Invasion of red blood cells by malaria parasites

The malaria parasite is the most important member of the Apicomplexa, a large and highly successful phylum of intracellular parasites. Invasion of host cells allows apicomplexan parasites access to a rich source of nutrients in a niche that is largely protected from host defenses. All Apicomplexa adopt a common mode of host-cell entry, but individual species incorporate unique features and utilize a specific set of ligand-receptor interactions. These adhesins ultimately connect to a parasite actin-based motor, which provides the power for invasion. While some Apicomplexa can invade many different host cells, the disease-associated blood-stage form of the malaria parasite is restricted to erythrocytes.     

The Trypanosoma cruzi-host-cell interplay: location, invasion, retention

Chagas disease is a debilitating human illness caused by infection with the protozoan Trypanosoma cruzi. A capacity to invade and replicate within many different cell types is a cornerstone of the remarkable fitness of this parasite. Although invasion occurs independently of actin polymerization, the host cell still participates in the process, often in unexpected ways. Recent surprising findings indicate that host-cell lysosomes are indispensable, either by directly mediating invasion or by retaining these highly motile parasites inside cells.     

Rhoptry organelles of the apicomplexa: Their role in host cell invasion and intracellular survival

Members of the phylum Apicomplexa are obligate intracellular parasites that invade erythrocytes, lymphocytes, macrophages or cells of the alimentary canal in various vertebrate species. Organelles within the apical complex of invasive stages facilitate host cell invasion. Parasites in this phylum cause some of the most debilitating diseases of medical and veterinary importance. These include malaria, toxoplasmosis, babesiosis, theileriosis (East Coast fever), and coccidiosis in poultry and livestock. In recent years, opportunistic infections caused by Cryptosporidium parvum, and recrudescent Toxoplasma gondii infections in AIDS patients have prompted intensified efforts in understanding the biology of these parasites. In this review, Tobili Sam-Yellowe examines the unifying and variant molecular features of rhoptry proteins, and addresses the role of multigene families in organelle function: the biogenesis of the rhoptries will also be examined, in an attempt to understand the sequence of events leading to successful packaging, modification and processing of proteins within the organelle.     

The apical organelles of malaria merozoites: host cell selection, invasion, host immunity and immune evasion

Malaria is caused by protozoan parasites belonging to the phylum Apicomplexa. These obligate intracellular parasites depend on the successful invasion of an appropriate host cell for their survival. This article is a broad overview of the molecular strategies employed by the merozoite, an invasive form of the malaria parasite, to successfully invade a suitable red blood cell.     

Trypanosoma cruzi: identification of a galactose-binding protein that binds to cell surface of human erythrocytes and is involved in cell invasion by the parasite

Trypanosoma cruzi must invade mammalian host cells to replicate and complete its life cycle. Almost all nucleated mammalian cells can be invaded by the parasite following a receptor-ligand recognition as an early prerequisite. In this work, we describe a 67-kDa lectin-like glycoprotein that binds to desialylated human erythrocyte membranes in a galactose-dependent way. This protein is present on the parasite surface in both infective and non-infective stages of T. cruzi. More interestingly, we demonstrate by lectin-immuno-histochemistry assays that the 67kDa protein is involved in the recognition of host-cell receptors in mouse cardiac tissue and human cardiac aortic endothelium and mammary artery tissue. Moreover, antibodies against the 67kDa glycoprotein inhibit in vitro host-cell invasion by 63%. These data suggest that the 67kDa glycoprotein in vivo is needed for host-cell invasion by T. cruzi.     

Plasmodium protease ROM1 is important for proper formation of the parasitophorous vacuole

Apicomplexans are obligate intracellular parasites that invade host cells by an active process leading to the formation of a non-fusogenic parasitophorous vacuole (PV) where the parasite replicates within the host cell. The rhomboid family of proteases cleaves substrates within their transmembrane domains and has been implicated in the invasion process. Although its exact function is unknown, Plasmodium ROM1 is hypothesized to play a role during invasion based on its microneme localization and its ability to cleave essential invasion adhesins. Using the rodent malaria model, Plasmodium yoelii, we carried out detailed quantitative analysis of pyrom1 deficient parasites during the Plasmodium lifecycle. Pyrom1(-) parasites are attenuated during erythrocytic and hepatic stages but progress normally through the mosquito vector with normal counts of oocyst and salivary gland sporozoites. Pyrom1 steady state mRNA levels are upregulated 20-fold in salivary gland sporozoites compared to blood stages. We show that pyrom1(-) sporozoites are capable of gliding motility and traversing host cells normally. Wildtype and pyrom1(-) sporozoites do not differ in the rate of entry into Hepa1-6 hepatocytes. Within the first twelve hours of hepatic development, however, only 50% pyrom1(-) parasites have developed into exoerythrocytic forms. Immunofluorescence microscopy using the PVM marker UIS4 and transmission electron microscopy reveal that the PV of a significant fraction of pyrom1(-) parasites are morphologically aberrant shortly after invasion. We propose a novel function for PyROM1 as a protease that promotes proper PV modification to allow parasite development and replication in a suitable environment within the mammalian host.     

The Thrombospondin-related Protein Family of Apicomplexan Parasites: The Gears of the Cell Invasion Machinery

A number of severe diseases of medical and veterinary importance are caused by parasites of the phylum Apicomplexa. These parasites invade host cells using similar subcellular structures, organelles and molecular species. Proteins containing one or more copies of the type I repeat of human platelet thrombospondin (TSP1), are crucial components of both locomotion and invasion machinery. Members of this family have been identified in Eimeria tenella, E. maxima, Toxoplasma gondii, Cryptosporidium parvum and in all Plasmodium species so far analysed. Here, Andrea Crisanti and colleagues discuss the structure, localization and current understanding of the function of TSP family members in the invasion of target cells by apicomplexan parasites.     

Signalling mechanisms involved in apical organelle discharge during host cell invasion by apicomplexan parasites

Malaria is caused by Plasmodium parasites, which belong to the phylum apicomplexa. The characteristic feature of apicomplexan parasites is the presence of apical organelles, referred to as micronemes and rhoptries, in the invasive stages of the parasite life cycle. Survival of these obligate intracellular parasites depends on successful invasion of host cells, which is mediated by specific molecular interactions between host receptors and parasite ligands that are commonly stored in these apical organelles. The timely release of these ligands from apical organelles to the parasite surface is crucial for receptor engagement and invasion. This article is a broad overview of the signalling mechanisms that control the regulated secretion of apical organelles during host cell invasion by apicomplexan parasites.     

Scanning Electron Microscopy of Toxoplasma gondii: Parasite Torsion and Host-Cell Responses during Invasion1

Scanning electron microscopy confirmed our previous finding that toxoplasmas actively invade mouse peritoneal cells that are inhibited from phagocytosis. The parasites entered cells with the conoid end first and sometimes showed a counter-clockwise torsion of the body during invasion. Counter-clockwise torsion was also noted in free toxoplasmas. Host-cell responses to active invasion varied with experimental conditions and with the type of host cell. Under adverse culture conditions for phagocytosis, normal macrophages formed rudimentary filopodia or lamellipodia around the tips of in vading toxoplasmas; macrophages subjected to hyperthermia before similar incubation with toxoplasmas showed little or no response to invasion. Normal and heat-treated lymphocytes showed little surface reaction to invasion, but occa ionally a flocculent collar was seen around the tip of an invading toxoplasma. Scanning electron microscopy provides clues to possil'e mechanisms of toxoplasma locomotion and host-cell invasion.     

Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells

Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite.     

Protein Kinase A Dependent Phosphorylation of Apical Membrane Antigen 1 Plays an Important Role in Erythrocyte Invasion by the Malaria Parasite

Apicomplexan parasites are obligate intracellular parasites that infect a variety of hosts, causing significant diseases in livestock and humans. The invasive forms of the parasites invade their host cells by gliding motility, an active process driven by parasite adhesion proteins and molecular motors. A crucial point during host cell invasion is the formation of a ring-shaped area of intimate contact between the parasite and the host known as a tight junction. As the invasive zoite propels itself into the host-cell, the junction moves down the length of the parasite. This process must be tightly regulated and signalling is likely to play a role in this event. One crucial protein for tight-junction formation is the apical membrane antigen 1 (AMA1). Here we have investigated the phosphorylation status of this key player in the invasion process in the human malaria parasite Plasmodium falciparum. We show that the cytoplasmic tail of P. falciparum AMA1 is phosphorylated at serine 610. We provide evidence that the enzyme responsible for serine 610 phosphorylation is the cAMP regulated protein kinase A (PfPKA). Importantly, mutation of AMA1 serine 610 to alanine abrogates phosphorylation of AMA1 in vivo and dramatically impedes invasion. In addition to shedding unexpected new light on AMA1 function, this work represents the first time PKA has been implicated in merozoite invasion.     

Toxoplasma gondii: microneme protein MIC2

The phylum Apicomplexa contains parasites responsible for a variety of diseases including malaria, cryptosporidiosis, and toxoplasmosis. One of the common features of these parasites is that they contain a set of apical organelles whose sequential secretion is required for the invasion of host cells. Microneme proteins are the main adhesins involved in the attachment to the host cell surface by apicomplexans. The microneme protein MIC2, produced by Toxoplasma gondii, is conserved in apicomplexans and serves as a model to understand the first steps of invasion by the phylum. New data about the structure-function relationship of MIC2 reinforce the critical role of this protein in the successful invasion of cells by Toxoplasma and reveal potential therapeutic targets that may be used to control toxoplasmosis.     

Be in motion . .

Most Apicomplexan are obligate intracellular parasites and at different steps of their life cycle they invade host cells. The invasive forms are generally called zoites and the majority of them largely depend on a unique form of gliding motility to invade cells. Although the parasite intracellular motor complex that drives gliding motility and/or invasion is shared across different parasite stages and species, the extracellular transmembrane adhesins required to recognize and bind host molecules are not only species‐ but also stage‐specific (even if homologues). This is not such a surprise as different parasite stages interact with different hosts or distinct host cells. In this issue, Siden‐Kiamos et al. shows that specificity extends into the parasite cell, affecting how motility is regulated. Why is specificity occurring at this level? And how important is it? These are critical issues that will be hopefully addressed in the near future.     

Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion

Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. Relatively little is known about the proteins that orchestrate host cell invasion by T. gondii or related apicomplexan parasites (including Plasmodium spp., which cause malaria), due to the difficulty of studying essential genes in these organisms. We have used a recently developed regulatable promoter to create a conditional knockout of T. gondii apical membrane antigen-1 (TgAMA1). TgAMA1 is a transmembrane protein that localizes to the parasite's micronemes, secretory organelles that discharge during invasion. AMA1 proteins are conserved among apicomplexan parasites and are of intense interest as malaria vaccine candidates. We show here that T. gondii tachyzoites depleted of TgAMA1 are severely compromised in their ability to invade host cells, providing direct genetic evidence that AMA1 functions during invasion. The TgAMA1 deficiency has no effect on microneme secretion or initial attachment of the parasite to the host cell, but it does inhibit secretion of the rhoptries, organelles whose discharge is coupled to active host cell penetration. The data suggest a model in which attachment of the parasite to the host cell occurs in two distinct stages, the second of which requires TgAMA1 and is involved in regulating rhoptry secretion.     

Do trematode parasites disrupt defence-cell signalling in their snail hosts?

More than a decade ago, it was postulated that components derived from trematode parasites block receptors on the defence cells of their snail intermediate hosts, thus preventing host-cell activation and parasite elimination. This phenomenon has still not been investigated extensively. However, recent work concerning the molecular regulation of the molluscan defence response provides a new framework for studies that focus on an extension of this original concept - subversion of host cell signalling by trematode parasites. The hypothesis is that, to facilitate survival and replication in their intermediate hosts, trematode parasites down regulate host defence responses by interfering with key signal-transduction pathways in snail defence cells.     

Accurate quantitation of Leishmania infection in cultured cells by flow cytometry

BACKGROUND: Leishmaniases are major parasitic diseases caused by protozoans that are obligate intracellular parasites during the mammalian phase of their life cycle. Quantitation of experimental mammalian cell infections is usually performed by time-consuming microscopic examination. In this report a flow cytometry (FCM)-based assay suitable for studying in vitro infections by L.amazonensis is presented. METHODS: Intense fluorescence staining of the amastigote forms with a stage- and species-specific monoclonal antibody was obtained after permeabilization of both the host-cell cytoplasmic membrane and the parasitophorous vacuole membrane by saponin treatment. RESULTS: Upon flow cytometry (FCM) analysis, parasitized cells separated sharply from the auto-fluorescence of the mammalian host cells, giving the assay a high degree of sensitivity and specificity. Ninety to 98% of cells in the more fluorescent population harbored parasites visible by phase-contrast and UV-light microscopy, while no parasites were observed in more than 95% of the cells in the population with background fluorescence. Comparisons of the FCM results with those from microscope counting and analysis of various dilutions of parasitized cells confirmed the reliability of the method. CONCLUSIONS: The FCM assay provided rapid quantitation of Leishmania infection either in mouse macrophages, the natural host cell in murine leishmaniasis, or in Chinese hamster ovary (CHO) cells, a non-macrophage cell line proposed as an in vitro model for studying host-parasite interactions. The protocol described here should be adaptable to studies involving other parasites residing in nucleated cells.     

Effect of the digenean parasites of fish on the fauna of Mediterranean lagoons

Attention is drawn to the effects of parasites on their hosts, taking as a model the digenean parasites of teleosts (hereafter: fish) from lagoons along the French Mediterranean coast. Because digeneans have a heteroxenic life cycle, their impact is not limited to the definitive host, which harbours the sexual adults, but is extended to the first host (mollusc) and to the second host ("invertebrate" or fish). Adult parasites, in order to ensure efficient sexual reproduction, never cause excessive damage to their definitive host, usually only exploiting the intestinal fluids; however, the host must intensify its search for prey, which results in a diminished fitness. Within the first host, 'larval' stages of digenean parasites invade the gonads, resulting in its castration, then exhaustion and eventually death. The diversion of energy from the second hosts towards the parasites forces them to intensify their search for food, resulting in decreased fitness and an increased risk of being eaten; in addition, manipulation of the host's behaviour by parasites drives this host into the food chain of the definitive host. In lagoons, many individuals of almost all species of fish and invertebrates act as first, second and/or definitive hosts for digeneans. Obviously, parasites have a severe impact on the population dynamics of key taxa, on the food web and therefore also on the functioning of the whole lagoon ecosystem. Yet this impact has been largely overlooked or underestimated in functioning models, by ecologists, who tend to prioritize more apparent trophic relationships.     

Kinetics of the initial rounds of cell division of Toxoplasma gondii

RH strain Toxoplasma gondii tachyzoites that had naturally lysed their host cells were allowed to infect new host cells for a limited amount of time; subsequent parasite cell divisions were observed closely. On the basis of 4 independent trials, the estimated time to first cell division was 9.8 hr postinfection (PI) and was quite variable (95% confidence interval [CI]: 3.1-16.5 hr PI). The estimated time to second cell division was 14.9 hr PI and was less variable (95% CI: 12.1-17.7 hr PI). Few parasites divided before 6 hr PI in these 4 trials. When tachyzoites were derived by forced lysis (scraping an infected host cell culture and passing it through 27-gauge needles), the first parasite cell division occurred much more rapidly than had been observed in any of the trials with parasites derived by natural lysis. When parasites derived by forced lysis were held away from host cells for 3 hr PI, the first cell division was delayed in a manner similar to that seen in parasites derived by natural lysis. No differences were observed in the timing of the second cell division of parasites derived by forced lysis whether or not they had been held away from cells. These studies demonstrate that the conditions to which tachyzoites are exposed during transit from one host cell to another can affect the kinetics of parasite cell division in the new host cell.     

The Plasmodium sporozoite journey: a rite of passage

Sporozoites are the most versatile of the invasive stages of the Plasmodium life cycle. During their passage within the mosquito vector and the vertebrate host, sporozoites display diverse behaviors, including gliding locomotion and invasion of, migration through and egress from target cells. At the end of the journey, sporozoites invade hepatocytes and transform into exoerythrocytic stages, marking the transition from the pre-erythrocytic to the erythrocytic part of the life cycle. This article discusses recent work, mostly done with rodent malaria parasites, that has contributed to a better understanding of the sporozoites' complex biology and which has opened up new avenues for future sporozoite research.