BMM induced to express major histocompatibility complex class II molecules can present defined antigens to specific T helper type 1 cells. As a model antigen, a membrane-bound acid phosphatase (MAP), a minor protein associated with intracellular vesicles in amastigotes, was either overexpressed at the surface of the parasites or overexpressed in a soluble form leading to antigen secretion into the parasitophorous vacuole. Presentation of MAP epitopes by these three types of amastigotes was then compared for macrophages containing live parasites or amastigotes inactivated by drug treatment. It is shown that surface-exposed and secreted MAP can be efficiently presented to T cells by macrophages harboring live amastigotes. Therefore, the parasitophorous vacuole communicates by vesicular membrane traffic with the plasmalemma of the host cell. The intracellular MAP of wild-type cells or the abundant lysosomal cysteine proteinases are not or only inefficiently presented, respectively. After killing of the parasites, abundant proteins such as overexpressed MAP and the cysteine proteinases efficiently stimulate T cells, while wild-type MAP levels are not effective. We conclude that intracellular proteins of intact amastigotes are not available for presentation, while after parasite inactivation, presentation depends on antigen abundance and possibly stability. The cell biological and possible immunological consequences of these results are discussed.     

Sporozoites of Eimeria acervulina within intestinal macrophages in normal experimental infections

Summary Sporozoites of Eimeria acervulina were observed in macrophages of the intestinal epithelium 5 and 6 days post-infection. These sporozoites lay within a well developed parasitophorous vacuole, were normal in structure and showed no signs of development. Macrophages harbouring sporozoites showed considerable structural changes, most pronounced being an absence of lysosomes, an enlarged nucleolus and extensive proliferation of the Golgi complex and endoplasmic reticulum. Possible mechanisms of survival and transport of sporozoites to preferred sites of development are discussed.Key to Lettering of Figures ER Endoplasmic reticulum - ERM Endoplasmic reticulum of macrophage - GOM Golgi zone of macrophage - INM Invaginations of macrophage cell membrane - LM Lipid globule of macrophage - LYM Lysosomes of macrophage - MI Mitochondria - MIM Mitochondria of macrophage - MN Micronemes - MP Micropore - N Nucleus - NM Nucleus of macrophage - PO Rhoptries (paired organelles) - PV Parasitophorous vacuole - RB Refractile body - V Vacuole This study was carried out in the Department of Poultry Research, Wye College (University of London), Wye, Kent, while the author was a Wellcome Trust Research Fellow.     

Diffusion of microinjected markers across the parasitophorous vacuole membrane in cells infected with Eimeria nieschulzi (Coccidia, Apicomplexa)

Cells infected with the intracellular parasite Eimeria nieschulzi were microinjected with lucifer yellow (457?Da), biocytin lucifer yellow (850?Da) and dextran-rhodamine (10,000?Da). Immediately after injection of a mixture of the markers into the host cell cytoplasm, a differential diffusion pattern was observed in trophozoites and schizonts. Lucifer yellow and biotin lucifer yellow were seen to enter the parasitophorous vacuole, whereas the dextran was excluded. Since these markers cannot permeate cell membranes, this suggests that the Eimerian parasitophorous vacuole acts as a molecular sieve. This method allows the visualization and further characterization of the parasitophorous vacuole in living cells.     

Ultrastructure of the development of a species ofEncephalitozoon cultured from the eye of an AIDS patient

Human fibroblast cell cultures inoculated with microsporidia-infected corneal scrapings from an AIDS patient were fixed in situ and examined by scanning and transmission electron microscopy. The parasite grew prolifically and all developmental stages were observed. Meronts underwent binary fission and the daughter cells transformed into clongate, chain-like sporonts that eventually separated into sporoblasts. The formation of components of the mature spores is described. The parasite, a species ofEncephalitozoon, underwent development both in the cytoplasm and within a parasitophorous vacuole, distinguishing it from the morphologically similar speciesE. cuniculi andE. hellem, both of which have been described from lesions in the human eye and have been reported to develop exclusively within a parasitophorous vacuole.This project was funded by Natural Sciences and Engineering Research Council of Canada Operating Grant 6965 (to S.S.D.)     

Effects of Molecular Liposomal Hybrid Compositions with Oxidized Dextrans and Isonicotinic Acid Hydrazide on Production of Granulocytic Macrophage Colony-Stimulating Factor by Macrophages

The effects of molecular liposomal hybrid compositions consisting of liposomes (200–450 nm) containing oxidized dextrans (dextranals; 35–60 kDa) conjugated with isonicotinic acid hydrazide (dextrazides), their components, and native dextrans on the production of granulocytic macrophage CSF by peritoneal macrophages were studied in vitro. Dextranals proved to be more potent inductors of granulocytic macrophage CSF than native dextrans. Conjugation of nicotinic acid hydrazide with dextranals did not modify their capacity to stimulate the production of granulocytic macrophage CSF. Liposomes in the molecular liposomal hybrid compositions did not attenuate the dextrazide capacity to stimulate the production of granulocytic macrophage CSF. Molecular liposomal compositions containing 60 kDa dextrazide exhibited the most potent stimulatory effect on macrophage production of granulocytic macrophage CSF.     

Fusion of Sendai virus with liposomes: dependence on the viral fusion protein (F) and the lipid composition of liposomes

The characteristics of fusion of the membrane of Sendai virus with that of liposomes has been investigated using two different methods to monitor the fusion reaction. The first method, which permits quantitation of lipid fused with virus, depends on separation by centrifugation of unfused liposomes from those fused with virus. The second involves the digestion after fusion of internal viral proteins by trypsin contained in liposomes; this assay is completely independent of exchange of lipid between liposomal and viral membranes in the absence of fusion. A fusion-inactive mutant virus, pa-cl, with an uncleaved F protein served as the appropriate control in these experiments. It was found that fusion of the virus with liposomes that contained no protein required cleavage of the F protein; such cleavage was previously shown to be required for fusion of the virus with cell membranes. This indicates the relevance of this model system for studies of fusion. Kinetic studies indicated that at neutral pH fusion was 88% complete in 10 min at 37 degrees. Investigation of the effects of liposomal lipid composition indicated that the presence of cholesterol in the liposomal membrane was required for fusion; a 0.3-0.4-mole fraction of cholesterol was optimal. The presence of neuraminic acid in the membrane was not essential for fusion. The results obtained are compatible with previous evidence suggesting a hydrophobic interaction between the cleaved F protein and the target membrane during fusion.     

Coexistence of heterogeneous populations ofToxoplasma gondii parasites within parasitophorous vacuoles of murine macrophages as revealed by a bradyzoite-specific monoclonal antibody

The expression of bradyzoite-specific antigens (Bsa) ofToxoplasma gondii was studied in murine bonemarrow-derived macrophages that had previously been infected with tachyzoites. Growth conditions that allowed only restricted replication ofToxoplasma gondii resulted in heterogeneous populations; (1) Bsa-positive and Bsa-negative parasites could be observed within one parasitophorous vacuole (heterogeneous vacuole community), and (2) homogeneous Bsa-positive and homogeneous Bsa-negative vacuole communities coexisted within one macrophage host cell. These observations suggest that stage conversion does not seem to be a synchromous event for a vacuole community.Parts of this paper were presented (by U.G.) at the first BIOMED Workshop onToxoplasma gondii Research in Europe, Würzburg, January 28, 1993; similar results were described by J.F. Dubremetz at the same workshop     

An ultrastructural study of the early development and tissue cyst formation of Toxoplasma gondii in the brains of mice

The ultrastructural features of the early development and tissue cyst formation of Toxoplasma gondii were examined in the brains of mice at various intervals from 7 days to 22 months post inoculation (PI). At 11 days PI toxoplasms, with the ultrastructural features of the proliferative (endozoite) form, were identified undergoing multiplication within both inflammatory and neural cells. Early tissue cyst formation was also observed, predominantly within neurons. By 21 days PI the proliferative forms had disappeared and only developing tissue cysts containing densely packed cystozoites were present. The proportion of dividing cystozoites decreased with increasing size and age of the cysts. The wall of the tissue cyst developed as an adaptation of the lining of the parasitophorous vacuole. In the majority of older cysts, numerous tubular structures were present beneath the cyst wall. All the cysts observed were retained within intact host cells. The only morphological change with increasing age was that a proportion of the older cysts contained loosely packed cystozoites in an electron lucent ground substance. There was no evidence of any degenerative changes within the cystozoites.Abbreviations C conoid - Cy cystozoite - D daughter anlage - En endozoite - HC host cell cytoplasm - HE host cell rough endoplasmic reticulum - HM host cell mitochondrion - HN host cell nucleus - M micronene - Me limiting membrane of parasitophorous vacuole/cyst wall - MP micropore - MT subpellicular microtubule - MV microvilli - N nucleus - NV neurosecretory vesicle - P polysaccharide granule - PL pellicle - PV parasitophorous vacuole - R rhoptry - Ri ribosome - S synapse - T tubular structure - K vesicle - W cyst wall     

Intracellular calcium and pH conditions of cultured cells infected with Eimeria bovis or E. separata

Loading of Eimeria bovis-infected Vero cells with membrane-permeant acetoxymethyl esters (AM-esters) of ion-sensitive dyes provided us with a noninvasive method for investigation of the permeability of the parasitophorous vacuole membrane (PVM) and simultaneous measurement of Ca²⁺ and H⁺ concentrations in different compartments of the infected cells. The distribution patterns of the cleaved membrane-impermeant dyes argue against the existence of nonselective pores in the PVM. There is also no indication of a parasitophorous duct connecting the vacuolar space with extracellular media. The pH inside the parasitophorous vacuole (PV) was lower than that in the cytoplasm of the host cell or the parasite, whereas the [Ca²⁺] in these compartments did not differ significantly. In HT29 cells infected with E. separata for 24 h the Ca²⁺ response to extracellular adenosine triphosphate (ATP) was significantly reduced, indicating influences on the host cell's intracellular signaling. Received: 15 October 1999 / Accepted: 8 November 1999     

The fine structure of <Emphasis Type="Italic">Garnia gonadati</Emphasis> and its association with the host cell

Most of the studies on the fine structure of protozoa of the Apicomplexa group have been carried out with members of the Toxoplasma, Eimeria, and Plasmodium genera. In the present study we analyzed the fine structure of Garnia gonadoti parasitizing the red blood cells of the Amazonian reptile Gonatodes humeralis (Reptilia: Lacertilia). Transmission electron microscopy of thin sections showed that G. gonadoti presented all structures characteristic of the group, including the apicoplast. However, four special features were observed: (1) absence of the hemozoin (malarial) pigment; (2) a group of microtubules associated with the mitochondrion; (3) a vacuole containing electron-dense material, which resembled the acidocalcisome described in trypanosomatids; and (4) a special array of the host-cell endoplasmic reticulum around the parasitophorous vacuole. Received: 4 January 2000 / Accepted: 30 May 2000