Gliding motility: an efficient mechanism for cell penetration.

An important group of animal and human pathogens, belonging to the phylum Apicomplexa, employs a novel form of motility, known as gliding, to move on solid substrates and to enter host cells. Gliding is dependent on the parasite cytoskeleton and involves a conserved family of secretory adhesins.

Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts.

Invasion of vertebrate cells by the protozoan Toxoplasma gondii is accompanied by regulated protein secretion from three distinct parasite organelles called micronemes, rhoptries, and dense granules. We have compared the kinetics of secretion from these different compartments during host cell invasion using immunofluorescence, immunoelectron microscopy, and quantitative immunoassays. Binding to the host cell triggered apical release of the micronemal protein MIC2 at the tight attachment zone that forms between the parasite and the host cell. In a second step, invagination of the host cell plasma membrane was initiated by discharge of the rhoptry protein ROP1 to form a nascent parasitophorous vacuole (PV). ROP1 was fully discharged into the vacuole by the time invasion was complete. In contrast to these very rapid early events, release of the dense granule markers GRA1 and NTPase was delayed until after the parasite was fully within the PV, eventually peaking at 20 min post-invasion. The sequential triggering of secretion from different organelles implies that their release is governed by separate signals and that their contents mediate distinct phases of intracellular parasitism.

Targeted disruption of expression site-associated gene-1 in bloodstream-form Trypanosoma brucei.

Each variant surface glycoprotein (Vsg) expression site (ES) in bloodstream-form Trypanosoma brucei is a polycistronic transcription unit containing several distinct expression site-associated genes (esag), in addition to a single vsg gene. esag1 genes from different ESs encode a highly polymorphic family of membrane-associated glycoproteins, whose function is unknown. In the hope of producing a phenotype that could indicate a function, we disrupted the esag1 genes in two ESs by targeted insertion of a hygromycin phosphotransferase gene. Our failure to produce an obvious phenotype prompted us to search for other esag1 transcripts. RNA from the mutant trypanosomes hybridized with an esag1-specific oligonucleotide. Cloning and sequencing of mRNA from both mutant and wild-type cells showed that several esag1 family members were expressed, each at a much lower level than the esag1 transcript from the active ES in wild-type trypanosomes. Long-range DNA mapping showed that these additional esag1 genes, some of which contained premature translation-termination codons, most probably originate from chromosomal-internal genes and pseudogenes. We have therefore been unable to determine whether esag1 is an essential gene, or what function it fulfils, or whether any competent Esag1 protein is expressed in the mutant trypanosomes.

Manipulation of the vsg co-transposed region increases expression-site switching in Trypanosoma brucei.

Disruption of a region of DNA in Trypanosoma brucei immediately upstream of the expressed telomere-proximal variant surface glycoprotein gene (vsg), known as the co-transposed region (CTR), can cause a dramatic increase in the rate at which the active expression site (ES) is switched off and a new ES is switched on. Deletion of most of the CTR in two ESs caused a greater than 100-fold increase in the rate of ES switching, to about 1.3 x 10(-4) per generation. A more dramatic effect was observed when the entire CTR and the 5' coding region of the expressed vsg221 were deleted. In this case a new ES was activated within a few cell divisions. This switch also occurred in cell lines where a second vsg had been inserted into the ES, prior to CTR deletion. These cell lines, which stably co-expressed the inserted and endogenous Vsgs, in equal amounts, did not differ from the wild-type in growth rate or switching frequency, suggesting that simultaneous expression of two Vsgs has no intrinsic effect. CTR deletion did not disturb the inserted vsg117. We tentatively conclude that it was not the disruption of the vsg221 in itself that destabilized the ES. All of the observed switches occurred without additional detectable DNA rearrangements in the switched ES. Deletion of the 70-bp repeats and/or a vsg pseudogene upstream of the CTR did not affect ES stability. Several speculative interpretations of these observation are offered, the most intriguing of which is that the CTR plays some role in modulating chromatin conformation at an ES.

A possible role for the 3'-untranslated region in developmental regulation in Trypanosoma brucei.

A series of Trypanosoma brucei transfection vectors was constructed in which transcription of the luciferase gene was driven by the procyclic acidic repetitive protein (procyclin) promoter. The untranslated regions surrounding the luciferase gene were derived from the actin, fructose bisphosphate aldolase, or PARP loci. Trans-splicing of the resulting transcripts occurred as expected, but the site of 3' polyadenylation was upstream of the position anticipated. The nature of the 3'-untranslated region was crucial to the level of expression in bloodstream forms.

The Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1) is a microneme protein secreted in response to elevated intracellular calcium levels.

A monoclonal antibody (MAb) has been generated against a novel 63 kDa surface/apical antigen of Toxoplasma gondii tachyzoites which is identified here as TgAMA-1, the Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1). Sequence analysis, phase partitioning in Triton X-114, and labeling of TgAMA-1 with iodonaphthalene azide all suggest that TgAMA-1 is a type I transmembrane protein. There is a high degree of sequence similarity between TgAMA-1 and Plasmodium AMA-1, most notably in the position of conserved cysteine residues within the protein's predicted extracellular domain. In contrast to full length Plasmodium AMA-1, which has previously been localized to the rhoptries, it is shown here by immunofluorescence and immunoelectron microscopy that intracellular TgAMA-1 is found in the micronemes. A 53 kDa N-terminal proteolytic fragment of TgAMA-1 is constitutively secreted from the parasite at 37 degrees C. As is the case with other microneme proteins, the proteolytic processing and secretion of TgAMA-1 is dramatically enhanced in response to treatments which increase intracellular calcium levels.

Molecular characterisation of an expressed sequence tag locus of Toxoplasma gondii encoding the micronemal protein MIC2.

The expressed sequence tag (EST) dataset of Toxoplasma gondii provides a wealth of information towards gene discovery. The complete cDNA and genomic sequence of EST tgc050 locus shows that it contains five copies of the conserved thrombospondin (TSP)-like motif present in a number of molecules with adhesive properties. A conserved region implicated with the adhesive characteristic of another group of proteins including several integrins, is also present in this molecule. The protein encoded by this sequence (rc50) is strongly recognised by monoclonal antibodies to MIC2. Affinity purified anti-rc50 antisera specifically reacted with a single protein of identical molecular mass as MIC2 and exclusively labeled the micronemes of T. gondii by cryo-immunoelectron microscopy. These results demonstrate that c50 encodes for MIC2, a previously characterised microneme protein of T. gondii. The extensive sequence similarity across multiple protein domains provides evidence that the protein encoded by this locus is the homologue to the Etp100 microneme protein of Eimeria tenella.

Molecular characterization of TgMIC5, a proteolytically processed antigen secreted from the micronemes of Toxoplasma gondii.

During invasion of host cells, Toxoplasma gondii discharges the contents of small, apically located secretory organelles called micronemes. Micronemal proteins are known to be necessary for both parasite motility and invasion of host cells. To further define the contents of Toxoplasma micronemes, we used cell fractionation and secretion-modulating drugs to identify six novel, putative micronemal proteins. In this paper we describe preliminary characterization of one of these novel proteins, TgMIC5. Molecular cloning and DNA sequence analysis of the TgMIC5 cDNA and gene revealed that it encodes a previously identified immunodominant antigen called H4. TgMIC5 also possesses a consensus sequence unique to members of the parvulin family of peptidyl-prolyl cis-trans isomerases (PPIases). TgMIC5 is expressed as a preproprotein, which is proteolytically processed to a proprotein by signal peptidase before being further processed to a mature protein of 22 kDa. Using a combination of protein secretion experiments, immunofluorescence and immunoelectron microscopy, we demonstrated that TgMIC2 is stored in the micronemes of T. gondii tachyzoites before it is secreted into the surrounding medium. Based on its homology with parvulin-like PPIases, TgMIC5 may assist in the folding of other micronemal proteins that function in invasion of host cells by T. gondii tachyzoites.

Armed and dangerous: Toxoplasma gondii uses an arsenal of secretory proteins to infect host cells.

Toxoplasma gondii is a protozoan parasite that infects a wide variety of warm-blooded animals and humans, in which it causes opportunistic disease. As an obligate intracellular parasite, T. gondii must invade a host cell to survive and replicate during infection. Recent studies suggest that T. gondii secretes a variety of proteins that appear to function during invasion or intracellular replication. These proteins originate from three distinct regulated secretory organelles called micronemes, rhoptries and dense granules. By discharging the contents of its secretory organelles at precise steps in invasion, T. gondii appears to timely deploy secretory proteins to their correct target destinations. Based on the timing of secretion and the characteristics of secretory proteins, an emerging theme is that T. gondii compartmentalizes its secretory proteins according to general function. Thus, it appears that micronemal proteins may function during parasite attachment to host cells, rhoptry proteins may facilitate parasite vacuole formation and host organellar association, and dense granule proteins likely promote intracellular replication, possibly by transporting and processing nutrients from the host cell. However, as more T. gondii secretory proteins are identified and characterized, it is likely that additional functions will be ascribed to each class of proteins secreted- by this fascinating invasive parasite.

Host cell manipulation by the human pathogen Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that can infect virtually any nucleated cell. During invasion Toxoplasma creates the parasitophorous vacuole, a subcellular compartment that acts as an interface between the parasite and host, and serves as a platform for modulation of host cell functions that support parasite replication and infection. Spatial reorganization of host organelles and cytoskeleton around the parasitophorous vacuole are observed following entry, and recent evidence suggests this interior redecorating promotes parasite nutrient acquisition. New findings also reveal that Toxoplasma manipulates host signaling pathways by deploying parasite kinases and a phosphatase, including at least two that infiltrate the host nucleus. Toxoplasma infection additionally controls several cellular pathways to establish an anti-apoptotic environment, and subverts immune cells as a conduit for dissemination. In this review we discuss these recent developments in understanding how Toxoplasma achieves widespread success as a human and animal parasite by manipulating its host.

Mapping of segmental antigenic determinants on structurally related variant surface glycoproteins of Trypanosoma brucei.

To study common and variant specific antigenic determinants on variant surface glycoproteins from Trypanosoma brucei, we have selected four serologically cross-reacting variant populations. Monoclonal antibodies were raised against the purified variant surface glycoproteins from each variant trypanosome population. Six monoclonal antibodies bind to segmental epitopes and one binds to a topographically assembled epitope. Amino acid compositions of these variant surface glycoproteins reveal striking conservation of certain residues including cysteine and charged amino acids. We also find that all seven monoclonal antibodies used in this study bind to protein determinants not exposed on the surface of the living trypanosome. Only one monoclonal antibody exhibits homologous specificity, while the remainder display cross-reactivity for three or all four variant surface glycoproteins. In addition, polyacrylamide gel electrophoresis peptide mapping and Western blots probed with each monoclonal antibody reveal significant peptide homologies. Furthermore, two pairs of monoclonal antibodies recognize two epitopes that are possibly immunodominant. The significance of these findings is discussed in terms of the structural similarities and differences among variant surface glycoproteins.

High-efficiency clonal growth of bloodstream- and insect-form Trypanosoma brucei on agarose plates.

This report describes a method for growing both bloodstream- and procyclic-form Trypanosoma brucei as colonies on agarose plates. Procyclic colonies, which took 2 weeks to develop, grew with approximately 17% plating efficiency on SDM-79/0.65% agarose supplemented with 20% (vol/vol) conditioned medium. Bloodstream forms were adapted to in vitro growth in liquid HMI-9 medium and then spread on HMI-9/0.65% agarose plates, where they grew to visible colonies in 3-5 days. Plating efficiencies were from 3 to 80%, depending upon the trypanosome variant and experiment. Colonies were proven to be the result of growth from a single cell and contained approximately 10(6) cells at maturity. Colonies were transferred to filters and probed for multicopy and single-copy genes. Potential uses of this method in conjunction with classical and reverse genetic approaches to studying trypanosomes are discussed.

Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii.

Apicomplexan parasites, including Toxoplasma gondii, apically attach to their host cells before invasion. Recent studies have implicated the contents of micronemes, which are small secretory organelles confined to the apical region of the parasite, in the process of host cell attachment. Here, we demonstrate that microneme discharge is regulated by parasite cytoplasmic free Ca2+ and that the micronemal contents, including the MIC2 adhesin, are released through the extreme apical tip of the parasite. Microneme secretion was triggered by Ca2+ ionophores in both the presence and the absence of external Ca2+, while chelation of intracellular Ca2+ prevented release. Mobilization of intracellular calcium with thapsagargin or NH4Cl also triggered microneme secretion, indicating that intracellular calcium stores are sufficient to stimulate release. Following activation of secretion by the Ca2+ ionophore A23187, MIC2 initially occupied the apical surface of the parasite, but was then rapidly treadmilled to the posterior end and released into the culture supernatant. This capping and release of MIC2 by ionophore-stimulated tachyzoites mimics the redistribution of MIC2 that occurs during attachment and penetration of host cells, and both events are dependent on the actin-myosin cytoskeleton of the parasite. These studies indicate that microneme release is a stimulus-coupled secretion system responsible for releasing adhesins involved in cell attachment.

The Toxoplasma adhesive protein MIC2 is proteolytically processed at multiple sites by two parasite-derived proteases.

MIC2 is an adhesive protein that participates in host cell invasion by the obligate intracellular parasite Toxoplasma gondii. Earlier studies established that MIC2 is secreted into the culture medium by extracellular parasites and that release is coincident with proteolytic modification. Since little is known about proteolytic processing of proteins secreted by T. gondii, we undertook this study to investigate the proteolytic events that accompany secretion of MIC2. We demonstrate that the C-terminal domain of MIC2 is removed by a protease, termed MPP1, when MIC2 is released into the culture supernatant. Additionally, prior to release, a second protease, termed MPP2, trims the N terminus of MIC2, resulting in the release of heterogeneously sized species of MIC2. Although MPP1 activity was unaffected by any of the protease inhibitors tested, MPP2 activity was blocked by a subset of serine and cysteine protease inhibitors. These results establish that MIC2 is proteolytically modified at multiple sites by two distinct enzymes that probably operate on the parasite surface.

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.

Ethanol and acetaldehyde elevate intracellular [Ca2+] and stimulate microneme discharge in Toxoplasma gondii.

One of the first steps in host-cell invasion by the protozoan parasite Toxoplasma gondii occurs when the parasite attaches by its apical end to the target host cell. The contents of apical secretory organelles called micronemes have recently been implicated in parasite apical attachment to host cells. Micronemes are regulated secretory vesicles that discharge in response to elevated parasite intracellular Ca(2+) levels ([Ca2+]i). In the present study we found that ethanol and related compounds produced a dose-dependent stimulation of microneme secretion. In addition, using fluorescence spectroscopy on tachyzoites loaded with the Ca(2+)-sensitive fluorescent dye fura-2, we demonstrated that ethanol stimulated microneme secretion by elevating parasite [Ca2+](i). Furthermore, sequential addition experiments with ethanol and other Ca(2+)-mobilizing drugs showed that ethanol probably elevated parasite [Ca2+](i) by mobilizing Ca(2+) from a thapsigargin-insensitive compartment of neutral pH. Earlier studies have shown that ethanol also elevates [Ca2+](i) in mammalian cells. Thus, because it is genetically tractable, T. gondii might be a convenient model organism for studying the Ca(2+)-elevating effects of alcohol in higher eukaryotes.

Secretion of micronemal proteins is associated with toxoplasma invasion of host cells.

Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of its pervasive cell invasion is not completely understood. Here, we demonstrate, using several independent assays, that Toxoplasma invasion of host cells is tightly coupled to the release of proteins stored within apical secretory granules called micronemes. Both microneme secretion and cell invasion were highly temperature dependent, and partial depletion of microneme resulted in a transient loss of infectivity. Chelation of parasite intracellular calcium strongly inhibited both microneme release and invasion of host cells, and this effect was partially reversed by raising intracellular calcium using the ionophore A23187. We also provide evidence that a staurosporine-sensitive kinase activity regulates microneme discharge and is required for parasite invasion of host cells. Additionally, we demonstrate that, during apical attachment to the host cell, the micronemal protein MIC2 is released at the junction between the parasite and the host cell. During invasion, MIC2 is successively translocated towards the posterior end of the parasite and is shed before entry of the parasite into the vacuole. Furthermore, we show that the full-length cellular form of MIC2, but not the proteolytically modified secreted form of MIC2, binds specifically to host cells. Collectively, these observations strongly imply that micronemal proteins play a role in Toxoplasma invasion of host cells.

Toxoplasma gondii uses sulfated proteoglycans for substrate and host cell attachment.

Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of this pervasive cell recognition is not understood. We demonstrate here that binding to the substratum and to host cells is partially mediated by interaction with sulfated glycosaminoglycans (GAGs). Addition of excess soluble GAGs blocked parasite attachment to serum-coated glass, thereby preventing gliding motility of extracellular parasites. Similarly, excess soluble GAGs decreased the attachment of parasites to human host cells from a variety of lineages, including monocytic, fibroblast, endothelial, epithelial, and macrophage cells. The inhibition of parasite attachment by GAGs was observed with heparin and heparan sulfate and also with chondroitin sulfates, indicating that the ligands for attachment are capable of recognizing a broad range of GAGs. The importance of sulfated proteoglycan recognition was further supported by the demonstration that GAG-deficient mutant host cells, and wild-type cells treated enzymatically to remove GAGs, were partially resistant to parasite invasion. Collectively, these studies reveal that sulfated proteoglycans are one determinant used for substrate and cell recognition by Toxoplasma. The widespread distribution of these receptors may contribute to the broad host and tissue ranges of this highly successful intracellular parasite.

A conserved subtilisin-like protein TgSUB1 in microneme organelles of Toxoplasma gondii.

Proteolytic processing plays a significant role in the process of invasion by the obligate intracellular parasite Toxoplasma gondii. We have cloned a gene, TgSUB1, encoding for a subtilisin-type serine protease found in T. gondii tachyzoites. TgSUB1 protein is homologous to other Apicomplexan and bacterial subtilisins and is processed within the secretory pathway of the parasite. Initial cleavage occurs in the endoplasmic reticulum, after which the protein is transported to micronemes, vesicles that secrete early during host cell invasion. Upon stimulation of microneme secretion, TgSUB1 is cleaved into smaller products that are secreted from the parasite. This secondary processing is inhibited by brefeldin A and serine protease inhibitors. TgSUB1 is a candidate processing enzyme for several microneme proteins cleaved within the secretory pathway or during invasion.