Inhibition of Nitric Oxide Production in Activated Macrophages Caused by Toxoplasma gondii Infection Occurs by Distinct Mechanisms in Different Mouse Macrophage Cell Lines.

Toxoplasma gondii, the causative agent of toxoplasmosis, is a widespread intracellular parasite able to infect virtually any nucleated cell. T. gondii infection of activated macrophages inhibits nitric oxide (NO) production; however, parasite effectors responsible for this block have not been defined. Macrophage populations are extremely heterogeneous, responding differently to stimuli and to parasite infection. Here we evaluated the inhibition of NO production caused by T. gondii infection of J774-A1 and RAW 264.7 macrophages and assessed the role of several known parasite virulence factors in this phenotype. Infection of activated macrophages from both macrophage lines reduced NO production, however, the mechanism of this decrease was different. Consistent with previous reports, infected J774-A1 macrophages had reduced iNOS expression and lower number of iNOS positive cells. In contrast, T. gondii infection of RAW 264.7 macrophages did not alter iNOS expression or the number of iNOS positive cells, and yet it led to lower levels of NO production. Deletion of a number of previously defined virulence factors including ROP kinases that disrupt innate immune factors, TgIST which blocks STAT1 activation, as well as the secretory trafficking proteins ASP5 and MYR1, did not alter the phenotype of decreased NO production. Taken together our findings indicate that T. gondii infection inhibits NO production of activated macrophages by different mechanisms that involve reduction of iNOS expression vs. iNOS impairment, and suggest that a novel parasite effector is involved in modulating this important host defense pathway.

Genotyping Toxoplasma gondii from wildlife in Pennsylvania and identification of natural recombinants virulent to mice.

Recent studies indicated the predominance of Toxoplasma gondii haplogroup 12 in wildlife in the USA. However, still little is known of the genetic diversity of this parasite circulating in wildlife. In the present study, we tested coyotes (Canis latrans), red foxes (Vulpes vulpes), white-tailed deer (Odocoileus virginianus), and geese (Branta canadensis) from the state of Pennsylvania for T. gondii infection. Antibodies to T. gondii were found in 160 of 367 animals, including 92 (34.5%) of 266 coyotes, 49 (62.0%) of 79 white-tailed deer, 17 (85.0%) of 20 red fox, and two of two Canada geese tested by the modified agglutination test (cut off titer 1:25). Tissues from 105 seropositive animals were bioassayed in mice, and viable T. gondii was isolated from 29 animals, including 10 of 53 coyotes, 11 of 16 foxes, 7 of 49 deer, and one of one goose. DNA isolated from culture-derived tachyzoites of these isolates was characterized initially using multilocus PCR-RFLP markers. Nine genotypes were revealed, including ToxoDB PCR-RFLP #1 (4 isolates), #2 (2 isolates), #3 (4 isolates), #4 (6 isolates), #5 (4 isolates), #54 (1 isolate), #141 (1 isolate), #143 (1 isolate), and #216 (6 isolates), indicating high genetic diversity of T. gondii in wildlife in Pennsylvania. Pathogenicity of six T. gondii isolates (5 of #216 and #141) was determined in outbred Swiss Webster mice. Three of #216 and the #141 isolates were acute virulent to mice, and the other 2 #216 isolates were intermediate virulent. To determine the extent of genetic variation of these as well as a few recently reported virulent isolates from wildlife in North America, intron sequences were generated. Analysis of intron sequences and PCR-RFLP genotyping results indicated that the #216 isolates are likely derived from recombination of the clonal type I and III lineages. To determine if T. gondii virulence can be predicted by typing, we genotyped a collection of strains using PCR-RFLP markers for polymorphic genes ROP5, ROP16, ROP18 and GRA15, which are known to interact with host immune response. The results showed that there is an association of genotypes of ROP5 and ROP18 with mouse-virulence, however, additional gene(s) may also contribute to virulence in distinct T. gondii genotypes.

Recent transcontinental sweep of Toxoplasma gondii driven by a single monomorphic chromosome.

Toxoplasma gondii is a highly prevalent protozoan parasite that infects a wide range of animals and threatens human health by contaminating food and water. A markedly limited number of clonal parasite lineages have been recognized as predominating in North American and European populations, whereas strains from South America are comparatively diverse. Here, we show that strains from North America and Europe share distinct genetic polymorphisms that are mutually exclusive from polymorphisms in strains from the south. A striking exception to this geographic segregation is a monomorphic version of one chromosome (Chr1a) that characterizes virtually all northern and many southern isolates. Using a combination of molecular phylogenetic and phenotypic analyses, we conclude that northern and southern parasite populations diverged from a common ancestor in isolation over a period of approximately 10(6) yr, and that the monomorphic Chr1a has swept each population within the past 10,000 years. Like its definitive feline hosts, T. gondii may have entered South America and diversified there after reestablishment of the Panamanian land bridge. Since then, recombination has been an infrequent but important force in generating new T. gondii genotypes. Genes unique to a monomorphic version of a single parasite chromosome may have facilitated a recent population sweep of a limited number of highly successful T. gondii lineages.

A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii.

Toxoplasma gondii strains differ dramatically in virulence despite being genetically very similar. Genetic mapping revealed two closely adjacent quantitative trait loci on parasite chromosome VIIa that control the extreme virulence of the type I lineage. Positional cloning identified the candidate virulence gene ROP18, a highly polymorphic serine-threonine kinase that was secreted into the host cell during parasite invasion. Transfection of the virulent ROP18 allele into a nonpathogenic type III strain increased growth and enhanced mortality by 4 to 5 logs. These attributes of ROP18 required kinase activity, which revealed that secretion of effectors is a major component of parasite virulence.

Polymorphic secreted kinases are key virulence factors in toxoplasmosis.

The majority of known Toxoplasma gondii isolates from Europe and North America belong to three clonal lines that differ dramatically in their virulence, depending on the host. To identify the responsible genes, we mapped virulence in F(1) progeny derived from crosses between type II and type III strains, which we introduced into mice. Five virulence (VIR) loci were thus identified, and for two of these, genetic complementation showed that a predicted protein kinase (ROP18 and ROP16, respectively) is the key molecule. Both are hypervariable rhoptry proteins that are secreted into the host cell upon invasion. These results suggest that secreted kinases unique to the Apicomplexa are crucial in the host-pathogen interaction.

Intracellular parasite invasion strategies.

Intracellular parasites use various strategies to invade cells and to subvert cellular signaling pathways and, thus, to gain a foothold against host defenses. Efficient cell entry, ability to exploit intracellular niches, and persistence make these parasites treacherous pathogens. Most intracellular parasites gain entry via host-mediated processes, but apicomplexans use a system of adhesion-based motility called "gliding" to actively penetrate host cells. Actin polymerization-dependent motility facilitates parasite migration across cellular barriers, enables dissemination within tissues, and powers invasion of host cells. Efficient invasion has brought widespread success to this group, which includes Toxoplasma, Plasmodium, and Cryptosporidium.

Actin filament polymerization regulates gliding motility by apicomplexan parasites.

Host cell entry by Toxoplasma gondii depends critically on actin filaments in the parasite, yet paradoxically, its actin is almost exclusively monomeric. In contrast to the absence of stable filaments in conventional samples, rapid-freeze electron microscopy revealed that actin filaments were formed beneath the plasma membrane of gliding parasites. To investigate the role of actin filaments in motility, we treated parasites with the filament-stabilizing drug jasplakinolide (JAS) and monitored the distribution of actin in live and fixed cells using yellow fluorescent protein (YFP)-actin. JAS treatment caused YFP-actin to redistribute to the apical and posterior ends, where filaments formed a spiral pattern subtending the plasma membrane. Although previous studies have suggested that JAS induces rigor, videomicroscopy demonstrated that JAS treatment increased the rate of parasite gliding by approximately threefold, indicating that filaments are rate limiting for motility. However, JAS also frequently reversed the normal direction of motility, disrupting forward migration and cell entry. Consistent with this alteration, subcortical filaments in JAS-treated parasites occurred in tangled plaques as opposed to the straight, roughly parallel orientation observed in control cells. These studies reveal that precisely controlled polymerization of actin filaments imparts the correct timing, duration, and directionality of gliding motility in the Apicomplexa.

Recent expansion of Toxoplasma through enhanced oral transmission.

The global predominance of three clonal Toxoplasma gondii lineages suggests that they are endowed with an exceptional trait responsible for their current parasitism of nearly all warm-blooded vertebrates. Genetic polymorphism analyses indicate that these clonal lineages emerged within the last 10,000 years after a single genetic cross. Comparison with ancient strains (approximately 1 million years) suggests that the success of the clonal lineages resulted from the concurrent acquisition of direct oral infectivity. This key adaptation circumvented sexual recombination, simultaneously promoting transmission through successive hosts, hence leading to clonal expansion. Thus, changes in complex life cycles can occur rapidly and can profoundly influence pathogenicity.

Redescription of Neospora caninum and its differentiation from related coccidia.

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Acute toxoplasmosis leads to lethal overproduction of Th1 cytokines.

Virulence in Toxoplasma gondii is strongly influenced by the genotype of the parasite. Type I strains uniformly cause rapid death in mice regardless of the host genotype or the challenge dose. In contrast, the outcome of infections with type II strains is highly dependent on the challenge dose and the genotype of the host. To understand the basis of acute virulence in toxoplasmosis, we compared low and high doses of the RH strain (type I) and the ME49/PTG strain (type II) of T. gondii in outbred mice. Differences in virulence were reflected in only modestly different growth rates in vivo, and both strains disseminated widely to different tissues. The key difference in the virulent RH strain was the ability to reach high tissue burdens rapidly following a low dose challenge. Lethal infections caused by type I (RH) or type II (PTG) strain infections were accompanied by extremely elevated levels of Th1 cytokines in the serum, including IFN-gamma, TNF-alpha, IL-12, and IL-18. Extensive liver damage and lymphoid degeneration accompanied the elevated levels of cytokines produced during lethal infection. Increased time of survival following lethal infection with the RH strain was provided by neutralization of IL-18, but not TNF-alpha or IFN-gamma. Nonlethal infections with a low dose of type II PTG strain parasites were characterized by a modest induction of Th1 cytokines that led to control of infection and minimal damage to host tissues. Our findings establish that overstimulation of immune responses that are normally necessary for protection is an important feature of acute toxoplasmosis.

Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole.

Rapid discharge of secretory organelles called rhoptries is tightly coupled with host cell entry by the protozoan parasite Toxoplasma gondii. Rhoptry contents were deposited in clusters of vesicles within the host cell cytosol and within the parasitophorous vacuole. To examine the fate of these rhoptry-derived secretory vesicles, we utilized cytochalasin D to prevent invasion, leading to accumulation of protein-rich vesicles in the host cell cytosol. These vesicles lack an internal parasite and are hence termed evacuoles. Like the mature parasite-containing vacuole, evacuoles became intimately associated with host cell mitochondria and endoplasmic reticulum, while remaining completely resistant to fusion with host cell endosomes and lysosomes. In contrast, evacuoles were recruited to pre-existing, parasite-containing vacuoles and were capable of fusing and delivering their contents to these compartments. Our findings indicate that a two-step process involving direct rhoptry secretion into the host cell cytoplasm followed by incorporation into the vacuole generates the parasitophorous vacuole occupied by TOXOPLASMA: The characteristic properties of the mature vacuole are likely to be determined by this early delivery of rhoptry components.

The toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains.

The initial stage of invasion by apicomplexan parasites involves the exocytosis of the micronemes-containing molecules that contribute to host cell attachment and penetration. MIC4 was previously described as a protein secreted by Toxoplasma gondii tachyzoites upon stimulation of micronemes exocytosis. We have microsequenced the mature protein, purified after discharge from micronemes and cloned the corresponding gene. The deduced amino acid sequence of MIC4 predicts a 61-kDa protein that contains 6 conserved apple domains. Apple domains are composed of six spacely conserved cysteine residues which form disulfide bridges and are also present in micronemal proteins from two closely related apicomplexan parasites, Sarcocystis muris and Eimeria species, and several mammalian serum proteins, including kallikrein. Here we show that MIC4 localizes in the micronemes of all the invasive forms of T. gondii, tachyzoites, bradyzoites, sporozoites, and merozoites. The protein is proteolytically processed both at the N and the C terminus only upon release from the organelle. MIC4 binds efficiently to host cells, and the adhesive motif maps in the most C-terminal apple domain.

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.

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.

Molecular characterization of a thrombospondin-related anonymous protein homologue in Neospora caninum.

Thrombospondin-related anonymous protein (TRAP) family members participate in attachment and invasion of host cells by apicomplexan parasites. A TRAP homologue in Neospora caninum strain Nc-1 (NcMIC2) was cloned, sequenced and found to be 61% identical (75% similar) at the amino acid level to Toxoplasma gondii MIC2 (TgMIC2). Similar to TgMIC2, the predicted amino acid sequence of NcMIC2 contains one integrin-like domain (I or A domain), five thrombospondin (TSP) repeats, a putative transmembrane spanning region and intracellular C-terminus, and was localized to micronemes by cryo-immunoelectron microscopy. The secretion of NcMIC2 was temperature dependent and was induced at or above 25 degrees C. The secreted form of NcMIC2 released into the medium was found to be proteolytically processed such that it lacked the C-terminal domain. Secretion of NcMIC2 was regulated by calcium, since several agents which raise intracellular calcium levels were shown to promote NcMIC2 secretion and chelation of [Ca(2+)](i) abrogated release. As a member of the growing family of apicomplexan TRAP proteins, NcMIC2 may play an important role in attachment and invasion by N. caninum into host cells.

Cell invasion by un-palatable parasites.

While some intracellular pathogens invade and replicate exclusively in phagocytic host cells, others have evolved mechanisms to stimulate their uptake by cells not equipped with a well-developed phagocytic machinery. A common mechanism utilized by bacteria involves the induction of macropinocytosis, or of other F-actin-driven processes which result in engulfment of the pathogen through formation of a plasma membrane-derived vacuole. Interestingly, this type of "induced phagocytosis" mechanism does not appear to be utilized by protozoan parasites, which are significantly larger than bacteria in size (about 5-10 microns in average length). Intracellular protozoa either restrict themselves to infecting "professional" phagocytes (one example is the trypanosomatid Leishmania), or utilize highly unusual mechanisms for gaining access to the intracellular environment. Here we discuss what has been revealed in recent years about the remarkable cell invasion strategies of two highly successful intracellular parasites: Toxoplasma gondii and Trypanosoma cruzi. Toxoplasma utilizes a distinct form of actin/myosin-dependent gliding motility to propel itself into mammalian cells, while T. cruzi invades by subverting a Ca(2+)-regulated lysosomal exocytic pathway.

Invasion by Toxoplasma gondii establishes a moving junction that selectively excludes host cell plasma membrane proteins on the basis of their membrane anchoring.

The protozoan parasite Toxoplasma gondii actively penetrates its host cell by squeezing through a moving junction that forms between the host cell plasma membrane and the parasite. During invasion, this junction selectively controls internalization of host cell plasma membrane components into the parasite-containing vacuole. Membrane lipids flowed past the junction, as shown by the presence of the glycosphingolipid G(M1) and the cationic lipid label 1. 1'-dihexadecyl-3-3'-3-3'-tetramethylindocarbocyanine (DiIC(16)). Glycosylphosphatidylinositol (GPI)-anchored surface proteins, such as Sca-1 and CD55, were also readily incorporated into the parasitophorous vacuole (PV). In contrast, host cell transmembrane proteins, including CD44, Na(+)/K(+) ATPase, and beta1-integrin, were excluded from the vacuole. To eliminate potential differences in sorting due to the extracellular domains, parasite invasion was examined in host cells transfected with recombinant forms of intercellular adhesion molecule 1 (ICAM-1, CD54) that differed in their mechanism of membrane anchoring. Wild-type ICAM-1, which contains a transmembrane domain, was excluded from the PV, whereas both GPI-anchored ICAM-1 and a mutant of ICAM-1 missing the cytoplasmic tail (ICAM-1-Cyt(-)) were readily incorporated into the PV membrane. Our results demonstrate that during host cell invasion, Toxoplasma selectively excludes host cell transmembrane proteins at the moving junction by a mechanism that depends on their anchoring in the membrane, thereby creating a nonfusigenic compartment.

Comparison of the major antigens of Neospora caninum and Toxoplasma gondii.

The Apicomplexa are a diverse group of parasitic protozoa with very ancient phylogenetic roots. Consistent with their phylogeny, the extant species share conserved proteins and traits that were found in their apicomplexan progenitor, but at the same time they have diverged to occupy different biological niches (e.g. host-range and cell type). Characterisation of gene and protein diversity is important for distinguishing between related parasites, for determining their phylogeny, and for providing insight into factors that determine host restriction, cell preference, and virulence. The value of molecular characterisations and comparisons between species is well illustrated by the close phylogenetic relationship between Neospora caninum and Toxoplasma gondii. These two organisms have nearly identical morphology and can cause similar pathology and disease. Consequently, N. caninum has often been incorrectly identified as T. gondii, thus demonstrating the need for studies addressing the molecular and antigenic composition of Neospora. In this review, we describe the major antigenic proteins that have been characterised in N. caninum. These show homology to T. gondii proteins, yet possess unique antigenic characteristics that distinguish them from their homologues and enable their use for specific serological diagnoses and parasite identification.

Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion.

Toxoplasma gondii is a member of the phylum Apicomplexa, a diverse group of intracellular parasites that share a unique form of gliding motility. Gliding is substrate dependent and occurs without apparent changes in cell shape and in the absence of traditional locomotory organelles. Here, we demonstrate that gliding is characterized by three distinct forms of motility: circular gliding, upright twirling, and helical rotation. Circular gliding commences while the crescent-shaped parasite lies on its right side, from where it moves in a counterclockwise manner at a rate of approximately 1.5 microm/s. Twirling occurs when the parasite rights itself vertically, remaining attached to the substrate by its posterior end and spinning clockwise. Helical gliding is similar to twirling except that it occurs while the parasite is positioned horizontally, resulting in forward movement that follows the path of a corkscrew. The parasite begins lying on its left side (where the convex side is defined as dorsal) and initiates a clockwise revolution along the long axis of the crescent-shaped body. Time-lapse video analyses indicated that helical gliding is a biphasic process. During the first 180(o) of the turn, the parasite moves forward one body length at a rate of approximately 1-3 microm/s. In the second phase, the parasite flips onto its left side, in the process undergoing little net forward motion. All three forms of motility were disrupted by inhibitors of actin filaments (cytochalasin D) and myosin ATPase (butanedione monoxime), indicating that they rely on an actinomyosin motor in the parasite. Gliding motility likely provides the force for active penetration of the host cell and may participate in dissemination within the host and thus is of both fundamental and practical interest.

Differential membrane targeting of the secretory proteins GRA4 and GRA6 within the parasitophorous vacuole formed by Toxoplasma gondii.

Following secretion into the parasitophorous vacuole, dense granule proteins, referred to as GRA proteins, are targeted to different locations including a complex of tubular membranes that are connected with the vacuolar membrane. To further define the formation of this intravacuolar network, we have investigated the secretion, trafficking and membrane association of GRA4 and GRA6 within the parasitophorous vacuole. In extracellular parasites, GRA4 and GRA6 were found exclusively in dense secretory granules where they were packaged primarily as soluble proteins. Following release into the vacuole, GRA6 was rapidly translocated to the posterior end of the parasite where, like previously reported for GRA2, it bound to a cluster of multi-lamellar vesicles that give rise to the network. In contrast, GRA4 was distributed throughout the lumen of the vacuole and only later became associated with the mature network that is found dispersed throughout the vacuole. Cell fractionation and treatment with denaturing agents established that the association of GRA4 with the network membranes was mediated by strong protein-protein interactions. In contrast, GRA6 was predominantly influenced by hydrophobic interactions, and a phosphorylated form of this protein present within the vacuole showed increased association with the network membranes. Cross-linking studies established that GRA4 and GRA6 specifically interact with GRA2 to form a multimeric complex that is stably associated with the intravacuolar network. Formation of this protein complex, which is based on both protein-protein and hydrophobic interactions, may participate in nutrient or protein transport within the vacuole.