Toxoplasma gondii micronemal protein MIC1 is a lactose-binding lectin.

Host cell invasion by Toxoplasma gondii is a multistep process with one of the first steps being the apical release of micronemal proteins that interact with host receptors. We demonstrate here that micronemal protein 1 (MIC1) is a lactose-binding lectin. MIC1 and MIC4 were recovered in the lactose-eluted (Lac(+)) fraction on affinity chromatography on immobilized lactose of the soluble antigen fraction from tachyzoites of the virulent RH strain. MIC1 and MIC4 were both identified by N-terminal microsequencing. MIC4 was also identified by sequencing cDNA clones isolated from an expression library following screening with mouse polyclonal anti-60/70 kDa (Lac(+) proteins) serum. This antiserum localized the Lac(+) proteins on the apical region of T. gondii tachyzoites by confocal microscopy. The Lac(+) fraction induced hemagglutination (mainly type A human erythrocytes), which was inhibited by beta-galactosides (3 mM lactose and 12 mM galactose) but not by up to 100 mM melibiose (alpha-galactoside), fucose, mannose, or glucose or 0.2 mg/ml heparin. The lectin activity of the Lac(+) preparation was attributed to MIC1, because blotted MIC1, but not native MIC4, bound human erythrocyte type A and fetuin. The copurification of MIC1 and MIC4 may have been due to their association, as reported by others. These data suggest that MIC1 may act through its lectin activity during T. gondii infection.     

Identification of trafficking determinants for polytopic rhomboid proteases in Toxoplasma gondii

Rhomboids (ROMs) constitute a family of polytopic serine proteases conserved throughout evolution. The obligate intracellular parasite Toxoplasma gondii possesses six genes coding for ROM-like proteases that are targeted to distinct subcellular compartments: TgROM1 localizes to regulated secretory organelles, micronemes, TgROM2 is present in the Golgi, while TgROM4 and TgROM5 are found in the pellicle of the parasite. The targeting mechanism/s of ROM proteins is an aspect that has not yet been assessed. The existence of TgROM family members localized to different subcellular compartments provides a convenient system to study their sorting mechanisms in a genetically tractable organism that possesses an elaborate secretory pathway and conserved trafficking machineries. In this study, we experimentally established the topology of TgROM1 and TgROM4 at the plasma membrane and applied domain-exchange and site-directed mutagenesis approaches to identify critical sorting determinants on the N-terminal cytosolic domains of TgROM2 and TgROM1 that confer their Golgi and post-Golgi localizations, respectively.     

Microneme rhomboid protease TgROM1 is required for efficient intracellular growth of Toxoplasma gondii

Rhomboids are serine proteases that cleave their substrates within the transmembrane domain. Toxoplasma gondii contains six rhomboids that are expressed in different life cycle stages and localized to different cellular compartments. Toxoplasma rhomboid protein 1 (TgROM1) has previously been shown to be active in vitro, and the orthologue in Plasmodium falciparum processes the essential microneme protein AMA1 in a heterologous system. We investigated the role of TgROM1 to determine its role during in vitro growth of T. gondii. TgROM1 was localized in the secretory pathway of the parasite, including the Golgi apparatus and micronemes, which contain adhesive proteins involved in invasion of host cells. However, unlike other micronemal proteins, TgROM1 was not released onto the parasite surface during cell invasion, suggesting it does not play a critical role in cell invasion. Suppression of TgROM1 using the tetracycline-regulatable system revealed that ROM1-deficient parasites were outcompeted by wild-type T. gondii. ROM1-deficient parasites showed only modest decrease in invasion but replicated more slowly than wild-type cells. Collectively, these results indicate that ROM1 is required for efficient intracellular growth by T. gondii.     

Structure of the micronemal protein 2 A/I domain from Toxoplasma gondii

Toxoplasma gondii is a widespread zoonotic pathogen capable of causing serious disease in humans and animals. As an obligate intracellular parasite, T. gondii relies on the orchestrated secretion of proteins from its apical complex organelles including the multimodular, transmembrane micronemal protein 2 (MIC2) that couples recognition of the host cell with cytoskeletal reorganization of the parasite to drive invasion. To probe the basis by which the von Willebrand Factor A (vWA)–Integrin like module of TgMIC2 engages the host cell, we solved the crystal structure of a truncated form of TgMIC2A/I (TgMIC2A/Ic) phased by iodide SIRAS and refined to a resolution of 2.05 Å. The TgMIC2A/Ic core is organized into a central twisted beta sheet flanked by α‐helices consistent with a canonical vWA fold. A restricted basic patch serves as the putative heparin binding site, but no heparin binding was detected in native gel shift assays. Furthermore, no metal was observed in the metal ion dependent adhesion site (MIDAS). Structural overlays with homologous A/I domains reveal a divergent organization of the MIDAS β4–α4 loop in TgMIC2A/Ic, which is stabilized through the burial of Phe195 into a deep pocket formed by Gly185. Intriguingly, Gly185 appears to be unique among A/I domains to TgMIC2A/I suggesting that the divergent loop conformation may also be unique to TgMIC2A/I. Although lacking the C‐terminal extension, the TgMIC2A/Ic structure reported here is the first of an A/I domain from an apicomplexan parasite and provides valuable insight into defining the molecular recognition of host cells by these widespread pathogens.     

Toxoplasma gondii protease TgSUB1 is required for cell surface processing of micronemal adhesive complexes and efficient adhesion of tachyzoites

Host cell invasion by Toxoplasma gondii is critically dependent upon adhesive proteins secreted from the micronemes. Proteolytic trimming of microneme contents occurs rapidly after their secretion onto the parasite surface and is proposed to regulate adhesive complex activation to enhance binding to host cell receptors. However, the proteases responsible and their exact function are still unknown. In this report, we show that T. gondii tachyzoites lacking the microneme subtilisin protease TgSUB1 have a profound defect in surface processing of secreted microneme proteins. Notably parasites lack protease activity responsible for proteolytic trimming of MIC2, MIC4 and M2AP after release onto the parasite surface. Although complementation with full‐length TgSUB1 restores processing, complementation of Δsub1 parasites with TgSUB1 lacking the GPI anchor (Δsub1::ΔGPISUB1) only partially restores microneme protein processing. Loss of TgSUB1 decreases cell attachment and in vitro gliding efficiency leading to lower initial rates of invasion. Δsub1 and Δsub1::ΔGPISUB1 parasites are also less virulent in mice. Thus TgSUB1 is involved in micronemal protein processing and regulation of adhesive properties of macromolecular adhesive complexes involved in host cell invasion.     

The rhoptry neck protein RON4 re-localizes at the moving junction during Toxoplasma gondii invasion

Host cell invasion in the Apicomplexa is unique in its dependency on a parasite actin-driven machinery and in the exclusion of most host cell membrane proteins during parasitophorous vacuole (PV) formation. This exclusion occurs at a junction between host cell and parasite plasma membranes that has been called the moving junction, a circumferential zone which forms at the apical tip of the parasite, moves backward and eventually pinches the PV from the host cell membrane. Despite having been described by electron microscopic studies 30 years ago, the molecular nature of this singular structure is still enigmatic. We have obtained a monoclonal antibody that recognizes the moving junction of invading tachyzoites of Toxoplasma gondii, in a pattern clearly distinct from those described so far for microneme and rhoptry proteins. The protein recognized by this antibody has been affinity purified. Mass spectrometry analysis showed that it is a rhoptry neck protein (RON4), a hypothetical protein with homologues restricted to Apicomplexa. Our findings reveals for the first time the participation of rhoptry neck proteins in moving junction formation and strongly suggest the conservation of this structure at the molecular level among Apicomplexa.     

Role of calcium during Toxoplasma gondii invasion and egress

Calcium is a ubiquitous signalling molecule involved in a large number of cellular processes in eukaryotic cells. In the obligate intracellular parasite, Toxoplasma gondii, for example, a rise in calcium concentration is associated with significant morphological changes, secretion of proteins involved in host cell invasion and rapid egress from the host cell. Recent findings indicate that calcium released from the parasite's intracellular pools is necessary and sufficient to induce some of the events critical for invasion and egress. In addition, ethanol, a powerful inducer of invasion-related events, is shown here to also induce rapid egress from the host cell, indicating that a common mechanism for calcium release might be involved during both invasion and egress.     

Functional analysis of Toxoplasma gondii protease inhibitor 1

We have characterized a Kazal family serine protease inhibitor, Toxoplasma gondii protease inhibitor 1 (TgPI-1), in the obligate intracellular parasite Toxoplasma gondii. TgPI-1 contains four inhibitor domains predicted to inhibit trypsin, chymotrypsin, and elastase. Antibodies against recombinant TgPI-1 detect two polypeptides, of 43 and 41 kDa, designated TgPI-1(43) and TgPI-1(41), in tachyzoites, bradyzoites, and sporozoites. TgPI-1(43) and TgPI-1(41) are secreted constitutively from dense granules into the excreted/secreted antigen fraction as well as the parasitophorous vacuole that T. gondii occupies during intracellular replication. Recombinant TgPI-1 inhibits trypsin, chymotrypsin, pancreatic elastase, and neutrophil elastase. Immunoprecipitation studies with anti-rTgPI-1 antibodies reveal that recombinant TgPI-1 forms a complex with trypsin that is dependent on interactions with the active site of the protease. TgPI-1 is the first anti-trypsin/chymotrypsin inhibitor to be identified in bradyzoites and sporozoites, stages of the parasite that would be exposed to proteolytic enzymes in the digestive tract of the host.     

Secretion from the rhoptries of Toxoplasma gondii during host-cell invasion

To determine whether the rhoptries of Toxoplasma gondii play a role in the invasion of host cells by this parasite, we inoculated toxoplasmas into the peritoneal cavities of normal mice and into macrophage cultures, fixed the specimens at various intervals thereafter, and analyzed them by electron microscopy. We found that during host-cell invasion, the rhoptry membrane fused with the anterior limiting membrane of the toxoplasma, producing an opening to the exterior. Since such openings were formed when the host-cell membrane was disrupted, it appears that the rhoptries may secrete a lytic product that facilitates invasion through the host-cell membrane. Such a "penetration-enhancing factor" was previously isolated from lysed toxoplasmas (Lycke and Norrby, 1966). Occasionally, when secretion was incomplete, masses of tubules were found in the rhoptries, sometimes as soon as 15 sec after the toxoplasms had been injected into mice. Similar tubules were found in the parasitophorous vacuole that was formed 10-15 min later, and such tubules are typical of vacuoles containing replicating parasites. Because these tubules are in continuity with the vacuolar membrane, it appears to be a hybrid membrane, composed in part of toxoplasma products. We speculate that the hybrid nature of the vacuolar membrane prevents it from fusing with the lysosomes of phagocytes and thereby contributes to the intracellular survival of the parasites.     

Signaling during the invasion of host cells by Toxoplasma gondii

Invasion of host cells is essential for the pathogenicity of Toxoplasma gondii. This review examines the signal transduction pathways that lead to the internalization of T. gondii. We demonstrate that extra- and intracellular Ca(2+) mobilization, Ca(2+)-calmodulin complex and phospholipase A(2) activities are required for T. gondii entry. T. gondii also causes the activation of mitogen-activated protein kinase in infected cells and modifies its ionic environment during its intracellular state. Thus, many of the signaling systems found in other eukaryotes are operative in Toxoplasma invasion.     

A novel galectin-like domain from Toxoplasma gondii micronemal protein 1 assists the folding, assembly, and transport of a cell adhesion complex

Immediately prior to invasion Toxoplasma gondii tachyzoites release a large number of micronemal proteins (TgMICs) that participate in host cell attachment and penetration. The TgMIC4-MIC1-MIC6 complex was the first to be identified in T. gondii and has been recently shown to be critical in invasion. This study establishes that the N-terminal thrombospondin type I repeat-like domains (TSR1-like) from TgMIC1 function as an independent adhesin as well as promoting association with TgMIC4. Using the newly solved three-dimensional structure of the C-terminal domain of TgMIC1 we have identified a novel Galectin-like fold that does not possess carbohydrate binding properties and redefines the architecture of TgMIC1. Instead, the TgMIC1 Galectin-like domain interacts and stabilizes TgMIC6, which provides the basis for a highly specific quality control mechanism for successful exit from the early secretory compartments and for subsequent trafficking of the complex to the micronemes.     

A Toxoplasma gondii leucine-rich repeat protein binds phosphatase type 1 protein and negatively regulates its activity

We have characterized the Toxoplasma gondii protein phosphatase type 1 (TgPP1) and a potential regulatory binding protein belonging to the leucine-rich repeat protein family, designated TgLRR1. TgLRR1 is capable of binding to TgPP1 to inhibit its activity and to override a G(2)/M cell cycle checkpoint in Xenopus oocytes. In the parasite, TgLRR1 mRNA and protein are both highly expressed in the rapidly replicating and virulent tachyzoites, while only low levels are detected in the slowly dividing and quiescent bradyzoites. TgPP1 mRNA and protein levels are equally abundant in tachyzoites and bradyzoites. Affinity pull down and immunoprecipitation experiments reveal that the TgLRR1-TgPP1 interaction takes place in the nuclear subcompartment of tachyzoites. These results are consistent with those of localization studies using both indirect immunofluorescence with specific polyclonal antibody and transient transfection of T. gondii vector expressing TgLRR1 and TgPP1. The inability to obtain stable transgenic tachyzoites suggested that overexpression of TgLRR1 and TgPP1 may impair the parasite's growth. Together with the activation of Xenopus oocyte meiosis reinitiation, these data indicate that TgLRR1 protein could play a role in the regulation of the T. gondii cell cycle through the modulation of phosphatase activity.     

Activation of the cellular mitogen-activated protein kinase pathways ERK,P38 and JNK during Toxoplasma gondii invasion

Host cell invasion is essential for the pathogenicity of the obligate intracellular protozoan parasite Toxoplasma gondii. In the present study, we evaluated the ability of T. gondii tachyzoites to trigger phosphorylation of the different mitogen-activated protein kinases (MAPK) in human monocytic cells THP1. Kinetic experiments show that the peak of extracellular-signal-regulated kinase (ERK1/2), P38 and cjun-NH2 terminal kinase (JNKs) phosphorylation occurs between 10 and 60 min. The use of specific inhibitors of ERK1/2, P38 and JNK1/2 phosphorylation indicates the specificity of MAPKs phosphorylation during invasion. Signaling through cellular and parasite mitogen-activated protein (MAP) kinase pathways appears to be critical for T. gondii invasion.     

A role for Toxoplasma gondii type 1 ser/thr protein phosphatase in host cell invasion

Host cell invasion by Toxoplasma gondii tachyzoites relies on many coordinated processes. The tachyzoite participates in invasion by providing an actomyosin-dependent force driving it into the nascent parasitophorous vacuole as well as by releasing molecules which contribute to the vacuole membrane. Exposure to type 1/2A protein phosphatase inhibitors, okadaic acid (OA) or tautomycin significantly impairs tachyzoite invasiveness. Furthermore, the tachyzoite extract contains a biochemically active type 1, but not a type 2A, serine-threonine protein phosphatase, which is immunologically related to eukaryotic phosphatase type 1 catalytic subunit. When tachyzoite extracts are incubated with a monoclonal antibody reactive to human type 1 catalytic subunit, other T. gondii molecules are coprecipitated among which one competes with the inhibitory toxin OA. Finally, in vitro phosphate labelling assays indicate that the biochemically characterized PP1 activity controls the phosphorylation of several proteins. Taken together, these data strongly suggest that the type 1 phosphatase activity detected in invasive tachyzoites is implicated in the control of the host cell invasion process.     

Attachment and invasion of host cells by Toxoplasma gondii

Recent studies indicate that Toxoplasma gondii attachment is mediated via a parasite ligand-host cell receptor interaction. Lloyd Kosper and Jose Mineo here survey factors involved in the attachment to and penetration and invasion of host cells by T. gondii.     

Toxoplasma gondii: redistribution of monoclonal antibodies on tachyzoites during host cell invasion

Immunodetection of protein P30, a major surface antigen of Toxoplasma gondii tachyzoites, by a specific monoclonal antibody has demonstrated a homogenous distribution of this antigen on the surface of intra- and extracellular tachyzoites at all stages of their endodyogenic development. On living zoites, no redistribution of anti-P30 was obtained, contrasting with the capping obtained with antiserum to T. gondii. Upon invasion of a host cell, however, most of the coat of anti-P30 was shed from preincubated zoites at the level of the moving junction governing the entry of the parasite into the host cell.     

CCR5 Controls Immune and Metabolic Functions during Toxoplasma gondii Infection

CCR5, an important receptor related to cell recruitment and inflammation, is expressed during experimental Toxoplasma gondii infection. However, its role in the immunopathology of toxoplasmosis is not clearly defined yet. Thus, we inoculated WT and CCR5^-/- mice with a sub lethal dose of the parasite by oral route. CCR5^-/- mice were extremely susceptible to infection, presenting higher parasite load and lower tissue expression of IL-12p40, IFN-γ, TNF, IL-6, iNOS, Foxp3, T-bet, GATA-3 and PPARα. Although both groups presented inflammation in the liver with prominent neutrophil infiltration, CCR5^-/- mice had extensive tissue damage with hepatocyte vacuolization, steatosis, elevated serum triglycerides and transaminases. PPARα agonist Gemfibrozil improved the vacuolization but did not rescue CCR5^-/- infected mice from high serum triglycerides levels and enhanced mortality. We also found intense inflammation in the ileum of CCR5^-/- infected mice, with epithelial ulceration, augmented CD4 and decreased frequency of NK cells in the gut lamina propria. Most interestingly, these findings were accompanied by an outstanding accumulation of neutrophils in the ileum, which seemed to be involved in the gut immunopathology, once the depletion of these cells was accompanied by reduced local damage. Altogether, these data demonstrated that CCR5 is essential to the control of T. gondii infection and to maintain the metabolic, hepatic and intestinal integrity. These findings add novel information on the disease pathogenesis and may be relevant for directing future approaches to the treatment of multi-deregulated diseases.     

The cathepsin B of Toxoplasma gondii,toxopain-1, is critical for parasite invasion and rhoptry protein processing

Cysteine proteinases play a major role in invasion and intracellular survival of a number of pathogenic parasites. We cloned a single copy gene, tgcp1, from Toxoplasma gondii and refolded recombinant enzyme to yield active proteinase. Substrate specificity of the enzyme and homology modeling identified the proteinase as a cathepsin B. Specific cysteine proteinase inhibitors interrupted invasion by tachyzoites. The T. gondii cathepsin B localized to rhoptries, secretory organelles required for parasite invasion into cells. Processing of the pro-rhoptry protein 2 to mature rhoptry proteins was delayed by incubation of extracellular parasites with a cathepsin B inhibitor prior to pulse-chase immunoprecipitation. Delivery of cathepsin B to mature rhoptries was impaired in organisms with disruptions in rhoptry formation by expression of a dominant negative micro1-adaptin. Similar disruption of rhoptry formation was observed when infected fibroblasts were treated with a specific inhibitor of cathepsin B, generating small and poorly developed rhoptries. This first evidence for localization of a cysteine proteinase to the unusual rhoptry secretory organelle of an apicomplexan parasite suggests that the rhoptries may be a prototype of a lysosome-related organelle and provides a critical link between cysteine proteinases and parasite invasion for this class of organism.     

Toxoplasma gondii: role of the phosphatidylcholine-specific phospholipase C during cell invasion and intracellular development

The effect of D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C, was investigated on cyst development of the Prugniaud strain of Toxoplasma gondii in vitro. Following treatment with the inhibitor 24 h after cell infection, cyst development was affected as assessed by staining with the bradyzoite-specific mAb CC2: the CC2-reactive antigen was shown to be differently located (in the wall versus the matrix under control conditions). This correlated with a decrease in parasite multiplication induced by D609. Pretreatment of the parasites with D609 inhibited their entry into the host cells, whereas pretreatment of the host cells enhanced the intracellular multiplication of the para sites, without any effect on cell invasion or cyst formation. Our results suggest a crucial role for phosphatidylcholine-specific phospholipase C in the pathophysiology of toxoplasmosis.     

A family of aspartic proteases and a novel, dynamic and cell-cycle-dependent protease localization in the secretory pathway of Toxoplasma gondii

Aspartic proteases are important virulence factors in pathogens like HIV, Candida albicans or Plasmodium falciparum . We report here the identification of seven putative aspartic proteases, TgASP1 to TgASP7, in the apicomplexan parasite Toxoplasma gondii . Bioinformatic and phylogenetic analysis of the TgASPs and other aspartic proteases from related Apicomplexa suggests the existence of five distinct groups of aspartic proteases with different evolutionary lineages. The members of each group share predicted biological features that validate the phylogeny. TgASP1 is expressed in tachyzoites, the rapidly dividing asexual stage of T. gondii . We present the proteolytic maturation and subcellular localization of this protease through the cell cycle. TgASP1 shows a novel punctate localization associated with the secretory system in non-dividing cells, and relocalizes dramatically and unambiguously to the nascent inner membrane complex of daughter cells at replication, before coalescing again at the end of division.