Cellular pathology in mouse embryonic brain cells following in vitro penetration by sporozoites ofEimeria papillata

The pathology that occurs in mouse embryonic brain (MEB) cells that have been penetrated by sporozoites ofEimeria papillata was studied by light and electron microscopy. At the light microscopy level the greatest number of intracellular parasites was seen at 15 and 45 min postinoculation (PI). The monolayer of MEB cells had begun to round up by 45 min PI, and by 60 min PI most of the cells were stripped from the coverslip. Little ultrastructural damage was seen in MEB cells just penetrated by the parasites at 15 min PI, and no host cell membrane was seen around the sporozoites that had just entered the cells. Flexing and bending of the sporozoites within the MEB cell caused vacuolization of cell cytoplasm and in some cases rupture of host cell membrane. Sporozoites leaving the host cells at 15 min PI caused a rupture of the host cell membrane at the apical end of the parasite, and both host cell membrane and cytoplasm were attached to the surface of the parasite. MEB cells still attached to coverslips at 45 min PI demonstrated complete degeneration.     

Toxoplasma gondii sporozoites form a transient parasitophorous vacuole that is impermeable and contains only a subset of dense-granule proteins.

Toxoplasma gondii sporozoites form two parasitophorous vacuoles during development within host cells, the first (PV1) during host cell invasion and the second (PV2) 18 to 24 h postinoculation. PV1 is structurally distinctive due to its large size, yet it lacks a tubulovesicular network (C. A. Speer, M. Tilley, M. Temple, J. A. Blixt, J. P. Dubey, and M. W. White, Mol. Biochem. Parasitol. 75:75-86, 1995). Confirming the finding that sporozoites have a different electron-dense-granule composition, we have now found that sporozoites within oocysts lack the mRNAs encoding the 5' nucleoside triphosphate hydrolases (NTPase). NTPase first appears 12 h postinfection. Other tachyzoite dense-granule proteins, GRA1, GRA2, GRA4, GRA5, and GRA6, were detected in oocyst extracts, and antibodies against these proteins stained granules in the sporozoite cytoplasm. In contrast to tachyzoite invasion of host cells, however, sporozoites did not exocytose the dense-granule proteins GRA1, GRA2, or GRA4 during PV1 formation. Even after NTPase induction, these proteins were retained within cytoplasmic granules rather than being secreted into PV1. Only GRA5 was secreted by the sporozoite during host cell invasion, becoming associated with the membrane surrounding PV1. Microinjection of sporozoite-infected cells with fluorescent dyes showed that PV1 is impermeable to fluorescent dyes with molecular masses as small as 330 Da, indicating that PV1 lacks channels through which molecules can pass from the host cytoplasm into the vacuole. By contrast, lucifer yellow rapidly diffused into PV2, demonstrating the presence of molecular channels. These studies indicate that PV1 and PV2 are morphologically, immunologically, and functionally distinct, and that PV2 appears to be identical to the tachyzoite vacuole. The inaccessibility of PV1 to host cell nutrients may explain why parasite replication does not occur in this vacuole.     

Binding of a Monoclonal Antibody to Sporozoites of Sarcocystis singaporensis Enhances Escape from the Parasitophorous Vacuole, Which Is Necessary for Intracellular Development

Early intracellular development in vitro of the cyst-forming protozoon Sarcocystis singaporensis and the influence of a monoclonal antibody on invasion, intracellular localization, and development of sporozoites were studied. As revealed by immunofluorescence using parasite-specific antibodies which labeled the parasitophorous vacuole membrane (PVM) and by ultrastructural analysis, sporozoites invaded pneumonocytes of the rat via formation of a parasitophorous vacuole (PV). About half of the sporozoites left this compartment within the first 8 h postinfection to enter the host cell cytosol. By semiquantitative analysis of acetyl-histone H4 expression of sporozoites, a marker linked to early gene expression of eukaryotic cells, we show (supported by ultrastructural analysis) that escape from the PV appears to be necessary for early intracellular development. More than 90% of sporozoites located in the cytosol expressed high levels of acetylated histone H4 in the nucleus, whereas only a quarter of the intravacuolar sporozoites exhibited a similar signal. As revealed by ultrastructural analysis, young schizonts all resided in the cytosol. Specific binding of a monoclonal antibody (11D5/H3) to sporozoites before invasion significantly enhanced their escape from the PV, whereas cell invasion itself remained unaffected. The antibody actually increased proliferation of the parasites in vitro, providing a further link between residence in the cytosol and successful intracellular development. Monoclonal antibody 11D5/H3 precipitated a major 58-kDa antigen from oocyst-sporocyst extracts and reacted with the cytoplasm and the surface of sporozoites in immunofluorescence assays. Collectively, the observed antibody-parasite interaction suggests the existence of a signaling event that influences intracellular development of Sarcocystis.     

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.     

Early hepatic stages of Plasmodium berghei: release of circumsporozoite protein and host cellular inflammatory response.

After injection of Plasmodium berghei sporozoites into Norway-Brown rats, we were able to localize these sporozoites and the early hepatic trophozoites developing from them in histological sections of the liver stained with a sensitive immunogold-silver procedure. Sporozoites invading hepatocytes released substantial quantities of circumsporozoite protein into the hepatocyte cytoplasm. This intrahepatic cytoplasmic distribution reached a maximal level at about 4 h post-sporozoite injection. As the hepatic parasites continued to differentiate, circumsporozoite protein became undetectable within the cytoplasm of the hepatocytes and became localized around the periphery of each parasite. There was generalized cellular inflammation within the liver of the host, which first became evident at around 4 h post-sporozoite injection and progressed to the formation of well-defined granulomas by 24 h. Such histopathological changes were not seen in rats injected with killed sporozoites, indicating that the cellular inflammation was induced by viable, infective sporozoites. We did not observe cellular infiltration specifically associated with any of the developing hepatic stages that we observed, even up to 28 h post-sporozoite inoculation. These results indicate that viable sporozoites induced rapid and generalized hepatic inflammation in host rats. However, sporozoites that successfully invaded hepatocytes and then proceeded to develop further did not appear to be the target of inflammatory cells until a period beginning at around 40 h post-sporozoite inoculation.     

Fluorescent <Emphasis Type="Italic">Eimeria bovis</Emphasis> sporozoites and meront stages in vitro: a helpful tool to study parasite–host cell interactions

A fluorescence-based technique was established to trace intracellular sporozoites of Eimeria bovis for tests on gliding motility, invasion, replication and quantification of infection rates in cultured bovine umbilical vein endothelial cells (BUVEC) by laser scanning confocal microscopy and flow cytometry (FCM) analyses. Employing the fluorescent dye 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), we determined its effects on sporozoites at various concentrations and duration of staining. More than 98% of sporozoites were labelled with the dye at a concentration of 2.5 μM. Staining was predominantly found in refractile bodies and presumptive micronemes. Upon infection of BUVEC, CFSE-labelled sporozoites developed into fluorescent immature macromeronts, which were traceable inside the cells until 22 days postinfection (p. i.). Consistent with a peripheral localisation of the fluorescence signal in macromeronts merozoites released from these lacked detectable fluorescence. As example of use, a multicolour FCM approach for the simultaneous determination of E. bovis infection and host cell surface molecule expression was established. The approach proved suitable to quantify major histocompatibility complex (MHC-I) and MHC-II expression, thereby clearly distinguishing between infected and uninfected BUVEC up to day 14 p. i. In conclusion, CFSE labelling of E. bovis sporozoites facilitates monitoring of intracellular stages in vitro and will be a highly useful tool for studying host cell responses towards parasite invasion.     

Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells.

Exoerythrocytic (EE) stages of Plasmodium berghei derived from irradiated sporozoites were cultured in vitro in HepG2 cells. They synthesized several antigens, predominantly but not exclusively those expressed by normal early erythrocytic schizonts. After invasion, over half the intracellular sporozoites, both normal and irradiated, appeared to die. After 24 h, in marked contrast to the normal parasites, EE parasites derived from irradiated sporozoites continued to break open, shedding their antigens into the cytoplasm of the infected host cells. Increasing radiation dosage, which has previously been shown to reduce the ability of irradiated sporozoites to protect animals, correlated with reduced de novo antigen synthesis by EE parasites derived from irradiated sporozoites.     

Gliding motility leads to active cellular invasion by Cryptosporidium parvum sporozoites

We examined gliding motility and cell invasion by an early-branching apicomplexan, Cryptosporidium parvum, which causes diarrheal disease in humans and animals. Real-time video microscopy demonstrated that C. parvum sporozoites undergo circular and helical gliding, two of the three stereotypical movements exhibited by Toxoplasma gondii tachyzoites. C. parvum sporozoites moved more rapidly than T. gondii sporozoites, which showed the same rates of motility as tachyzoites. Motility by C. parvum sporozoites was prevented by latrunculin B and cytochalasin D, drugs that depolymerize the parasite actin cytoskeleton, and by the myosin inhibitor 2,3-butanedione monoxime. Imaging of the initial events in cell entry by Cryptosporidium revealed that invasion occurs rapidly; however, the parasite does not enter deep into the cytosol but rather remains at the cell surface in a membrane-bound compartment. Invasion did not stimulate rearrangement of the host cell cytoskeleton and was inhibited by cytochalasin D, even in host cells that were resistant to the drug. Our studies demonstrate that C. parvum relies on a conserved actin-myosin motor for motility and active penetration of its host cell, thus establishing that this is a widely conserved feature of the Apicomplexa.     

Induction of secretion and surface capping of microneme proteins in Eimeria tenella

Micronemes are secretory organelles of the invasive stages of apicomplexan parasites and contain proteins that are important for parasite motility and host cell invasion. We have examined the induction of microneme secretion in the coccidian Eimeria tenella. When sporozoites were added to MDBK cells in culture, microneme proteins were secreted, capped backwards over the parasite surface and deposited onto underlying host cells from the posterior end of gliding parasites. Induction of secretion was also achieved by the addition of foetal calf serum, or purified albumin, to extracellular sporozoites. Microneme secretion per se was not dependent on parasites being able to move or to invade host cells. However, in the presence of cytochalasin D, which disrupts actin polymerisation and prevents parasite movement, microneme proteins were secreted from the apical tip but were not capped backwards over the sporozoite surface. These observations support the hypothesis that microneme proteins function as ligands which, when secreted out onto the parasite surface, form a link, either directly or indirectly, between the sub-pellicular actin–myosin cytoskeletal motor of the parasite and the surface of target host cells.     

OspE2 of Shigella sonnei is required for the maintenance of cell architecture of bacterium-infected cells

The OspE2 product of Shigella spp., the expression of which is regulated by the mxiE gene, is secreted through a type III secretion system into host cells. We investigated the function of OspE2 of Shigella sonnei by using cultured epithelial cells. Cells invaded by an ospE2 deletion mutant altered their morphology into the rounding shape, which was not due to cell death, whereas cells invaded by the wild-type strain kept their cell shape intact. The ospE2 mutation did not affect initial cell entry and multiplication in cells, but the mutant formed smaller-than-normal plaques on cell monolayers, indicating a deficiency in cell-to-cell spread by the bacteria. An mxiE deletion mutant also showed changes in cell morphology and deficiency in bacterial spread to adjacent cells. In cells invaded by the ospE2 mutant, disturbance of actin stress fibers was prominent at 3 h after invasion. Analysis of OspE2 localization indicated that the OspE2 protein accumulated on focal contact-like structures in the infected host cells. These results suggest that colocalization of the OspE2 protein in the focal contacts of infected cells may function to maintain an intact cell morphology. The morphological change induced by invasion of the ospE2 mutant may affect secondary bacterial transmission.     

Actin cytoskeleton-dependent and -independent host cell invasion by Trypanosoma cruzi is mediated by distinct parasite surface molecules

The disassembly of host cell actin cytoskeleton as a facilitator of Trypanosoma cruzi invasion has been reported by some authors, while other workers claim that it instead inhibits internalization of the parasite. In this study we aimed at elucidating the basis of this discrepancy. We performed experiments with metacyclic trypomastigotes of T. cruzi strains G and CL, which differ markedly in infectivity and enter target cells by engaging the surface molecules gp35/50 and gp82, respectively, which have signaling activity. Treatment of HeLa cells with the F-actin-disrupting drug cytochalasin D or latrunculin B inhibited the invasion by strain G but not the invasion by strain CL. In contrast to cells penetrated by strain CL, which were previously shown to have a disrupted actin cytoskeleton architecture, no such alteration was observed in HeLa cells invaded by strain G, and parasites were found to be closely associated with target cell actin. Coinfection with enteroinvasive Escherichia coli (EIEC), which recruits host cell actin for internalization, drastically reduced entry of strain CL into HeLa cells but not entry of strain G. In contrast to gp82 in its recombinant form, which induces disruption of F-actin and inhibits EIEC invasion, purified mucin-like gp35/50 molecules promoted an increase in EIEC uptake by HeLa cells. These data, plus the finding that drugs that interfere with mammalian cell signaling differentially affect the internalization of metacyclic forms of strains G and CL, indicate that the host cell invasion mediated by gp35/50 is associated with signaling events that favor actin recruitment, in contrast to gp82-dependent invasion, which triggers the signaling pathways leading to disassembly of F-actin.     

Electron microscopic studies on the interaction of rat Kupffer cells andPlasmodium berghei sporozoites

The interactions betweenPlasmodium berghei sporozoites and Kupffer cells in rat liver were studied by transmission electron microscopy. Between 10 and 45 min after inoculation, sporozoites were found in the process of entering Kupffer cells and inside phagolysosomes. The sporozoites entered the Kupffer cells by phagocytosis as determined by the presence of pseudopods and local accumulations of aggregated microfilaments and the resulting exclusion of other organelles in the phagocyte cytoplasm beneath the attached parasite. Sporozoites were taken up either with their anterior end first, or backwards. Scanning electron microscopy of in vitro sporozoite Kupffer cell interaction confirmed these observations. It was concluded that sporozoites are taken up in a normal phagocytic way by the Kupffer cells, regardless of their initial place of contact or position. Thirty min after inoculation sporozoites found in phagolysosomes were still morphologically intact but after 45 min we could encounter completely digested sporozoites.     

Extracellular calcium deficiency and ryanodine inhibit Eimeria tenella sporozoite invasion in vitro

An in vitro assay system with Eimeria tenella sporozoites was used to study the effects of extracellular calcium and active agents affecting the invasion of parasites into host cells. At concentrations of 900M Ca²⁺ and less the invasion rates were distinctly decreased. Ryanodine, a herbal alkaloid known for binding to internal Ca²⁺ channels (ryanodine receptors), showed an inhibitory effect on E. tenella sporozoite invasion. Preincubation tests and staining with a fluorescent derivative of ryanodine assured that the compound bound specifically to the sporozoites and affected them rather than the host cells.All experiments described in this publication comply with the current laws of Germany     

An electron and immunoelectron microscopic study of dengue-2 virus infection of cultured mosquito cells: Maturation events

Summary The maturation process of dengue-2 virus in C6/36 mosquito cells was studied by electron microscopy at 12, 16, 24, 48, and 78 hours postinoculation (p.i.) and by immunoelectron microscopy at 48 and 78 hours p.i. Maturing virions appeared within cytoplasmic vacuoles and on the surface of infected cells from 24 hours p.i. onward in close topographical relationship to the dense particles that occurred concurrently in the cytoplasm. The dense particles measured 25 to 35 nm in diameter; the mature virions measured 50 to 55 nm in diameter, with a dense core measuring 30 to 35 nm in diameter covered by a 10 nm-thick membrane envelope. The morphological observations indicated that the dense particles were dengue nucleocapsids assembled in the cytoplasm and that they apparently budded into the vacuolar lumens and the extracellular space at the vacuolar and plasma membranes, acquiring membrane envelopes and becoming mature virions in the process. The virions that budded into the vacuolar lumens were released extracellularly by exocytosis. In the samples tested with dengue-2 polyclonal antibodies, intense immunostaining occurred at the sites of virus budding on the cell surface; host cell membrane and cytoplasm adjacent to the budding virions stained less intensely. In the samples tested with a dengue-2 monoclonal antibody specific for the envelope glycoprotein, budding virions stained rather exclusively, with no staining occurring in adjacent host membrane or cytoplasm.With 10 Figures     

A merozoite-specific 22-kDa rhoptry protein of the coccidium Eimeria nieschulzi (Sporozoa,Coccidia) is exocytosed in the parasitophorous vacuole upon host cell invasion

A monoclonal antibody (Mab D12A4) was used to follow the genesis and fate of rhoptries from early first-generation merogony through second-generation merozoites of the rat coccidium Eimeria nieschulzi. The epitope recognized by Mab D12A4 belongs to a 22-kDa protein which can be localized first in developing meronts 25 h post-infection. Early rhoptries appear as distinct granules in the cytoplasm of developing meronts and elongate into mature organelles before merozoite release. The 22-kDa protein is found in the parasitophorous vacuole after host cell invasion. Western blotting and immunofluorescence showed that the 22-kDa rhoptry protein is expressed in schizonts and merozoites but not in sporozoites. Received: 9 August 1997 / Accepted: 14 October 1997     

Immunity to Plasmodium berghei exoerythrocytic forms derived from irradiated sporozoites

 The nature of immunity generated by Plasmodium berghei exoerythrocytic (EE) stages developing from irradiated sporozoites was studied using in vivo parameters of host protection on immunization with irradiated sporozoites and in vitro parameters of inhibition of sporozoite invasion and EE form development by serum antibodies from immunized mice. On in vivo challenge of immunized mice by sporozoites, protection was observed in an irradiation-dose-dependent manner. This finding stresses that protection is dependent on the irradiation dose of sporozoites that allows sporozoite penetration yet controls EE form development within the liver. Using the human hepatoma line Hep G2 as host cells in vitro, we observed that serum antibodies raised in mice immunized with irradiated sporozoites reacted with sporozoite- and hepatic-stage parasites in an immunofluorescent antibody test (IFAT). No reactivity was observed with blood-stage parasites. Serum antibodies from mice immunized with 6- to 18-krad-irradiated sporozoites inhibited sporozoite invasion and caused severe inhibition of EE form development in hepatoma cells, pointing to the antigenic content of EE forms developing from irradiated sporozoites (irra EE forms) as critical immunogens. Moreover, in an enzyme-linked immunosorbent assay (ELISA), serum antibodies raised to 12-krad-irradiated sporozoites showed reactivity to synthetic peptides representing the conserved Region II sequences of the P. falciparum circumsporozoite (CS) protein as well as the P. falciparum liver-stage-specific antigen (LSA-1)-based repeat sequences, thus implicating an important role for both the sporozoite and the hepatic stage in protection. Received: 21 June 1995 / Accepted: 27 Oktober 1995     

Apical organelle discharge by Cryptosporidium parvum is temperature, cytoskeleton, and intracellular calcium dependent and required for host cell invasion

The apical organelles in apicomplexan parasites are characteristic secretory vesicles containing complex mixtures of molecules. While apical organelle discharge has been demonstrated to be involved in the cellular invasion of some apicomplexan parasites, including Toxoplasma gondii and Plasmodium spp., the mechanisms of apical organelle discharge by Cryptosporidium parvum sporozoites and its role in host cell invasion are unclear. Here we show that the discharge of C. parvum apical organelles occurs in a temperature-dependent fashion. The inhibition of parasite actin and tubulin polymerization by cytochalasin D and colchicines, respectively, inhibited parasite apical organelle discharge. Chelation of the parasite's intracellular calcium also inhibited apical organelle discharge, and this process was partially reversed by raising the intracellular calcium concentration by use of the ionophore A23187. The inhibition of parasite cytoskeleton polymerization by cytochalasin D and colchicine and the depletion of intracellular calcium also decreased the gliding motility of C. parvum sporozoites. Importantly, the inhibition of apical organelle discharge by C. parvum sporozoites blocked parasite invasion of, but not attachment to, host cells (i.e., cultured human cholangiocytes). Moreover, the translocation of a parasite protein, CP2, to the host cell membrane at the region of the host cell-parasite interface was detected; an antibody to CP2 decreased the C. parvum invasion of cholangiocytes. These data demonstrate that the discharge of C. parvum sporozoite apical organelle contents occurs and that it is temperature, intracellular calcium, and cytoskeleton dependent and required for host cell invasion, confirming that apical organelles play a central role in C. parvum entry into host cells.     

Cell immortalization enhances Listeria monocytogenes invasion

Recent outbreaks of human listeriosis have emphasized the importance of food in the etiology of epidemic listeriosis, suggesting that the gastrointestinal tract is the natural site of entry for Listeria monocytogenes into the organism. L. monocytogenes invasion of finite cell lines derived from the porcine ileum exhibited a 100-fold lower penetration level, without any intracellular multiplication, when compared to CaCo-2 cells, a widely used in vitro model for L. monocytogenes invasion. Same results were obtained with both pig kidney primary cells and mouse kidney finite cell lines. To demonstrate that cell immortalization enhances L. monocytogenes invasion, finite cell lines from porcine ileum and from murine kidney were immortalized by Simian virus 40 (SV40) large T oncogene. Unlike their untransformed counterparts, the immortal cells obtained were invaded by L. monocytogenes, as observed for CaCo-2 cells as well as for spontaneously immortal human (HeLa) and murine (3T3) cell lines. Extensive electron microscopy examinations of porcine epithelioid cells infected by L. monocytogenes showed numerous bacteria within the immortal cells, whereas neither intracellular bacteria nor any bacterial antigen were revealed inside finite cell lines. These data suggested that L. monocytogenes were not destroyed inside finite cell lines but only poorly entered the finite or primary cells. Speculating that L. monocytogenes invasion is under control of differentiation or proliferation of the cells, only an enterocyte subset at a defined state of differentiation or expressing particular receptors could be invaded in vivo.