Parasitophorous Membrane Research Articles

Investigations on the Novel Antimalarial Ferroquine in Biomimetic Solutions Using Deep UV Resonance Raman Spectroscopy and Density Functional Theory

Deep ultraviolet (DUV) resonance Raman experiments are performed, investigating the novel, promising antimalarial ferroquine (FQ). Two buffered aqueous solutions with pH values of 5.13 and 7.00 are used, simulating the acidic and neutral conditions inside a parasite's digestive vacuole and cytosol, respectively. To imitate the different polarities of the membranes and interior, the buffer's 1,4-dioxane content was increased. These experimental conditions should mimic the transport of the drug inside malaria-infected erythrocytes through parasitophorous membranes. Supporting density functional theory (DFT) calculations on the drug's micro-speciation were performed, which could be nicely assigned to shifts in the peak positions of resonantly enhanced high-wavenumber Raman signals at λexc = 257 nm. FQ is fully protonated in polar mixtures like the host interior and the parasite's cytoplasm or digestive vacuole (DV) and is only present as a free base in nonpolar ones, such as the host's and parasitophorous membranes. Additionally, the limit of detection (LoD) of FQ at vacuolic pH values was determined using DUV excitation wavelengths at 244 and 257 nm. By applying the resonant laser line at λexc = 257 nm, a minimal FQ concentration of 3.1 μM was detected, whereas the pre-resonant excitation wavelength 244 nm provides an LoD of 6.9 μM. These values were all up to one order of magnitude lower than the concentration found for the food vacuole of a parasitized erythrocyte.

Massive Muscular Infection by aSarcocystisSpecies in a South American Rattlesnake (Crotalus durissus terrificus)

Massive numbers of sarcocysts of a previously undescribed species of Sarcocystis were observed in the skeletal muscles throughout the body of an adult, female South American rattlesnake (Crotalus durissus terrificus). Examination of tissue sections by light microscopy demonstrated that sarcocysts were present in 20 to 40% of muscle fibers from 5 sampled locations. Sarcocysts were not present in cardiac muscle, smooth muscle, or other organs. Sarcocysts were 0.05-0.15 mm wide, had variable length depending on the viewed orientation and size of the muscle fiber, and had a sarcocyst wall less than 1-μm thick. Sarcocysts were subdivided by septa and had central degeneration in older sarcocysts. Host induced secondary encapsulation or an inflammatory response was not present. By transmission electron microscopy (TEM), the sarcocyst wall was Type I, with a parasitophorous membrane of approximately 100 nanometers in width arranged in an undulating pattern and intermittently folded inward in a branching pattern. The sarcocysts contained metrocytes in different stages of development and mature bradyzoites. The nucleic acid sequence from a section of the 18S small subunit rRNA gene was most closely related to S. mucosa that uses marsupials as intermediate hosts and has an unknown definitive host. This is apparently the third report of muscular Sarcocystis infection in snakes and is the first to describe the ultrastructure of the sarcocysts and use sequencing methods to aid in identification.

The ETRAMP Family Member SEP2 Is Expressed throughout Plasmodium berghei Life Cycle and Is Released during Sporozoite Gliding Motility

The early transcribed membrane proteins ETRAMPs belong to a family of small, transmembrane molecules unique to Plasmodium parasite, which share a signal peptide followed by a short lysine-rich stretch, a transmembrane domain and a variable, highly charged C-terminal region. ETRAMPs are usually expressed in a stage-specific manner. In the blood stages they localize to the parasitophorous vacuole membrane and, in described cases, to vesicle-like structures exported to the host erythrocyte cytosol. Two family members of the rodent parasite Plasmodium berghei, uis3 and uis4, localize to secretory organelles of sporozoites and to the parasitophorous membrane vacuole of the liver stages. By the use of specific antibodies and the generation of transgenic lines, we showed that the P. berghei ETRAMP family member SEP2 is abundantly expressed in gametocytes as well as in mosquito and liver stages. In intracellular parasite stages, SEP2 is routed to the parasitophorous vacuole membrane while, in invasive ookinete and sporozoite stages, it localizes to the parasite surface. To date SEP2 is the only ETRAMP protein detected throughout the parasite life cycle. Furthermore, SEP2 is also released during gliding motility of salivary gland sporozoites. A limited number of proteins are known to be involved in this key function and the best characterized, the CSP and TRAP, are both promising transmission-blocking candidates. Our results suggest that ETRAMP members may be viewed as new potential candidates for malaria control.

New roles for perforins and proteases in apicomplexan egress

Egress is a pivotal step in the life cycle of intracellular pathogens initiating the transition from an expiring host cell to a fresh target cell. While much attention has been focused on understanding cell invasion by intracellular pathogens, recent work is providing a new appreciation of mechanisms and therapeutic potential of microbial egress. This review highlights recent insight into cell egress by apicomplexan parasites and emerging contributions of membranolytic and proteolytic secretory products, along with host proteases. New findings suggest that Toxoplasma gondii secretes a pore-forming protein, TgPLP1, during egress that facilitates parasite escape from the cell by perforating the parasitophorous membrane. Also, in a cascade of proteolytic events, Plasmodium falciparum late-stage schizonts activate and secrete a subtilisin, PfSUB1, which processes enigmatic putative proteases called serine-repeat antigens that contribute to merozoite egress. A new report also suggests that calcium-activated host proteases called calpains aid parasite exit, possibly by acting upon the host cytoskeleton. Together these discoveries reveal important new molecular players involved in the principal steps of egress by apicomplexans.

Preparation of pure and intact Plasmodium falciparum plasma membrane vesicles and partial characterisation of the plasma membrane ATPase

BackgroundIn host erythrocytes, the malaria parasite must contend with ion and drug transport across three membranes; its own plasma membrane, the parasitophorous membrane and the host plasma membrane. Isolation of pure and intact Plasmodium falciparum plasma membrane would provide a suitable model to elucidate the possible role played by the parasite plasma membrane in ion balance and drug transport.ResultsThis study describes a procedure for isolating parasite plasma membrane from P. falciparum-infected erythrocytes. With this method, the trophozoites released by saponin treatment were cleansed of erythrocyte membranes using anti-erythrocyte antibodies fixed to polystyrene beads. These trophozoites were then biotinylated and the parasite plasma membrane was disrupted by nitrogen cavitation. This process allows the membranes to reform into vesicles. The magnetic streptavidin beads bind specifically to the biotinylated parasite plasma membrane vesicles facilitating their recovery with a magnet. These vesicles can then be easily released from the magnetic beads by treatment with dithiotreithol. The parasite plasma membrane showed optimal ATPase activity at 2 mM ATP and 2 mM Mg2+. It was also found that Ca2+ could not substitute for Mg2+ ATPase activity in parasite plasma membranes whereas activity was completely preserved when Mn2+ was used instead of Mg2+. Other nucleoside triphosphates tested were hydrolysed as efficiently as ATP, while the nucleoside monophosphate AMP was not.ConclusionsWe have described the successful isolation of intact P. falciparum plasma membrane vesicles free of contaminating organelles and determined the experimental conditions for optimum ATPase activity.

Cytochemical localisation of calcium ATPase activity during the erythrocytic cell cycle of Plasmodium falciparum

Using a cytochemical technique, we evaluated the levels of Ca 2+-ATPase activity in the plasmatic and in the parasitophorous vacuole membrane through the different developmental stages of the Plasmodium falciparum parasitised erythrocyte. We found that the activity is detectable and remains unaltered in the plasma membrane throughout the 48 h cell cycle. However, in the parasitophorous membrane, although the activity was very similar to that measured in the plasma membrane of the young stages (younger than 20-h-old parasites), it diminished gradually with maturation and in schizonts it was almost undetectable. These data suggest that the plasma membrane Ca 2+-ATPase is important in the maintenance of a low erythrocyte cytoplasmic Ca 2+ concentration, and that in addition it could be a way to supply the vital cation to the parasite at the beginning of the infection, when other transport mechanisms have not yet developed.

Plasmodium invasion of mosquito cells: hawk or dove? Response from Mota and Rodríguez

Hepatocyte invasion by Plasmodium sporozoites is mediated by invagination of the host plasma membrane to form a parasitophorous vacuole which surrounds the invading sporozoite, and within which the parasite will replicate. Our recent observations 1xMigration of Plasmodium sporozoites through cells before infection. Mota, M.M. et al. Science. 2001; 291: 141–144Crossref | PubMed | Scopus (275)See all References1 show that Plasmodium sporozoites have an alternative mechanism for entering cells. Sporozoites migrate through several cells in the host before stopping to replicate inside a hepatocyte 1xMigration of Plasmodium sporozoites through cells before infection. Mota, M.M. et al. Science. 2001; 291: 141–144Crossref | PubMed | Scopus (275)See all

GASTRIC SPIRAL BACTERIA AND INTRAMUSCULAR SARCOCYSTS IN AFRICAN LIONS FROM NAMIBIA

Six juvenile male, one adult male, and three adult, female African lions (Panthera leo) from Etosha National Park, Republic of Namibia were presented for necropsy. Two of four adults and one of six juveniles had moderate numbers of gastric spiral bacteria. Additionally, four of four adults had sarcocysts. All juveniles had enteric Sarcocystis sp. oocysts, but no sarcocysts. The gastric spiral bacteria were located extracellularly in fundic and pyloric glands, and also apparently intracellularly within parietal cells in the fundic region. The organisms were 4 to 8 microns long, 0.63 micron wide, with a periodicity of 0.60 micron. The bacteria had blunted ends with multiple flagella. No periplasmic fibrils were observed. The histologic and ultrastructural characteristics of the bacteria were considered most consistent with species in the genus Helicobacter or incompletely identified Helicobacter-like organisms. Gastric inflammation did not differ significantly between infected and uninfected individuals. The bacteria may be commensals, or an opportunistic pathogen. The sarcocysts were observed in hindlimb skeletal muscle of four individuals, with one individual also containing a single sarcocyst within glossal musculature. All observed cysts were mature, and were contained within individual myocytes. The cyst wall consisted of a 44 to 66 nm, granular, electron dense parasitophorous membrane with subjacent, 0.8 to 1.3 microns thick, granular and fibrillar ground substance which also extended into the cyst interior as thin septa. The membrane was folded and lined irregularly spaced, 0.8 to 1.3 microns tall villi centrally containing ground substance. The membrane was continuous in the villar projections, but divided into discrete aggregations of the electron dense material between the villi. Bradyzoites within the interior of the cyst were 3 by 12 microns. The sarcocysts were determined to be Sarcocystis felis based on the characteristic ultrastructural appearance of the cyst wall.

Natural History and Biology of Cryptosporidium parvum

The taxonomy of the genus Cryptosporidium remains ambiguous, because the current criteria for speciation are insufficient to validate the 6-8 named species. Cross-transmission experiments have shown varying and conflicting results, and the limited genetic data available do not necessarily support currently proposed species designations. The reasons for this ambiguity lie with the ubiquitous nature of Cryptosporidium, probably infecting all vertebrates and variety of tissues therein, and the absence of reference strains with defined virulence attributes that can be linked to genetic markers for comparative analysis. The inability to classify oocysts or confidently to identify their origin, implicate oocysts from all sources as hazardous to humans. Another major issue is the unusual degree of resistance that Cryptosporidium has shown to antiprotozoan and antimicrobial agents. The intracellular but extracytoplasmic domain the parasite occupies is in itself a significant barrier to drug entry. In support of this we outline how the intracellular niche of this parasite differs from the related Apicomplexans, Plasmodium and Toxoplasma, and delineate why the feeder organelle membrane, rather than, or in addition to, the parasitophorous membrane, is the major portal of nutrient entry for Cryptosporidium. The broad conclusion is that anticryptosporidial agents will have to enter the parasite via the multiple apical membranes that camouflage the parasite, or via the host cell, possibly transported by vesicles to the feeder organelle membrane. This may have major implications for rational drug discovery and design.

Extracellular development of erythrocytic stages ofPlasmodium falciparum

Continuous culture of the erythrocytic cycle ofPlasmodium falciparum within human erythrocytes maintained under appropriate conditionsin vitro has facilitated a large body of fundamental investigations with this important pathogen. It has revealed complex relationships between the developing intracellular parasite and its host erythrocyte. In pursuit of further understanding of these relationships, we have now developed, and here describe, methods that support extracellular development of the erythrocytic stages ofP. falciparum. Since only 1 to 2% of an inoculum of free merozoites can develop extracellularly through the entire schizogonic cycle to again form merozoites, the method does not support continuous extracellular culture. The work shows that the parasitophorous membrane and vacuole are not essential. Development of the parasites beyond the ring stage does however require constituents of the erythrocyte membrane.

Transport pathways in the malaria-infected erythrocyte their characterization and their use as potential targets for chemotherapy

The intraerythrocytic malarial parasite is involved in an extremely intensive anabolic activity while it resides in its metabolically quiescent host cell. The necessary fast uptake of nutrients and the discharge of waste products are guaranteed by parasite-induced alterations of the constitutive transporters of the host cell and the production of new parallel pathways. The membrane of the host cell thus becomes permeable to phospholipids, purine bases and nucleosides, small non-electrolytes, anions and cations. While the new pathways are quantitatively unimportant for the translocation of a particular solute, classical inhibitors of native transporters can be used to inhibit parasite growth. Several compounds were found to inhibit effectively the new pathways and, consequently, parasite growth. The pathways have also been used to introduce cytotoxic agents. The parasitophorous membrane consists of channels that are highly permeable to small solutes and display no ion selectivity. Transport of some cations and anions across the parasite membrane is rapid and insensitive to classical inhibitors, and in some cases it is mediated by specific antiporters that respond to their respective inhibitors. Macromolecules have been shown to reach the parasitophorous space through a duct contiguous with the host cell membrane, and subsequently to be endocytosed at the parasite membrane. The simultaneous presence of the parasitophorous membrane channels and the duct, however, is incompatible with experimental evidence. No specific inhibitors have been found as yet that would efficiently inhibit transport through the channels or the duct.

Transport pathways in the malaria-infected erythrocyte: characterization and their use as potential targets for chemotherapy.

The intraerythrocytic malarial parasite is involved in an extremely intensive anabolic activity while it resides in its metabolically quiescent host cell. The necessary fast uptake of nutrients and the discharge of waste product, are guaranteed by parasite-induced alterations of the constitutive transporters of the host cell and the production of new parallel pathways. The membrane of the host cell thus becomes permeable to phospholipids, purine bases and nucleosides, small non-electrolytes, anions and cations. When the new pathways are quantitatively unimportant, classical inhibitors of native transporters can be used to inhibit parasite growth. Several compounds were found to effectively inhibit the new pathways and consequently, parasite growth. The pathways have also been used to introduce cytotoxic agents. The parasitophorous membrane consists of channels which are highly permeable to small solutes and display no ion selectivity. Transport of some cations and anions across the parasite membrane is rapid and insensitive to classical inhibitors, and in some cases it is mediated by specific antiporters which respond to their respective inhibitors. Macromolecules have been shown to reach the parasitophorous space through a duct contiguous with the host cell membrane, and subsequently to be endocytosed at the parasite membrane. The simultaneous presence of the parasitophorous membrane channels and the duct, however, is incompatible with experimental evidences. No specific inhibitors were found as yet that would efficiently inhibit transport through the channels or the duct.

Extracellular (axenic) development in vitro of the erythrocytic cycle of Plasmodium falciparum.

Merozoites of the erythrocytic stage of Plasmodium falciparum were suspended in erythrocyte sonicate medium with ATP and pyruvate and mixed with Matrigel to form a soft gel. The gel was overlaid with complete medium; this was replaced with fresh medium at 12, 24, and 36 hr. At these times and also at 45 hr rhodamine 123 was added to some cultures and gels were sampled. Viable extracellular forms showing rhodamine fluorescence were seen: rings at 12 hr, trophozoites and early schizonts with pigment at 36 hr, and late schizonts with developing merozoites at 45 hr. These merozoites were shown to be infective to erythrocytes added to the cultures at 45 hr. Electron micrographs of 36-hr trophozoites show the organisms to have only their single plasma membrane; no parasitophorous membrane is evident. We conclude that the complex process of entry, the intactness of the host erythrocyte, and the parasitophorous membrane are not essential to the development of a merozoite through its complete asexual cycle.

Lipid traffic between high density lipoproteins and Plasmodium falciparum-infected red blood cells.

Several intraerythrocytic growth cycles of Plasmodium falciparum could be achieved in vitro using a serum free medium supplemented only with a human high density lipoprotein (HDL) fraction (d = 1.063-1.210). The parasitemia obtained was similar to that in standard culture medium containing human serum. The parasite development was incomplete with the low density lipoprotein (LDL) fraction and did not occur with the VLDL fraction. The lipid traffic from HDL to the infected erythrocytes was demonstrated by pulse labeling experiments using HDL loaded with either fluorescent NBD-phosphatidylcholine (NBD-PC) or radioactive [3H]palmitoyl-PC. At 37 degrees C, the lipid probes rapidly accumulated in the infected cells. After incubation in HDL medium containing labeled PC, a subsequent incubation in medium with either an excess of native HDL or 20% human serum induced the disappearance of the label from the erythrocyte plasma membrane but not from the intraerythrocytic parasite. Internalization of lipids did not occur at 4 degrees C. The mechanism involved a unidirectional flux of lipids but no endocytosis. The absence of labeling of P. falciparum, with HDL previously [125I]iodinated on their apolipoproteins or with antibodies against the apolipoproteins AI and AII by immunofluorescence and immunoblotting, confirmed that no endocytosis of the HDL was involved. A possible pathway of lipid transport could be a membrane flux since fluorescence videomicroscopy showed numerous organelles labeled with NBD-PC moving between the erythrocyte and the parasitophorous membranes. TLC analysis showed that a partial conversion of the PC to phosphatidylethanolamine was observed in P. falciparum-infected red cells after pulse with [3H]palmitoyl-PC-HDL. The intensity of the lipid traffic was stage dependent with a maximum at the trophozoite and young schizont stages (38th h of the erythrocyte life cycle). We conclude that the HDL fraction appears to be a major lipid source for Plasmodium growth.

Initial extracellular development in vitro of erythrocytic stages of malaria parasites (Plasmodium falciparum).

Merozoites of Plasmodium falciparum placed in culture medium with a 50% erythrocyte extract and supplemented with ATP and pyruvate differentiated extracellularly into early trophic forms. Erythrocyte extract prepared by sonication was found superior to extract prepared by freezing and thawing. Under the best conditions, up to 30% of the merozoites showed some development after 16 hr of incubation and approximately 5% developed into larger forms, often ring-shaped and occasionally showing pigment. The small as well as the larger forms took up the fluorescent dye rhodamine 123. Under similar conditions, partial further development was also obtained of young rings freed from their host cells. Again, the sonicated erythrocyte extract gave better development than the frozen-thawed extract, and ATP with pyruvate had a marked favorable effect. These parasites had both a plasma membrane and a surrounding closely apposed parasitophorous membrane, whereas the forms derived by extracellular development of merozoites had only their plasma membrane. We conclude that initial development requires neither an intact erythrocyte nor a parasitophorous membrane.

Movement of a falciparum malaria protein through the erythrocyte cytoplasm to the erythrocyte membrane is associated with lysis of the erythrocyte and release of gametes.

Erythrocytes containing mature gametocytes of Plasmodium falciparum circulate in the blood until they are ingested by a mosquito, an event that triggers gametogenesis and lysis of the infected erythrocyte. It was previously shown that a parasite protein (Pf155/RESA) accumulates in the erythrocyte cytoplasm next to the parasitophorous vacuolar membrane (S. Uni, A. Masuda, M. J. Stewart, R. Nussenzweig, and M. Aikawa, Am. J. Trop. Med. Hyg., 36:481-488, 1987). Using a monoclonal antibody to Pf155/RESA and rabbit sera to two different repeat peptides of Pf155/RESA, we have studied the location of Pf155/RESA after induction of gametogenesis. Five minutes after triggering gametogenesis, the parasitophorous membrane no longer surrounded the parasite, bringing the parasite membrane in contact with the erythrocyte cytoplasm. Clear spaces appeared throughout the hemoglobin-rich host cytoplasm; Pf155/RESA was now localized in the cytoplasm directly surrounding the spaces. No membrane existed between the spaces and the erythrocyte cytoplasm. The spaces with surrounding Pf155/RESA protein extended to the erythrocyte membrane. After lysis of the erythrocyte membrane (15 min after triggering gametogenesis), the protein was distributed along the erythrocyte membrane and throughout the space between the gamete and the erythrocyte membrane. The mechanism by which Pf155/RESA remained aggregated around the spaces and its role in erythrocyte lysis are unknown. It is of interest that the parasite appeared to use the same molecule during invasion of erythrocytes and during release of gametes from infected erythrocytes.

Plasmodium simium: Ultrastructure of erythrocytic phase

An electron microscopic study of Plasmodium simium infections in the squirrel monkey has supplied information on the ultrastructure of erythrocytic trophozoites, schizonts, merozoites, and gametocytes, in addition to an unusual form of host cell pathology. In general, the structural features, as well as certain specialized functions, e.g., hemoglobin ingestion and utilization, nuclear and cytoplasmic division, were found to be similar to those described for other malarial parasites. Some striking features were noted, however. A highly asynchronous mode of merozoite production was observed within single segmenting parasites in spite of the overall developmental synchrony displayed by the population as a whole. Secondly, during parasite segmentation, newly formed merozoites are connected to one another, as well as to the parasitophorous membrane, by periodic surface strands. It is speculated that these interparasite bridges serve as structural support to the segmenting parasite. When merozoites are matured fully, these interconnections break, leaving a uniform array of short surface bristles. In addition, a number of different pathological changes in host cell structure have been noted. Localized surface discontinuities appear in region of infected cells where apical regions of developing or fully mature merozoites are abutted against the plasma membrane. These profiles suggest that these specialized apical regions of the merozoite function in release as well as in host cell penetration. More generalized surface pathology occurs within parasitized erythrocytes in the form of surface blebs, surface clefts, and associated cytoplasmic microvesicles. The severity of this pathology increases as the intraerythrocytic parasite matures. Topographically these altered cells have a “berry-like” surface texture which makes them quite distinctive when viewed by scanning electron microscopy.