Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole

The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear, as the PV avoids fusion with host organelles. In this study, we explore the host Golgi–PV interaction and evaluate the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous ministacks, which surround the PV, and hijacks host Golgi–derived vesicles within the PV. These vesicles, marked with Rab14, Rab30, or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite''s growth. Thus our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles.     

Peculiarities of host cholesterol transport to the unique intracellular vacuole containing Toxoplasma

The intracellular protozoan Toxoplasma gondii is auxotrophic for low-density lipoprotein (LDL)-derived cholesterol (C). We previously showed that T. gondii scavenges this essential lipid from host endolysosomal compartments and that C delivery to the parasitophorous vacuole (PV) does not require transit through host Golgi or endoplasmic reticulum. In this study, we explore the itinerary of C from the host endolysosomes to the PV. Labeled C incorporated into LDL is rapidly detected in intravacuolar parasites and partially esterified by the parasites. In contrast to diverse mammalian organelles, the post-endolysosomal transfer of C to the PV does not involve the host plasma membrane as an intermediate. Nevertheless, the PV membrane is accessible to extracellular sterol acceptors, suggesting C trafficking from intracellular parasites to host plasma membrane. C movement to the PV requires temperatures permissive for vesicular transport, metabolic energy and functional microtubules. Host caveolae vesicles and the sterol carrier protein-2 do not participate in this process. Proteolytic treatment of purified PV or free parasites abolishes C acquisition by the parasites. Altogether, these results support a vesicular transport system from host endolysosomes to the PV, and a requirement for PV membrane and parasite plasma membrane proteins in C delivery to T. gondii.     

Toxoplasma gondii manipulates host cell signaling pathways via its secreted effector molecules

The obligate intracellular parasite Toxoplasma gondii secretes a vast variety of effector molecules from organelles known as rhoptries (ROPs) and dense granules (GRAs). ROP proteins are released into the cytosol of the host cell where they are directed to the cell nucleus or to the parasitophorous vacuole (PV) membrane. ROPs secrete proteins that enable host cell penetration and vacuole formation by the parasites, as well as hijacking host-immune responses. After invading host cells, T. gondii multiplies within a PV that is maintained by the parasite proteins secreted from GRAs. Most GRA proteins remain within the PV, but some are known to access the host cytosol across the PV membrane, and a few are able to traffic into the host-cell nucleus. These effectors bind to host cell proteins and affect host cell signaling pathways to favor the parasite. Studies on host–pathogen interactions have identified many infection-altered host signal transductions. Notably, the relationship between individual parasite effector molecules and the specific targeting of host-signaling pathways is being elucidated through the advent of forward and reverse genetic strategies. Understanding the complex nature of the host–pathogen interactions underlying how the host-signaling pathway is manipulated by parasite effectors may lead to new molecular biological knowledge and novel therapeutic methods for toxoplasmosis. In this review, we discuss how T. gondii modulates cell signaling pathways in the host to favor its survival.     

Rabbit antibodies against Toxoplasma Hsp20 are able to reduce parasite invasion and gliding motility in Toxoplasma gondii and parasite invasion in Neospora caninum

Toxoplasma gondii Hsp20 is a pellicle-associated functional chaperone whose biological role is still unknown. Hsp20 is present in different apicomplexan parasites, showing a high degree of conservation across the phylum, with Neospora caninum Hsp20 presenting an 82% identity to that of T. gondii. Hence rabbit anti-T. gondii Hsp20 serum was able to recognize the N. caninum counterpart. Interestingly, both N. caninum and T. gondii Hsp20 localized to the inner membrane complex and to the plasma membrane. Incubation of T. gondii and N. caninum tachyzoites with an anti-TgHsp20 serum reduced parasite invasion at rates of 57.23% and 54.7%, respectively. This anti-serum also reduced T. gondii gliding 48.7%. Together, all this data support a role for Hsp20 in parasite invasion and gliding motility.     

Antibody reaction of human anti-Toxoplasma gondii positive and negative sera with Neospora caninum antigens

Anti-Neospora caninum antibody was detected in anti-Toxoplasma gondii positive and negative human sera by ELISA, western blot and immunofluorescence assay (IFA). Twelve cases out of 172 (6.7%) Toxoplasma-positive sera cross-reacted with both T. gondii and N. caninum antigens, and one out of 110 Toxoplasma-negative sera reacted with N. caninum antigen by ELISA. By western blot, all 12 sera reacted with T. gondii antigens with various banding patterns but specifically at 30 kDa (SAG1) and 22 kDa (SAG2) bands. With N. caninum antigen, the number of reactive bands was reduced, however a 43 kDa band reacted in three cases in Toxoplasma-positive sera in addition to one in Toxoplasma-negative control sera. All sera of the Toxoplasma-positive group labeled surface membrane of T. gondii, but reacted differently with N. caninum. Fluorescence was detected in surface membrane, subcellular organelles, or both in N. caninum. And one case in the Toxoplasma-negative group also reacted with N. caninum strongly in subcellular organelles. This suggested that the antibody against N. caninum may be present in human sera although the positive rate was very low in this study. The possibility of human infection with N. caninum remains to be evaluated further.     

Vesicles Bearing Toxoplasma Apicoplast Membrane Proteins Persist Following Loss of the Relict Plastid or Golgi Body Disruption

Toxoplasma gondii and malaria parasites contain a unique and essential relict plastid called the apicoplast. Most apicoplast proteins are encoded in the nucleus and are transported to the organelle via the endoplasmic reticulum (ER). Three trafficking routes have been proposed for apicoplast membrane proteins: (i) vesicular trafficking from the ER to the Golgi and then to the apicoplast, (ii) contiguity between the ER membrane and the apicoplast allowing direct flow of proteins, and (iii) vesicular transport directly from the ER to the apicoplast. Previously, we identified a set of membrane proteins of the T. gondii apicoplast which were also detected in large vesicles near the organelle. Data presented here show that the large vesicles bearing apicoplast membrane proteins are not the major carriers of luminal proteins. The vesicles continue to appear in parasites which have lost their plastid due to mis-segregation, indicating that the vesicles are not derived from the apicoplast. To test for a role of the Golgi body in vesicle formation, parasites were treated with brefeldin A or transiently transfected with a dominant-negative mutant of Sar1, a GTPase required for ER to Golgi trafficking. The immunofluorescence patterns showed little change. These findings were confirmed using stable transfectants, which expressed the toxic dominant-negative sar1 following Cre-loxP mediated promoter juxtaposition. Our data support the hypothesis that the large vesicles do not mediate the trafficking of luminal proteins to the apicoplast. The results further show that the large vesicles bearing apicoplast membrane proteins continue to be observed in the absence of Golgi and plastid function. These data raise the possibility that the apicoplast proteome is generated by two novel ER to plastid trafficking pathways, plus the small set of proteins encoded by the apicoplast genome.     

Fierce Competition between Toxoplasma and Chlamydia for Host Cell Structures in Dually Infected Cells

The prokaryote Chlamydia trachomatis and the protozoan Toxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similar modus operandi to colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected by C. trachomatis and T. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and that Toxoplasma holds a significant competitive advantage over Chlamydia. Here we have expanded our coinfection studies by examining the respective abilities of Chlamydia and Toxoplasma to co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However, Toxoplasma outcompetes Chlamydia to the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected with Chlamydia, the centrosomes become associated with the chlamydial inclusion, while the Toxoplasma parasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by both Chlamydia and Toxoplasma depends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen''s benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients.     

3D reconstruction of Trypanosoma cruzi-macrophage interaction shows the recruitment of host cell organelles towards parasitophorous vacuoles during its biogenesis

Trypanosoma cruzi has a complex life cycle where two infective developmental stages, known as trypomastigote and amastigote, can be found in the vertebrate host. Both forms can invade a large variety of cellular types and induce the formation of a parasitophorous vacuole (PV), that, posteriorly, disassembles and releases the parasites into the host cell cytoplasm. The biogenesis of T. cruzi PVs has not been analyzed in professional phagocytic cells. We investigated the biogenesis of PVs containing trypomastigotes or amastigotes in peritoneal macrophages. We observed the presence of profiles of the endoplasmic reticulum and lysosomes from the host cell near PVs at early stages of interaction in both developmental stages, suggesting that both organelles may participate as possible membrane donors for the formation of the PVs. The Golgi complex, however, was observed only near already formed PVs. Electron microscopy tomography and FIB-SEM microscopy followed by 3D reconstruction of entire PVs containing amastigotes or trypomastigotes confirmed the presence of both endoplasmic reticulum and lysosomes in the initial stages of PV formation. In addition, Golgi complex and mitochondria localize around PVs during their biogenesis. Taken together these observations provide a whole view of the invasion process in a professional phagocytic cell.     

Silencing of GRA10 protein expression inhibits Toxoplasma gondii intracellular growth and development

Toxoplasma gondii dense granule proteins (GRAs) are secreted abundantly in both the tachyzoite and bradyzoite stages of the parasite and are known to localize to various compartments of the parasitophorous vacuole (PV) that interfaces with the host cell milieu. Thus, GRAs may play significant roles in the biogenesis of the PV that is important for survival of intracellular T. gondii. GRA10 is a dense granule protein whose role in T. gondii has not yet been characterized. Therefore, in this study, we endeavored to determine the role of GRA10 in the growth and survival of intracellular T. gondii by using phosphorodiamidate morpholino oligomers (PPMOs) antisense knockdown approach to disrupt the translation of GRA10 mRNA in the parasites. We expressed and purified a truncated recombinant GRA10 protein to generate anti-GRA10 polyclonal antibodies that we used to characterize GRA10 in T. gondii. We found that GRA10 is a soluble, dense granule-associated protein that is secreted into the parasite cytosol and the parasitophorous vacuole milieu. Using in vitro cultures, we found that knockdown of GRA10 results in severe inhibition of T. gondii growth in human fibroblasts and in ovine monocytic cells. Together, our findings define GRA10 as a dense granule protein that plays a significant role in the growth and propagation of intracellular T. gondii in human fibroblasts and in ovine monocytic cells.     

Exploitation of auxotrophies and metabolic defects in Toxoplasma as therapeutic approaches

Like any obligate intracellular pathogen, the parasite Toxoplasma gondii has lost its capacity for living independently of another organism. Toxoplasma lacks many genes that encode for entire metabolic pathways and has, in return, expanded genes that promote nutrient scavenging to meet its basic metabolic requirements. Although sequestrated in a parasitophorous vacuole and thus insulated from the nutrient-rich host cytosol and organelles by a membrane, T. gondii has evolved efficient strategies to acquire essential metabolites from mammalian cells. This review explores the natural auxotrophies and nutrient scavenging activities of the parasite, emphasising unique transport systems and salvage pathways. We describe the mechanisms deployed by Toxoplasma to modify its parasitophorous vacuole to gain access to host cytosolic molecules and to hijack host organelles to retrieve their nutrient content. From a therapeutic perspective, we survey the different possibilities to starve T. gondii by nutrient depletion or disruption of salvage pathways.     

Toxoplasma gondii Vps11, a subunit of HOPS and CORVET tethering complexes,is essential for the biogenesis of secretory organelles

Apicomplexan parasites harbour unique secretory organelles (dense granules, rhoptries and micronemes) that play essential functions in host infection. Toxoplasma gondii parasites seem to possess an atypical endosome‐like compartment, which contains an assortment of proteins that appear to be involved in vesicular sorting and trafficking towards secretory organelles. Recent studies highlighted the essential roles of many regulators such as Rab5A, Rab5C, sortilin‐like receptor and syntaxin‐6 in secretory organelle biogenesis. However, little is known about the protein complexes that recruit Rab‐GTPases and SNAREs for membrane tethering in Apicomplexa. In mammals and yeast, transport, tethering and fusion of vesicles from early endosomes to lysosomes and the vacuole, respectively, are mediated by CORVET and HOPS complexes, both built on the same Vps‐C core that includes Vps11 protein. Here, we show that a T. gondii Vps11 orthologue is essential for the biogenesis or proper subcellular localization of secretory organelle proteins. TgVps11 is a dynamic protein that associates with Golgi endosomal‐related compartments, the vacuole and immature apical secretory organelles. Conditional knock‐down of TgVps11 disrupts biogenesis of dense granules, rhoptries and micronemes. As a consequence, parasite motility, invasion, egress and intracellular growth are affected. This phenotype was confirmed with additional knock‐down mutants of the HOPS complex. In conclusion, we show that apicomplexan parasites use canonical regulators of the endolysosome system to accomplish essential parasite‐specific functions in the biogenesis of their unique secretory organelles.     

Development of competitive ELISA for neosporosis by employing immunoproteomics

In this study, proteomics was used to explore the antigenic proteins that are involved in cross-reactivity during serodiagnosis between Neospora caninum (N. caninum) and Toxoplasma gondii (T. gondii). Competitive enzyme-linked immunosorbent assay (C-ELISA) developed by proteomics shed a new light on the infection of N. caninum. Cross-reactivity of antigenic proteins between N. caninum and T. gondii tachyzoites was explored by using the conventional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (1-DE) and two-dimensional gel electrophoresis (2-DE) immunoblot. The proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The protein expression patterns in the immunoblot profiles of N. caninum were similar to bovine, chicken, and rabbit anti-N. caninum serum, but they were not similar to rabbit anti-T. gondii serum. Band at 79 kDa, HSP70, and actin on immunoblot profiles reacted, in general, with bovine, chicken, and rabbit anti-N. caninum serum and also with rabbit anti-T. gondii serum, respectively. Whereas the band at 144 kDa, and NCDG-1 were detected on bovine, chicken, and rabbit anti-N. caninum immunoblot profiles, they were not observed on rabbit anti-T. gondii immunoblot profile. These specific antigenic proteins were recorded as species-specific proteins of N. caninum against T. gondii. Based on the proteome analysis, C-ELISA was developed to screen the cattle infected with N. caninum by using N. caninum tachyzoite lysate as a coating antigen and chicken anti-N. caninum immunoglobulin (Ig)Y as a competitor. C-ELISA was able to detect the antibody of N. caninum without cross-reactivity with T. gondii. Furthermore, it achieved a fine diagnostic performance in the cases of 162 bovine sera.     

TgGRA23, a novel Toxoplasma gondii dense granule protein associated with the parasitophorous vacuole membrane and intravacuolar network

Toxoplasma gondii is an intracellular protozoan parasite, which relies on a specialized compartment, the parasitophorous vacuole (PV), to survive within host cells. Dense granules within the parasite release a large variety of proteins to maintain the integrity of the vacuole structure. Here, we identified a novel dense granule protein in T. gondii, TgGRA23, which is a homolog of the Sarcocystis muris dense granule protein, SmDG32. Recombinant TgGRA23 (rTgGRA23) expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein was used to raise antisera in mice and rabbits. Immunoblotting showed that antisera from the immunized mice and rabbits reacted with parasite lysates to yield a 21-kDa native protein. In addition, immuno-electron microscopic examination showed that TgGRA23 resides in the dense granules, PV membrane and intravacuolar network of the parasite. To confirm the precise subcellular localization of TgGRA23 in T. gondii, an immunofluorescent antibody test was performed using dense granule markers. Notably, TgGRA23 co-localized with other dense granule proteins including TgGRA4 and TgGRA7, in the extracellular-stage parasites. Biochemical experiments indicated that TgGRA23 is insoluble and may form an electrostatic complex that is resistant to non-ionic detergents. Furthermore, specific antibodies to TgGRA23 were detected during the chronic stage of Toxoplasma infection in mice. Our results suggest that TgGRA23 is an as yet unknown member of the T. gondii dense granule proteins, and that it may be involved in remodeling or maintenance of the PV.     

Toxoplasma gondii exploits the host ESCRT machinery for parasite uptake of host cytosolic proteins

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole (PV), the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the host Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV-1 virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV-1 Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of TgGRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PV membrane (PVM). These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission.     

Serological evidence of Toxoplasma gondii and Neospora caninum infection in black-boned sheep and goats in southwest China

Toxoplasma gondii and Neospora caninum are two closely related protozoan parasites which can cause abortion and significant economic losses in sheep and goats. However, it is yet to know whether black-bone sheep and goats are infected with T. gondii and N. caninum in China. In the present investigation, the seroprevalence and risk factors of T. gondii and N. caninum infections in black-boned sheep and goats were investigated in Yunnan Province, subtropical southwest China between July and August of 2017. A total of 481 serum samples were tested for T. gondii antibodies using the Modified Agglutination Test (MAT), and 468 serum samples were examined for N. caninum antibodies by indirect Enzyme-Linked Immunosorbent Assay (iELISA). The overall seroprevalence of T. gondii in black-boned sheep and goats was 36.80% (177/481, 95% CI 32.49–41.11), and 40 out of 468 serum samples were N. caninum-seropositive (8.55%, 95% CI 6.02–11.08). There was significant difference in the seroprevalence of T. gondii infection in different regions (χ² = 19.869, df = 2, P<0.01). As for the seroprevalence of N. caninum infection, region (χ² = 8.558, df = 2, P<0.05), age (χ² = 16.631, df = 3, P < 0.01), gender (χ² = 11.219, df = 1, P < 0.01) and species (χ² = 8.673, df = 1, P < 0.01) were the risk factors. In addition, the seroprevalence of coinfection of T. gondii and N. caninum in black-boned sheep and goats was 3.63% (17/468, 95% CI 1.94–5.32). To our knowledge, this is the first report of T. gondii and N. caninum seroprevalence in black-boned sheep and goats in China, which provided base-line data for the execution of control strategies and measures against T. gondii and N. caninum infection in black-boned sheep and goats.     

ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP₂ and PIP₃ to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.     

Serosurvey of Toxoplasma gondii,Neospora caninum and Sarcocystis neurona in raptors and risk factor analysis

Raptors are carnivorous birds with great hunting ability. Toxoplasma gondii, Neospora caninum and Sarcocystis spp. are intracellular Apicomplexan protozoans which infect a wide range of intermediate hosts, including birds. The aims of this study were to evaluate the serological reactivity of captive raptors serum to T. gondii, N. caninum and S. neurona antigens and identify possible risk factors associated with the infection. From August 2014 to September 2015, blood samples from 72 raptors were collected and serum samples were tested by immunofluorescence antibody test (IFAT). Antigen slides were prepared using tachyzoites of T. gondii and N. caninum and using merozoites of S. neurona. Serum samples were tested at the following cut-off dilutions: 1:16 for T. gondii and 1:50 for N. caninum and S. neurona. An anti-chicken IgY antibody conjugated with FITC was used as a secondary antibody at 1:50 dilution. Out of the 72 raptors serum tested by IFAT, 2.7% reacted to N. caninum, 8.3% to T. gondii and 11.1% to S. neurona antigens. The region in which the sample was collected, the reason the raptors were kept in captivity and diet were statistically associated with seropositivity to T. gondii and the use of the birds and diet were statistically associated with seropositivity to N. caninum and S. neurona (p ≤ 0.05). We highlight the occurrence of these protozoans in birds of prey and the importance of good hygiene and feeding management of these birds in captivity to reduce the risk of protozoal infections.     

Toxoplasma gondii-Induced Activation of EGFR Prevents Autophagy Protein-Mediated Killing of the Parasite

Toxoplasma gondii resides in an intracellular compartment (parasitophorous vacuole) that excludes transmembrane molecules required for endosome - lysosome recruitment. Thus, the parasite survives by avoiding lysosomal degradation. However, autophagy can re-route the parasitophorous vacuole to the lysosomes and cause parasite killing. This raises the possibility that T. gondii may deploy a strategy to prevent autophagic targeting to maintain the non-fusogenic nature of the vacuole. We report that T. gondii activated EGFR in endothelial cells, retinal pigment epithelial cells and microglia. Blockade of EGFR or its downstream molecule, Akt, caused targeting of the parasite by LC3⁺ structures, vacuole-lysosomal fusion, lysosomal degradation and killing of the parasite that were dependent on the autophagy proteins Atg7 and Beclin 1. Disassembly of GPCR or inhibition of metalloproteinases did not prevent EGFR-Akt activation. T. gondii micronemal proteins (MICs) containing EGF domains (EGF-MICs; MIC3 and MIC6) appeared to promote EGFR activation. Parasites defective in EGF-MICs (MIC1 ko, deficient in MIC1 and secretion of MIC6; MIC3 ko, deficient in MIC3; and MIC1-3 ko, deficient in MIC1, MIC3 and secretion of MIC6) caused impaired EGFR-Akt activation and recombinant EGF-MICs (MIC3 and MIC6) caused EGFR-Akt activation. In cells treated with autophagy stimulators (CD154, rapamycin) EGFR signaling inhibited LC3 accumulation around the parasite. Moreover, increased LC3 accumulation and parasite killing were noted in CD154-activated cells infected with MIC1-3 ko parasites. Finally, recombinant MIC3 and MIC6 inhibited parasite killing triggered by CD154 particularly against MIC1-3 ko parasites. Thus, our findings identified EGFR activation as a strategy used by T. gondii to maintain the non-fusogenic nature of the parasitophorous vacuole and suggest that EGF-MICs have a novel role in affecting signaling in host cells to promote parasite survival.     

Members of a Novel Protein Family Containing Microneme Adhesive Repeat Domains Act as Sialic Acid-binding Lectins during Host Cell Invasion by Apicomplexan Parasites

Numerous intracellular pathogens exploit cell surface glycoconjugates for host cell recognition and entry. Unlike bacteria and viruses, Toxoplasma gondii and other parasites of the phylum Apicomplexa actively invade host cells, and this process critically depends on adhesins (microneme proteins) released onto the parasite surface from intracellular organelles called micronemes (MIC). The microneme adhesive repeat (MAR) domain of T. gondii MIC1 (TgMIC1) recognizes sialic acid (Sia), a key determinant on the host cell surface for invasion by this pathogen. By complementation and invasion assays, we demonstrate that TgMIC1 is one important player in Sia-dependent invasion and that another novel Sia-binding lectin, designated TgMIC13, is also involved. Using BLAST searches, we identify a family of MAR-containing proteins in enteroparasitic coccidians, a subclass of apicomplexans, including T. gondii, suggesting that all these parasites exploit sialylated glycoconjugates on host cells as determinants for enteric invasion. Furthermore, this protein family might provide a basis for the broad host cell range observed for coccidians that form tissue cysts during chronic infection. Carbohydrate microarray analyses, corroborated by structural considerations, show that TgMIC13, TgMIC1, and its homologue Neospora caninum MIC1 (NcMIC1) share a preference for α2–3- over α2–6-linked sialyl-N-acetyllactosamine sequences. However, the three lectins also display differences in binding preferences. Intense binding of TgMIC13 to α2–9-linked disialyl sequence reported on embryonal cells and relatively strong binding to 4-O-acetylated-Sia found on gut epithelium and binding of NcMIC1 to 6′sulfo-sialyl Lewis^x might have implications for tissue tropism.