The roles of intramembrane proteases in protozoan parasites

AbstractIntramembrane proteolysis is widely conserved throughout different forms of life, with three major types of proteases being known for their ability to cleave peptide bonds directly within the transmembrane domains of their substrates. Although intramembrane proteases have been extensively studied in humans and model organisms, they have only more recently been investigated in protozoan parasites, where they turn out to play important and sometimes unexpected roles. Signal peptide peptidases are involved in endoplasmic reticulum (ER) quality control and signal peptide degradation from exported proteins. Recent studies suggest that repurposing inhibitors developed for blocking presenilins may be useful for inhibiting the growth of Plasmodium, and possibly other protozoan parasites, by blocking signal peptide peptidases. Rhomboid proteases, originally described in the fly, are also widespread in parasites, and are especially expanded in apicomplexans. Their study in parasites has revealed novel roles that expand our understanding of how these proteases function. Within this diverse group of parasites, rhomboid proteases contribute to processing of adhesins involved in attachment, invasion, intracellular replication, phagocytosis, and immune evasion, placing them at the vertex of host–parasite interactions. This article is part of a Special Issue entitled: Intramembrane Proteases

Characterization of the Modified Phagocytic Vacuole Occupied by Intracellular Toxoplasma Gondii (Macrophage, Protozoa).

Toxoplasma gondii is a protozoan parasite which resides in modified endocytic compartments of host cells. Extensive intracellular replication and host cell lysis leads to acute toxoplasmosis which is eventually limited by the host immune response to a chronic state of infection. The features of this unique intracellular compartment, which enables Toxoplasma to survive in macrophages, are reported here. Although Toxoplasma survives in normal macrophages, activated macrophages from immune animals rapidly killed Toxoplasma by production of oxygen radicals and intermediates generated during parasite invasion. In addition, activated macrophages inhibited Toxoplasma growth by an oxygen-independent mechanism. Qualitative and quantitative features of oxygen intermediate detoxifying enzymes, catalase and superoxide dismutase, were described from two strains of Toxoplasma. These enzymes did not appear to be the basis for differences in strain virulence, but may contribute to intracellular survival in normal macrophages. A newly recognized microbicidal mechanism involves the rapid acidification during the formation of endocytic compartments. Live Toxoplasma entered into modified phagocytic vacuoles in normal macrophages that do not show characteristic acidification, but remain at near neutral pH. In contrast, the enhanced acidification capacity of activated macrophages and of normal macrophages in the presence of specific antibody may contribute to the toxoplasmacidal response of these cells. The unique modified endocytic vacuole occupied by Toxoplasma is characterized by an accumulation of membrane-like tubules which may contribute to growth of the vacuole membrane. Purification of this significant host parasite interface confirmed that the tubules are parasite derived, are comprised of membrane vesicles which are responsive to calcium levels, and contain proteins recognized by mouse anti-Toxoplasma sera

Defining stage-specific activity of potent new inhibitors of Cryptosporidium parvum growth in vitro

Currently, nitazoxanide is the only FDA-approved treatment for cryptosporidiosis; unfortunately, it is ineffective in immunocompromised patients, has varied efficacy in immunocompetent individuals, and is not approved in infants under 1 year of age. Identifying new inhibitors for the treatment of cryptosporidiosis requires standardized and quantifiable in vitro assays for assessing potency, selectivity, timing of activity, and reversibility. Here, we provide new protocols for defining which stages of the life cycle are susceptible to four highly active compound classes that likely inhibit different targets in the parasite. We also utilize a newly developed long-term culture system to define assays for monitoring reversibility as a means of defining cidal activity as a function of concentration and time of treatment. These assays should provide valuable in vitro parameters to establish conditions for efficacious in vivo treatment.Cryptosporidium parvum and Cryptosporidium hominis have emerged as major enteric pathogens of infants in the developing world, in addition to their known importance in immunocompromised adults. Although there has been recent progress in identifying new small molecules that inhibit Cryptosporidium sp. growth in vitro or in animal models, we lack information about their mechanism of action, potency across the life cycle, and cidal versus static activities. Here, we explored four potent classes of compounds that include inhibitors that likely target phosphatidylinositol 4 kinase (PI4K), phenylalanine-tRNA synthetase (PheRS), and several potent inhibitors with unknown mechanisms of action. We utilized monoclonal antibodies and gene expression probes for staging life cycle development to define the timing of when inhibitors were active during the life cycle of Cryptosporidium parvum grown in vitro. These different classes of inhibitors targeted different stages of the life cycle, including compounds that blocked replication (PheRS inhibitors), prevented the segmentation of daughter cells and thus blocked egress (PI4K inhibitors), or affected sexual-stage development (a piperazine compound of unknown mechanism). Long-term cultivation of C. parvum in epithelial cell monolayers derived from intestinal stem cells was used to distinguish between cidal and static activities based on the ability of parasites to recover from treatment. Collectively, these approaches should aid in identifying mechanisms of action and for designing in vivo efficacy studies based on time-dependent concentrations needed to achieve cidal activity

Toxoplasma actin is required for efficient host cell invasion

ABSTRACT Apicomplexan parasites actively invade host cells using a mechanism predicted to be powered by a parasite actin-dependent myosin motor. In the model apicomplexan Toxoplasma gondii, inducible knockout of the actin gene, ACT1, was re-cently demonstrated to limit but not completely abolish invasion. This observation has led to the provocative suggestion that T. gondii possesses alternative, ACT1-independent invasion pathways. Here, we dissected the residual invasive ability ofact1 parasites. Surprisingly, we were able to detect residual ACT1 protein in inducibleact1 parasites as long as 5 days after ACT1 deletion. We further found that the longeract1 parasites were propagated after ACT1 deletion, the more severe an invasion defect was observed. Both findings are consistent with the quantity of residual ACT1 retained inact1 parasites being responsi-ble for their invasive ability. Furthermore, invasion by theact1 parasites was also sensitive to the actin polymerization inhibi-tor cytochalasin D. Finally, there was no clear defect in attachment to host cells or moving junction formation byact1 para-sites. However,act1 parasites often exhibited delayed entry into host cells, suggesting a defect specific to the penetration stage of invasion. Overall, our results support a model where residual ACT1 protein retained in inducibleact1 parasites facilitates their limited invasive ability and confirm that parasite actin is essential for efficient penetration into host cells during invasion. IMPORTANCE The prevailing model for apicomplexan invasion has recently been suggested to require major revision, based on studies where core components of the invasionmachinery were genetically disrupted using a Cre-Lox-based inducible knockout system. For the myosin component of the motor thought to power invasion, an alternative parasite myosin was recently demon

Functional analysis of the role of Toxoplasma gondii nucleoside triphosphate hydrolases I and II in acute mouse virulence and immune suppression

Bioluminescent reporter assays have been widely used to study the effect of Toxoplasma gondii on host gene expression. In the present study, we extend these studies by engineering novel reporter cell lines containing a gamma-activated sequence (GAS) element driving firefly luciferase (FLUC). In RAW264.7 macrophages, T. gondii type I strain (GT1) infection blocked interferon gamma (IFN-γ)-induced FLUC activity to a significantly greater extent than infection by type II (ME49) and type III (CTG) strains. Quantitative trait locus (QTL) analysis of progeny from a prior genetic cross identified a genomic region on chromosome XII that correlated with the observed strain-dependent phenotype. This QTL region contains two isoforms of the T. gondii enzyme nucleoside triphosphate hydrolase (NTPase) that were the prime candidates for mediating the observed strain-specific effect. Using reverse genetic analysis we show that deletion of NTPase I from a type I strain (RH) background restored the higher luciferase levels seen in the type II (ME49) strain. Rather than an effect on IFN-γ-dependent transcription, our data suggest that NTPase I was responsible for the strain-dependent difference in FLUC activity due to hydrolysis of ATP. We further show that NTPases I and II were not essential for tachyzoite growth in vitro or virulence in mice. Our study reveals that although T. gondii NTPases are not essential for immune evasion, they can affect ATP-dependent reporters. Importantly, this limitation was overcome using an ATP-independent Gaussia luciferase, which provides a more appropriate reporter for use with T. gondii infection studies

Dendritic cells and Cryptosporidium: From recognition to restriction

Host immune responses are required for the efficient control of cryptosporidiosis. Immunity agains

Comparative genome analysis reveals a conserved family of actin-like proteins in apicomplexan parasites

BACKGROUND: The phylum Apicomplexa is an early-branching eukaryotic lineage that contains a number of important human and animal pathogens. Their complex life cycles and unique cytoskeletal features distinguish them from other model eukaryotes. Apicomplexans rely on actin-based motility for cell invasion, yet the regulation of this system remains largely unknown. Consequently, we focused our efforts on identifying actin-related proteins in the recently completed genomes of Toxoplasma gondii, Plasmodium spp., Cryptosporidium spp., and Theileria spp. RESULTS: Comparative genomic and phylogenetic studies of apicomplexan genomes reveals that most contain only a single conventional actin and yet they each have 8–10 additional actin-related proteins. Among these are a highly conserved Arp1 protein (likely part of a conserved dynactin complex), and Arp4 and Arp6 homologues (subunits of the chromatin-remodeling machinery). In contrast, apicomplexans lack canonical Arp2 or Arp3 proteins, suggesting they lost the Arp2/3 actin polymerization complex on their evolutionary path towards intracellular parasitism. Seven of these actin-like proteins (ALPs) are novel to apicomplexans. They show no phylogenetic associations to the known Arp groups and likely serve functions specific to this important group of intracellular parasites. CONCLUSION: The large diversity of actin-like proteins in apicomplexans suggests that the actin protein family has diverged to fulfill various roles in the unique biology of intracellular parasites. Conserved Arps likely participate in vesicular transport and gene expression, while apicomplexan-specific ALPs may control unique biological traits such as actin-based gliding motility

The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat

The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2 encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rodents less fearful of their definitive Felidae hosts. However, L-DOPA is also a structural precursor of melanins, secondary quinones, and dityrosine protein crosslinks, which are produced by many species. For example, dityrosine crosslinks are abundant in the oocyst walls of Eimeria and T. gondii, although their structural role has not been demonstrated, Here, we investigated the biology of AAH knockout parasites in the sexual reproductive cycle within cats. We found that ablation of the AAH genes resulted in reduced infection in the cat, lower oocyst yields, and decreased rates of sporulation. Our findings suggest that the AAH genes play a predominant role during infection in the gut of the definitive feline host

Transepithelial Migration of Toxoplasma gondii Is Linked to Parasite Motility and Virulence

After oral ingestion, Toxoplasma gondii crosses the intestinal epithelium, disseminates into the deep tissues, and traverses biological barriers such as the placenta and the blood-brain barrier to reach sites where it causes severe pathology. To examine the cellular basis of these processes, migration of T. gondii was studied in vitro using polarized host cell monolayers and extracellular matrix. Transmigration required active parasite motility and the highly virulent type I strains consistently exhibited a superior migratory capacity than the nonvirulent type II and type III strains. Type I strain parasites also demonstrated a greater capacity for transmigration across mouse intestine ex vivo, and directly penetrated into the lamina propria and vascular endothelium. A subpopulation of virulent type I parasites exhibited a long distance migration (LDM) phenotype in vitro, that was not expressed by nonvirulent type II and type III strains. Cloning of parasites expressing the LDM phenotype resulted in substantial increase of migratory capacity in vitro and in vivo. The potential to up-regulate migratory capacity in T. gondii likely plays an important role in establishing new infections and in dissemination upon reactivation of chronic infections