Host cell egress and invasion induce marked relocations of glycolytic enzymes in Toxoplasma gondii tachyzoites

Apicomplexan parasites are dependent on an F-actin and myosin-based motility system for their invasion into and escape from animal host cells, as well as for their general motility. In Toxoplasma gondii and Plasmodium species, the actin filaments and myosin motor required for this process are located in a narrow space between the parasite plasma membrane and the underlying inner membrane complex, a set of flattened cisternae that covers most the cytoplasmic face of the plasma membrane. Here we show that the energy required for Toxoplasma motility is derived mostly, if not entirely, from glycolysis and lactic acid production. We also demonstrate that the glycolytic enzymes of Toxoplasma tachyzoites undergo a striking relocation from the parasites' cytoplasm to their pellicles upon Toxoplasma egress from host cells. Specifically, it appears that the glycolytic enzymes are translocated to the cytoplasmic face of the inner membrane complex as well as to the space between the plasma membrane and inner membrane complex. The glycolytic enzymes remain pellicle-associated during extended incubations of parasites in the extracellular milieu and do not revert to a cytoplasmic location until well after parasites have completed invasion of new host cells. Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K(+)] experienced during egress and invasion, a signal that requires changes of [Ca(2+)](c) in the parasite during egress. Enzyme translocation is, however, not dependent on either F-actin or intact microtubules. Our observations indicate that Toxoplasma gondii is capable of relocating its main source of energy between its cytoplasm and pellicle in response to exit from or entry into host cells. We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites.     

A role for coccidian cGMP-dependent protein kinase in motility and invasion

The coccidian parasite cGMP-dependent protein kinase is the primary target of a novel coccidiostat, the trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl] pyridine (compound 1), which effectively controls the proliferation of Eimeria tenella and Toxoplasma gondii parasites in animal models. The efficacy of compound 1 in parasite-specific metabolic assays of infected host cell monolayers is critically dependent on the timing of compound addition. Simultaneous addition of compound with extracellular E. tenella sporozoites or T. gondii tachyzoites inhibited [3H]-uracil uptake in a dose-dependent manner, while minimal efficacy was observed if compound addition was delayed, suggesting a block in host cell invasion. Immunofluorescence assays confirmed that compound 1 blocks the attachment of Eimeria sporozoites or Toxoplasma tachyzoites to host cells and inhibits parasite invasion and gliding motility. Compound 1 also inhibits the secretion of micronemal adhesins (E. tenella MIC1, MIC2 and T. gondii MIC2), an activity closely linked to invasion and motility in apicomplexan parasites. The inhibition of T. gondii MIC2 adhesin secretion by compound 1 was not reversed by treatment with calcium ionophores or by ethanol (a microneme secretagogue), suggesting a block downstream of calcium-dependent events commonly associated with the discharge of the microneme organelle in tachyzoites. Transgenic Toxoplasma strains expressing cGMP-dependent protein kinase mutant alleles that are refractory to compound 1 (including cGMP-dependent protein kinase knock-out lines complemented by such mutants) were used as tools to validate the potential role of cGMP-dependent protein kinase in invasion and motility. In these strains, parasite adhesin secretion, gliding motility, host cell attachment and invasion displayed a reduced sensitivity to compound 1. These data clearly demonstrate that cGMP-dependent protein kinase performs an important role in the host-parasite interaction.     

Versatility in the acquisition of energy and carbon sources by the Apicomplexa

Members of the phylum Apicomplexa are motile and rapidly dividing intracellular parasites, able to occupy a large spectrum of niches by infecting diverse hosts and invading various cell types. As obligate intracellular parasites, most apicomplexans only survive for a short period extracellularly, and, during this time, have a high energy demand to power gliding motility and invasion into new host cells. Similarly, these fast‐replicating intracellular parasites are critically dependent on host‐cell nutrients as energy and carbon sources, noticeably for the extensive membrane biogenesis imposed during growth and division. To access host‐cell metabolites, the apicomplexans Toxoplasma gondii and Plasmodium falciparum have evolved strategies that exquisitely reflect adaptation to their respective niches. In the present review, we summarize and compare some recent findings regarding the energetic metabolism and carbon sources used by these two genetically tractable apicomplexans during host‐cell invasion and intracellular growth and replication.     

Overexpression of a cytosolic pyrophosphatase (TgPPase) reveals a regulatory role of PP(i) in glycolysis for Toxoplasma gondii

PP(i) is a critical element of cellular metabolism as both an energy donor and as an allosteric regulator of several metabolic pathways. The apicomplexan parasite Toxoplasma gondii uses PP(i) in place of ATP as an energy donor in at least two reactions: the glycolytic PP(i)-dependent PFK (phosphofructokinase) and V-H(+)-PPase [vacuolar H(+)-translocating PPase (pyrophosphatase)]. In the present study, we report the cloning, expression and characterization of cytosolic TgPPase (T. gondii soluble PPase). Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a family I soluble PPase. Overexpression of the enzyme in extracellular tachyzoites led to a 6-fold decrease in the cytosolic concentration of PP(i) relative to wild-type strain RH tachyzoites. Unexpectedly, this subsequent reduction in PP(i) was associated with a higher glycolytic flux in the overexpressing mutants, as evidenced by higher rates of proton and lactate extrusion. In addition to elevated glycolytic flux, TgPPase-overexpressing tachyzoites also possessed higher ATP concentrations relative to wild-type RH parasites. These results implicate PP(i) as having a significant regulatory role in glycolysis and, potentially, other downstream processes that regulate growth and cell division.     

Modulation of mammalian sperm motility by quercetin

The flavonoid quercetin inhibits collective motility of ejaculated ram spermatozoa in the first 2 hr of incubation; during the next 3-4 hr motility is stimulated. To explain this interesting effect, we followed the influence of quercetin on sperm glycolysis, extracellular pH, ATP content, mitochondrial respiration, and lipid peroxidation. The collective motility of untreated cells is decreased to about 40% of the original motility during two hours of incubation. During this time, the rate of glycolysis is constant, respiration rate is increasing, there is no change in ATP content, the rate of lipid peroxidation is very slow, and the extracellular pH became very acidic (pH 5.5). It is concluded that motility is decreased due to this acidification. This acidification is prevented to some extent by quercetin, which indirectly inhibits glycolysis. Quercetin inhibits motility due to the inhibition of the plasma membrane calcium pump, as we showed previously (Breitbart et al., J Biol Chem 260:11548-11553, 1985). The motility of untreated cells is arrested after 3.5 hr of incubation, whereas quercetin-treated cells show high motility, which continues for additional 2-3 hr. After 3.5 hr, the control cells show no glycolytic activity, ATP content and respiration rates are decreased, and rate of lipid peroxidation is highly increased. At this time, quercetin-treated cells show no glycolytic activity, only a small decrease in ATP content and respiratory rate, and a very low rate of lipid peroxidation. Based on these data it is concluded that sperm motility after 3.5 hr of incubation is dependent mainly on mitochondrial respiration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycolysis is important for optimal asexual growth and formation of mature tissue cysts by Toxoplasma gondii

Toxoplasma gondii can grow and replicate using either glucose or glutamine as the major carbon source. Here, we have studied the essentiality of glycolysis in the tachyzoite and bradyzoite stages of T. gondii, using transgenic parasites that lack a functional hexokinase gene (Δhk) in RH (Type-1) and Prugniaud (Type-II) strain parasites. Tachyzoite stage Δhk parasites exhibit a fitness defect similar to that reported previously for the major glucose transporter mutant, and remain virulent in mice. However, although Prugniaud strain Δhk tachyzoites were capable of transforming into bradyzoites in vitro, they were severely compromised in their ability to make mature bradyzoite cysts in the brain tissue of mice. Isotopic labelling studies reveal that glucose-deprived tacyzoites utilise glutamine to replenish glycolytic and pentose phosphate pathway intermediates via gluconeogenesis. Interestingly, while glutamine-deprived intracellular Δhk tachyzoites continued to replicate, extracellular parasites were unable to efficiently invade host cells. Further, studies on mutant tachyzoites lacking a functional phosphoenolpyruvate carboxykinase (Δpepck1) revealed that glutaminolysis is the sole source of gluconeogenic flux in glucose-deprived parasites. In addition, glutaminolysis is essential for sustaining oxidative phosphorylation in Δhk parasites, while wild type (wt) and Δpepck1 parasites can obtain ATP from either glycolysis or oxidative phosphorylation. This study provides insights into the role of nutrient metabolism during asexual propagation and development of T. gondii, and validates the versatile nature of central carbon and energy metabolism in this parasite.     

Conservation of a gliding motility and cell invasion machinery in Apicomplexan parasites

Most Apicomplexan parasites, including the human pathogens Plasmodium, Toxoplasma, and Cryptosporidium, actively invade host cells and display gliding motility, both actions powered by parasite microfilaments. In Plasmodium sporozoites, thrombospondin-related anonymous protein (TRAP), a member of a group of Apicomplexan transmembrane proteins that have common adhesion domains, is necessary for gliding motility and infection of the vertebrate host. Here, we provide genetic evidence that TRAP is directly involved in a capping process that drives both sporozoite gliding and cell invasion. We also demonstrate that TRAP-related proteins in other Apicomplexa fulfill the same function and that their cytoplasmic tails interact with homologous partners in the respective parasite. Therefore, a mechanism of surface redistribution of TRAP-related proteins driving gliding locomotion and cell invasion is conserved among Apicomplexan parasites.     

Gliding motility: the molecules behind the motion

In apicomplexan parasites, gliding motility and host cell invasion are driven by an actomyosin-based system. Recent studies have characterized several components of the gliding motility apparatus and have provided new insight into the molecular architecture of this locomotory system.     

Gliding motility of Babesia bovis merozoites visualized by time-lapse video microscopy

Background

Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed “gliding motility”. However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite.

Methodology/Principal Findings

Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion.

Conclusions/Significance

This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding.     

Concerted action of two formins in gliding motility and host cell invasion by Toxoplasma gondii

The invasive forms of apicomplexan parasites share a conserved form of gliding motility that powers parasite migration across biological barriers, host cell invasion and egress from infected cells. Previous studies have established that the duration and direction of gliding motility are determined by actin polymerization; however, regulators of actin dynamics in apicomplexans remain poorly characterized. In the absence of a complete ARP2/3 complex, the formin homology 2 domain containing proteins and the accessory protein profilin are presumed to orchestrate actin polymerization during host cell invasion. Here, we have undertaken the biochemical and functional characterization of two Toxoplasma gondii formins and established that they act in concert as actin nucleators during invasion. The importance of TgFRM1 for parasite motility has been assessed by conditional gene disruption. The contribution of each formin individually and jointly was revealed by an approach based upon the expression of dominant mutants with modified FH2 domains impaired in actin binding but still able to dimerize with their respective endogenous formin. These mutated FH2 domains were fused to the ligand-controlled destabilization domain (DD-FKBP) to achieve conditional expression. This strategy proved unique in identifying the non-redundant and critical roles of both formins in invasion. These findings provide new insights into how controlled actin polymerization drives the directional movement required for productive penetration of parasites into host cells.     

Neospora caninum protein disulfide isomerase is involved in tachyzoite-host cell interaction

We have previously shown that treatment of Neospora caninum tachyzoites with the aspartyl protease inhibitor pepstatin A reduces host cell invasion [Naguleswaran, A., Muller, N., Hemphill, A., 2003. Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions. Exp. Parasitol. 104, 149-158]. Pepstatin A-affinity-chromatography led to the isolation of a major band of approximately 52 kDa which was identified as a homologue of a previously described Toxoplasma gondii putative protein disulfide isomerase (TgPDI) through tandem mass spectrometry. A BLAST search against N. caninum expressed sequence tags (ESTs) on the ApiDots server using TgPDI cDNA as query sequence revealed a 2251 bp PDI-like consensus (NcPDI), which shows 94% identity to the T. gondii homologue. In N. caninum tachyzoites, NcPDI was found mainly in the soluble hydrophilic fraction. Immunofluorescence showed that expression of NcPDI was dramatically down-regulated in the bradyzoite stage, and immunogold-EM on tachyzoites localised the protein to the cytoplasm, mostly in close vicinity to the nuclear membrane, to the micronemes, and to the parasite cell surface. However, NcPDI was absent in rhoptries and dense granules. Preincubation of tachyzoites with the sulfhydryl blocker 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (pCMBA), and with the PDI inhibitor bacitracin reduced adhesion of parasites to host cells. In addition, incubation of N. caninum tachyzoites with affinity-purified anti-NcPDI antibodies reduced host cell adhesion. PDIs catalyse the formation, reduction or isomerisation of disulfide bonds. Many major components of the adhesion and invasion machinery of apicomplexan parasites are cysteine-rich and dependent on correct folding via disulfide bond formation. Thus, our data points towards an important role for surface-associated NcPDI in Neospora-host cell interaction.     

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.     

Toxoplasma profilin is essential for host cell invasion and TLR11-dependent induction of an interleukin-12 response

Apicomplexan parasites exhibit actin-dependent gliding motility that is essential for migration across biological barriers and host cell invasion. Profilins are key contributors to actin polymerization, and the parasite Toxoplasma gondii possesses a profilin-like protein that is recognized by Toll-like receptor TLR11 in the host innate immune system. Here, we show by conditional disruption of the corresponding gene that T.gondii profilin, while not required for intracellular growth, is indispensable for gliding motility, host cell invasion, active egress from host cells, and virulence in mice. Furthermore, parasites lacking profilin are unable to induce TLR11-dependent production in vitro and in vivo of the defensive host cytokine interleukin-12. Thus, profilin is an essential element of two aspects of T. gondii infection. Like bacterial flagellin, profilin plays a role in motility while serving as a microbial ligand recognized by the host innate immune system.     

Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that actively invades mammalian cells using a unique form of gliding motility that critically depends on actin filaments in the parasite. To determine if parasite motility is driven by a myosin motor, we examined the distribution of myosin and tested the effects of specific inhibitors on gliding and host cell invasion. A single 90 kDa isoform of myosin was detected in parasite lysates using an antisera that recognizes a highly conserved myosin peptide. Myosin was localized in T. gondii beneath the plasma membrane in a circumferential pattern that overlapped with the distribution of actin. The myosin ATPase inhibitor, butanedione monoxime (BDM), reversibly inhibited gliding motility across serum-coated slides. The myosin light-chain kinase inhibitor, KT5926, also blocked parasite motility and greatly reduced host cell attachment; however, these effects were primarily caused by its ability to block the secretion of microneme proteins, which are involved in cell attachment. In contrast, while BDM partially reduced cell attachment, it prevented invasion even under conditions in which microneme secretion was not affected, indicating a potential role for myosin in cell entry. Collectively, these results indicate that myosin(s) probably participate(s) in powering gliding motility, a process that is essential for cell invasion by T. gondii .     

A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites

Apicomplexan parasites constitute one of the most significant groups of pathogens infecting humans and animals. The liver stage sporozoites of Plasmodium spp. and tachyzoites of Toxoplasma gondii, the causative agents of malaria and toxoplasmosis, respectively, use a unique mode of locomotion termed gliding motility to invade host cells and cross cell substrates. This amoeboid-like movement uses a parasite adhesin from the thrombospondin-related anonymous protein (TRAP) family and a set of proteins linking the extracellular adhesin, via an actin-myosin motor, to the inner membrane complex. The Plasmodium blood stage merozoite, however, does not exhibit gliding motility. Here we show that homologues of the key proteins that make up the motor complex, including the recently identified glideosome-associated proteins 45 and 50 (GAP40 and GAP50), are present in P. falciparum merozoites and appear to function in erythrocyte invasion. Furthermore, we identify a merozoite TRAP homologue, termed MTRAP, a micronemal protein that shares key features with TRAP, including a thrombospondin repeat domain, a putative rhomboid-protease cleavage site, and a cytoplasmic tail that, in vitro, binds the actin-binding protein aldolase. Analysis of other parasite genomes shows that the components of this motor complex are conserved across diverse Apicomplexan genera. Conservation of the motor complex suggests that a common molecular mechanism underlies all Apicomplexan motility, which, given its unique properties, highlights a number of novel targets for drug intervention to treat major diseases of humans and livestock.     

Calcium Signaling throughout the Toxoplasma gondii Lytic Cycle: A STUDY USING GENETICALLY ENCODED CALCIUM INDICATORS*

Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca²⁺ oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca²⁺ enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca²⁺ changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca²⁺ oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca²⁺ influx. This is the first study showing, in real time, Ca²⁺ signals preceding egress and their direct link with motility, an essential virulence trait.     

RNA granules present only in extracellular toxoplasma gondii increase parasite viability

Toxoplasma gondii is an obligate intracellular parasite. When searching for a new cell to invade, the parasites have to confront the stress of being exposed to the extracellular environment. The mechanisms by which T. gondii survives outside the host cells are poorly understood. In this work we show that extracellular parasites form mRNA aggregates with characteristics of stress granules. Intracellular tachyzoites or bradyzoites do not form mRNA granules. We tested different stimuli that trigger granule formation in vitro and discovered that a buffer that mimics the host cell cytosol ionic composition (high potassium) strongly induces granule formation, suggesting that the granules arise when the parasites come in contact with the host cell cytosol during egress. We examined the importance of granule formation for parasite viability and show that the parasite populations that are able to form granules have a growth advantage, increased invasion, and decreased apoptosis in the extracellular environment. Overall, granule formation improves the fitness of extracellular parasites and increases the efficiency of the lytic cycle.     

Gliding motility and cell invasion by Apicomplexa: insights from the Plasmodium sporozoite

Apicomplexa constitute one of the largest phyla of protozoa. Most Apicomplexa, including those pathogenic to humans, are obligate intracellular parasites. Their extracellular forms, which are highly polarized and elongated cells, share two unique abilities: they glide on solid substrates without changing their shape and reach an intracellular compartment without active participation from the host cell. There is now ample ultrastructural evidence that these processes result from the backward movement of extracellular interactions along the anteroposterior axis of the parasite. Recent work in several Apicomplexa, including genetic studies in the Plasmodium sporozoite, has provided molecular support for this 'capping' model. It appears that the same machinery drives both gliding motility and host cell invasion. The cytoplasmic motor, a transmembrane bridge and surface ligands essential for cell invasion are conserved among the main apicomplexan pathogens.     

Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions

This paper describes an adhesion/invasion assay, based on combined pyrrolidine dithiocarbamate (PDTC) and antibody treatment of parasites followed by quantitative real-time PCR. This PDTC-PCR assay can be used to comparatively assess the participation of host cell- and parasite-associated components during host cell adhesion and entry by Neospora caninum and Toxoplasma gondii tachyzoites, respectively, and is potentially applicable to any other apicomplexan parasite. The assay allows to determine the parasite invasion rate in relation to the overall number of parasites which interact with host cells in any given experiment, and thus represents a significant improvement to conventional microscopic assays in terms of accuracy and reproducibility. Using this assay it was possible to show that adhesion and invasion of N. caninum tachyzoites are two distinct and separated events, in that N. caninum tachyzoites preferentially utilise host cell surface chondroitin sulphates for adhesion, but not for the host cell invasion process. Application of the PDTC-PCR assay also demonstrated that N. caninum and T. gondii tachyzoites differ largely with regard to the functional involvement of proteases in adhesion and invasion of host cells. Thus, although phylogenetically closely related, N. caninum and T. gondii are biologically quite different and exhibit distinct dissimilarities with regard to host cell interactions.