Ultrastructure of early stages of Rozella allomycis (Cryptomycota) infection of its host, Allomyces macrogynus (Blastocladiomycota)

This study reconstructs early stages of Rozella allomycis endoparasitic infection of its host, Allomyces macrogynus. Young thalli of A. macrogynus were inoculated with suspensions of R. allomycis zoospores and allowed to develop for 120 h. Infected thalli at intervals were fixed for electron microscopy and observed. Zoospores were attracted to host thalli, encysted on their surfaces, and penetrated their walls with an infection tube. The parasite cyst discharged its protoplast through an infection tube, which invaginated the host plasma membrane. The host plasma membrane then surrounded the parasite protoplast and formed a compartment confining it inside host cytoplasm. The earliest host-parasite interface within host cytoplasm consisted of two membranes, the outer layer the host plasma membrane and the inner layer the parasite plasma membrane. At first a wide space separated the two membranes and no material was observed within this space. Later, as the endoparasite thallus expanded within the compartment, the two membranes became closely appressed. As the endoparasite thallus continued to enlarge, the interface developed into three membrane layers. Thus, host plasma membrane surrounded the parasite protoplast initially without the parasite having to pierce the host plasma membrane for entry. Significantly, host-derived membrane was at the interface throughout development.     

Plasmodium falciparum SURFIN4.1 forms an intermediate complex with PTEX components and Pf113 during export to the red blood cell

Plasmodium falciparum malaria parasites export several hundred proteins to the cytoplasm of infected red blood cells (RBCs) to modify the cell environment suitable for their growth. A Plasmodium translocon of exported proteins (PTEX) is necessary for both soluble and integral membrane proteins to cross the parasitophorous vacuole (PV) membrane surrounding the parasite inside the RBC. However, the molecular composition of the translocation complex for integral membrane proteins is not fully characterized, especially at the parasite plasma membrane. To examine the translocation complex, here we used mini-SURFIN_4.1, consisting of a short N-terminal region, a transmembrane region, and a cytoplasmic region of an exported integral membrane protein SURFIN_4.1. We found that mini-SURFIN_4.1 forms a translocation intermediate complex with core PTEX components, EXP2, HSP101, and PTEX150. We also found that several proteins are exposed to the PV space, including Pf113, an uncharacterized PTEX-associated protein. We determined that Pf113 localizes in dense granules at the merozoite stage and on the parasite periphery after RBC invasion. Using an inducible translocon-clogged mini-SURFIN_4.1, we found that a stable translocation intermediate complex forms at the parasite plasma membrane and contains EXP2 and a processed form of Pf113. These results suggest a potential role of Pf113 for the translocation step of mini-SURFIN_4.1, providing further insights into the translocation mechanisms for parasite integral membrane proteins.     

Formation of a close junction during invasion of erythrocytes by Toxoplasma gondii in vitro

Michel R., Schupp K., Raether W. and Bierther F.W. 1980. Formation of a close junction during invasion of erythrocytes by Toxoplasma gondii in vitro. International Journal for Parasitology10: 309–313. During the investigation of erythrocyte entry by Toxoplasma trophozoites the formation of a close junction between the erythrocyte and the invading parasite at the site of contact could be observed. The erythrocyte membrane and the outer membrane of the parasite pellicle show increased electron density in the circumferential attachment zone at the orifice of the erythrocyte invagination. The erythrocyte membrane has a thickness in this area of 7–8.5 nm. This membrane differentiation can be observed at the early stages of invasion when the tip of the apical end of the parasite induces a small erythrocyte invagination.It is still preserved in further advanced stages, when the parasite enters the enlarging invagination of the erythrocyte. As the morphological features resemble those observed during merozoite invasion into erythrocytes we consider our findings may have a similar significance for the mechanism of invasion by Toxoplasma as for the erythrocyte entry by malaria parasites.     

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.     

Characterization of Metabolically Quiescent Leishmania Parasites in Murine Lesions Using Heavy Water Labeling

Information on the growth rate and metabolism of microbial pathogens that cause long-term chronic infections is limited, reflecting the absence of suitable tools for measuring these parameters in vivo. Here, we have measured the replication and physiological state of Leishmania mexicana parasites in murine inflammatory lesions using ²H₂O labeling. Infected BALB/c mice were labeled with ²H₂O for up to 4 months, and the turnover of parasite DNA, RNA, protein and membrane lipids estimated from the rate of deuterium enrichment in constituent pentose sugars, amino acids, and fatty acids, respectively. We show that the replication rate of parasite stages in these tissues is very slow (doubling time of ~12 days), but remarkably constant throughout lesion development. Lesion parasites also exhibit markedly lower rates of RNA synthesis, protein turnover and membrane lipid synthesis than parasite stages isolated from ex vivo infected macrophages or cultured in vitro, suggesting that formation of lesions induces parasites to enter a semi-quiescent physiological state. Significantly, the determined parasite growth rate accounts for the overall increase in parasite burden indicating that parasite death and turnover of infected host cells in these lesions is minimal. We propose that the Leishmania response to lesion formation is an important adaptive strategy that minimizes macrophage activation, providing a permissive environment that supports progressive expansion of parasite burden. This labeling approach can be used to measure the dynamics of other host-microbe interactions in situ.     

The Role of Palmitoylation for Protein Recruitment to the Inner Membrane Complex of the Malaria Parasite

To survive and persist within its human host, the malaria parasite Plasmodium falciparum utilizes a battery of lineage-specific innovations to invade and multiply in human erythrocytes. With central roles in invasion and cytokinesis, the inner membrane complex, a Golgi-derived double membrane structure underlying the plasma membrane of the parasite, represents a unique and unifying structure characteristic to all organisms belonging to a large phylogenetic group called Alveolata. More than 30 structurally and phylogenetically distinct proteins are embedded in the IMC, where a portion of these proteins displays N-terminal acylation motifs. Although N-terminal myristoylation is catalyzed co-translationally within the cytoplasm of the parasite, palmitoylation takes place at membranes and is mediated by palmitoyl acyltransferases (PATs). Here, we identify a PAT (PfDHHC1) that is exclusively localized to the IMC. Systematic phylogenetic analysis of the alveolate PAT family reveals PfDHHC1 to be a member of a highly conserved, apicomplexan-specific clade of PATs. We show that during schizogony this enzyme has an identical distribution like two dual-acylated, IMC-localized proteins (PfISP1 and PfISP3). We used these proteins to probe into specific sequence requirements for IMC-specific membrane recruitment and their interaction with differentially localized PATs of the parasite.     

Characterization of Plasmodium vivax Early Transcribed Membrane Protein 11.2 and Exported Protein 1

In Plasmodium, the membrane of intracellular parasites is initially formed during invasion as an invagination of the red blood cell surface, which forms a barrier between the parasite and infected red blood cells in asexual blood stage parasites. The membrane proteins of intracellular parasites of Plasmodium species have been identified such as early-transcribed membrane proteins (ETRAMPs) and exported proteins (EXPs). However, there is little or no information regarding the intracellular parasite membrane in Plasmodium vivax. In the present study, recombinant PvETRAMP11.2 (PVX_003565) and PvEXP1 (PVX_091700) were expressed and evaluated antigenicity tests using sera from P. vivax-infected patients. A large proportion of infected individuals presented with IgG antibody responses against PvETRAMP11.2 (76.8%) and PvEXP1 (69.6%). Both of the recombinant proteins elicited high antibody titers capable of recognizing parasites of vivax malaria patients. PvETRAMP11.2 partially co-localized with PvEXP1 on the intracellular membranes of immature schizont. Moreover, they were also detected at the apical organelles of newly formed merozoites of mature schizont. We first proposed that these proteins might be synthesized in the preceding schizont stage, localized on the parasite membranes and apical organelles of infected erythrocytes, and induced high IgG antibody responses in patients.     

Phosphatidylthreonine and Lipid-Mediated Control of Parasite Virulence

The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer.     

The overexpression of a new ABC transporter in Leishmania is related to phospholipid trafficking and reduced infectivity

This paper reports the characterization of a new ABC transporter (LtrABC1.1), related to the human ABCA subfamily, in the protozoan parasite Leishmania tropica. LtrABC1.1 is a tandem duplicated gene flanked by inverted repeats. LtrABC1.1 is expressed mainly in the flagellar pocket of the parasite. Drug resistance studies in Leishmania overexpressing LtrABC1.1 showed the transporter not to confer resistance to a range of unrelated drugs. LtrABC1.1 appears to be involved in lipid movements across the plasma membrane of the parasite since overexpression reduces the accumulation of fluorescent phospholipid analogues. The activity of this protein may also affect membrane movement processes since secreted acid phosphatase (SAP) activity was significantly lower in promastigotes overexpressing LtrABC1.1. In vitro infection experiments with macrophages indicated LtrABC1.1-transfected parasites to be significantly less infective. Together, these results suggest that this new ABC transporter could play a role in lipid movements across the plasma membrane, and that its activity might influence vesicle trafficking. This is the first ABCA-like transporter described in unicellular eukaryotes.     

Plasmodium lophurae: Immunization of Pekin ducklings with different antigen preparations

Pekin ducklings were vaccinated with Freund's complete adjuvant plus free Plasmodium lophurae parasites, erythrocytes infected with P. lophurae schizonts, or parasite membrane vesicles. Approximately 50% of the vaccinated ducklings were resistant to challenge with this malarial parasite. However, little protection was afforded by immunization of ducklings with a parasite-specific histidine-rich protein.     

Plasmodium vivax Tryptophan Rich Antigen PvTRAg36.6 Interacts with PvETRAMP and PvTRAg56.6 Interacts with PvMSP7 during Erythrocytic Stages of the Parasite

Plasmodium vivax is most wide spread and a neglected malaria parasite. There is a lack of information on parasite biology of this species. Genome of this parasite encodes for the largest number of tryptophan-rich proteins belonging to ‘Pv-fam-a’ family and some of them are potential drug/vaccine targets but their functional role(s) largely remains unexplored. Using bacterial and yeast two hybrid systems, we have identified the interacting partners for two of the P. vivax tryptophan-rich antigens called PvTRAg36.6 and PvTRAg56.2. The PvTRAg36.6 interacts with early transcribed membrane protein (ETRAMP) of P.vivax. It is apically localized in merozoites but in early stages it is seen in parasite periphery suggesting its likely involvement in parasitophorous vacuole membrane (PVM) development or maintenance. On the other hand, PvTRAg56.2 interacts with P.vivax merozoite surface protein7 (PvMSP7) and is localized on merozoite surface. Co-localization of PvTRAg56.2 with PvMSP1 and its molecular interaction with PvMSP7 probably suggest that, PvTRAg56.2 is part of MSP-complex, and might assist or stabilize the protein complex at the merozoite surface. In conclusion, the PvTRAg proteins have different sub cellular localizations and specific associated functions during intra-erythrocytic developmental cycle.     

The Protozoan Parasite Toxoplasma gondii Targets Proteins to Dense Granules and the Vacuolar Space Using Both Conserved and Unusual Mechanisms

All known proteins that accumulate in the vacuolar space surrounding the obligate intracellular protozoan parasite Toxoplasma gondii are derived from parasite dense granules. To determine if constitutive secretory vesicles could also mediate delivery to the vacuolar space, T. gondii was stably transfected with soluble Escherichia coli alkaline phosphatase and E. coli β-lactamase. Surprisingly, both foreign secretory reporters were delivered quantitatively into parasite dense granules and efficiently secreted into the vacuolar space. Addition of a glycosylphosphatidylinositol membrane anchor rerouted alkaline phosphatase to the parasite surface. Alkaline phosphatase fused to the transmembrane domain and cytoplasmic tail from the endogenous dense granule protein GRA4 localized to dense granules. The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent upon the cytoplasmic tail, but not upon a tyrosine-based motif within the tail. Alkaline phosphatase fused to the vesicular stomatitis virus G protein transmembrane domain and cytoplasmic tail localized primarily to the Golgi, although staining of dense granules and the intravacuolar network was also detected; truncating the cytoplasmic tail decreased Golgi staining and increased delivery to dense granules but blocked delivery to the intravacuolar network. Targeting of secreted proteins to T. gondii dense granules and the plasma membrane uses general mechanisms identified in higher eukaryotic cells but is simplified and exaggerated in scope, while targeting of secreted proteins beyond the boundaries of the parasite involves unusual sorting events.     

Characterization of coproporphyrinogen III oxidase in Plasmodium falciparum cytosol

A unique hybrid pathway has been proposed for de novo heme biosynthesis in Plasmodium falciparum involving three different compartments of the parasite, namely mitochondrion, apicoplast and cytosol. While parasite mitochondrion and apicoplast have been shown to harbor key enzymes of the pathway, there has been no experimental evidence for the involvement of parasite cytosol in heme biosynthesis. In this study, a recombinant P. falciparum coproporphyrinogen III oxidase (rPfCPO) was produced in E. coli and confirmed to be active under aerobic conditions. rPfCPO behaved as a monomer of 61 kDa molecular mass in gel filtration analysis. Immunofluorescence studies using antibodies to rPfCPO suggested that the enzyme was present in the parasite cytosol. These results were confirmed by detection of enzyme activity only in the parasite soluble fraction. Western blot analysis with anti-rPfCPO antibodies also revealed a 58 kDa protein only in this fraction and not in the membrane fraction. The cytosolic presence of PfCPO provides evidence for a hybrid heme-biosynthetic pathway in the malarial parasite.     

Clonal and atypical Toxoplasma strain differences in virulence vary with mouse sub-species

The severe virulence of Toxoplasma gondii in classical laboratory inbred mouse strains contradicts the hypothesis that house mice (Mus musculus) are the most important intermediate hosts for its transmission and evolution because death of the mouse before parasite transmission equals death of the parasite. However, the classical laboratory inbred mouse strains (Mus musculus domesticus), commonly used to test Toxoplasma strain differences in virulence, do not capture the genetic diversity within Mus musculus. Thus, it is possible that Toxoplasma strains that are severely virulent in laboratory inbred mice are avirulent in some other mouse sub-species. Here, we present insight into the responses of individual mouse strains, representing strains of the genetically divergent Mus musculus musculus, Mus musculus castaneus and Mus musculus domesticus, to infection with individual clonal and atypical Toxoplasma strains. We observed that, unlike M. m. domesticus, M. m. musculus and M. m. castaneus are resistant to the clonal Toxoplasma strains. For M. m. musculus, we show that this is due to a locus on chromosome 11 that includes the genes that encode the interferon gamma (IFNG)-inducible immunity-related GTPases (Irgs) that can kill the parasite by localising and subsequently vesiculating the parasitophorous vacuole membrane. However, despite the localization of known effector Irgs to the Toxoplasma parasitophorous vacuole membrane, we observed that some atypical Toxoplasma strains are virulent in all the mouse strains tested. The virulence of these atypical strains in M. m. musculus could not be attributed to individual rhoptry protein 5 (ROP5) alleles, a secreted parasite pseudokinase that antagonises the canonical effector Irgs and is indispensable for parasite virulence in laboratory inbred mice (M. m. domesticus). We conclude that murine resistance to Toxoplasma is modulated by complex interactions between host and parasite genotypes and may be independent of known effector Irgs on murine chromosome 11.     

A single aquaporin gene encodes a water/glycerol/urea facilitator in Toxoplasma gondii with similarity to plant tonoplast intrinsic proteins

We describe a single aquaporin gene in Toxoplasma gondii which, surprisingly, has only 28% sequence similarity to the aquaglyceroporin of another apicomplexan parasite, Plasmodium falciparum. Sequence comparisons showed 47% similarity to water-specific plant aquaporins and the conservation of typical pore-forming residues. We established that the Toxoplasma aquaporin protein is a bifunctional membrane pore with intermediate water and high glycerol permeability. Furthermore, we identified hydroxyurea, an antineoplastic agent with inhibitory effects on parasite proliferation, as a permeant of this channel.     

Toxoplasma gondii: Ultrastructure study of the entry of tachyzoites into mammalian cells

Toxoplama gondii (Apicomplexa: Coccidia), an obligatory intracellular parasite with a unique capacity to invade virtually all nucleated cell type from warm-blooded vertebrate hosts. Despite the efficiency with which Toxoplasma enters its host cell, it remains unresolved if invasion occurs by direct penetration of the parasite or through phagocytosis. In the present work, electron microscopic study was designed to examine the entry process of Toxoplasma (RH strain) into macrophages and non phagocytic-host cells (Hela cells) and to observe the ultrastructure changes associated with intracellular parasitism. The results showed that both active invasion and phagocytosis were occurred and revealed that invasion is an ordered process that initiates with binding of the parasite at its apical end followed by tight-fitting invagination of the host cell membrane and a prominent constriction in the parasite at the site of penetration. The process ended by the professional parasitophorous vacuole that is distinct at the outset from those formed by phagocytosis in which once Toxoplasma triggered, phagocytic uptake can proceed by capture of the parasite within a loose fitting vacuole formed by localized membrane ruffling. The cytopathic effects of the parasite on macrophages and Hela cells were demonstrated within 5–15 h post-inoculation in the form of degenerative mitochondria, swelling Golgi apparatus and widening of endoplasmic reticulum indicating intracellular oedema. These changes were exaggerated and several cells were found dead after 48–72 h.     

Leishmania tropica: Surface antigens of intracellular and flagellate forms

Promastigotes and amastigotes of Leishmania tropica were surface-radioiodinated using the lactoperoxidase technique. Detergent lysates of the labeled organisms were analyzed by two-dimensional gel electrophoresis. Analysis of radioiodinated promastigote membrane proteins revealed six major and some minor acidic polypeptides. Analysis of the amastigote membrane proteins revealed six major proteins, mostly acidic, and some poorly resolved basic proteins. Four of the major membrane proteins appeared to be common to the two parasitic forms (M_r 67,000, M_r 50,000, M_r 68,000, and M_r 80,000). These polypeptides were recognized by antipromastigote antibodies as well as antibodies from CBA/H mice that had recovered from infection. Peptide mapping confirmed their homology in the two parasite forms. One polypeptide appeared to be specific for the promastigote (M_r 50,000) and two polypeptides appeared to be specific for the amastigote form of the parasite (M_r 94,000 and M_r 43,000).     

Identification and Functional Analysis of the Primary Pantothenate Transporter,PfPAT, of the Human Malaria Parasite Plasmodium falciparum

The human malaria parasite Plasmodium falciparum is absolutely dependent on the acquisition of host pantothenate for its development within human erythrocytes. Although the biochemical properties of this transport have been characterized, the molecular identity of the parasite-encoded pantothenate transporter remains unknown. Here we report the identification and functional characterization of the first protozoan pantothenate transporter, PfPAT, from P. falciparum. We show using cell biological, biochemical, and genetic analyses that this transporter is localized to the parasite plasma membrane and plays an essential role in parasite intraerythrocytic development. We have targeted PfPAT to the yeast plasma membrane and showed that the transporter complements the growth defect of the yeast fen2Δ pantothenate transporter-deficient mutant and mediates the entry of the fungicide drug, fenpropimorph. Our studies in P. falciparum revealed that fenpropimorph inhibits the intraerythrocytic development of both chloroquine- and pyrimethamine-resistant P. falciparum strains with potency equal or better than that of currently available pantothenate analogs. The essential function of PfPAT and its ability to deliver both pantothenate and fenpropimorph makes it an attractive target for the development and delivery of new classes of antimalarial drugs.     

A scanning electron microscope study of a microsporidian with a pansporoblast: Thelohania maenadis

Air drying is not adequate for the preservation of the pansporoblastic membrane of Thelohania maenadis (Protozoa, Microsporidia), a parasite of the crabs Carcinus mediterraneus and Carcinus maenas. Freeze-drying and critical point drying preserve the pansporoblast membrane and reveal that isolated spores of the microsporidian are covered with a thick hairy coat. This coat originates as secretory product within the pansporoblast cavity.     

Reduced glycerol incorporation into phospholipids contributes to impaired intra-erythrocytic growth of glycerol kinase knockout Plasmodium falciparum parasites

Background

Malaria is a devastating disease and Plasmodium falciparum is the most lethal parasite infecting humans. Understanding the biology of this parasite is vital in identifying potential novel drug targets. During every 48-hour intra-erythrocytic asexual replication cycle, a single parasite can produce up to 32 progeny. This extensive proliferation implies that parasites require substantial amounts of lipid precursors for membrane biogenesis. Glycerol kinase is a highly conserved enzyme that functions at the interface of lipid synthesis and carbohydrate metabolism. P. falciparum glycerol kinase catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate, a major phospholipid precursor.

Methods

The P. falciparum glycerol kinase gene was disrupted using double crossover homologous DNA recombination to generate a knockout parasite line. Southern hybridization and mRNA analysis were used to verify gene disruption. Parasite growth rates were monitored by flow cytometry. Radiolabelling studies were used to assess incorporation of glycerol into parasite phospholipids.

Results

Disruption of the P. falciparum glycerol kinase gene produced viable parasites, but their growth was significantly reduced to 56.5 ± 1.8% when compared to wild type parasites. ¹⁴C-glycerol incorporation into the major phospholipids of the parasite membrane, phosphatidylcholine and phosphatidylethanolamine, was 48.4 ± 10.8% and 53.1 ± 5.7% relative to an equivalent number of wild type parasites.

Conclusions

P. falciparum glycerol kinase is required for optimal intra-erythrocytic asexual parasite development. Exogenous glycerol may be used as an alternative carbon source for P. falciparum phospholipid biogenesis, despite the lack of glycerol kinase to generate glycerol-3-phosphate.

General significance

These studies provide new insight into glycerolipid metabolism in P. falciparum.