Deciphering protein prenylation in endocytic trafficking in Toxoplasma gondii.

Toxoplasma gondii is a widespread intracellular protozoan pathogen infecting virtually all warm-blooded animals. This parasite acquires host-derived resources to support its replication inside a membrane-bound parasitophorous vacuole within infected host cells. Previous research has discovered that Toxoplasma actively endocytoses host proteins and transports them to a lysosome-equivalent structure for digestion. However, few molecular determinants required for trafficking of host-derived material within the parasite were known. A recent study (Q.-Q. Wang, M. Sun, T. Tang, D.-H. Lai, et al., mBio 14:e01309-23, 2023, https://doi.org/10.1128/mbio.01309-23) identified a critical role for membrane anchoring of proteins via prenylation in the trafficking of endocytosed host proteins by Toxoplasma, including an essential Toxoplasma ortholog of Rab1B. The authors also found that TgRab1 is crucial for protein trafficking of the rhoptry secretory organelles, indicating a dual role in endocytic and exocytic protein trafficking. This study sets the stage for further dissecting endomembrane trafficking in Toxoplasma, along with potentially exploiting protein prenylation as a target for therapeutic development.

Toxoplasma gondii harbors a hypoxia-responsive coproporphyrinogen dehydrogenase-like protein.

Toxoplasma gondii is an apicomplexan parasite that is the cause of toxoplasmosis, a potentially lethal disease for immunocompromised individuals. During in vivo infection, the parasites encounter various growth environments, such as hypoxia. Therefore, the metabolic enzymes in the parasites must adapt to such changes to fulfill their nutritional requirements. Toxoplasma can de novo biosynthesize some nutrients, such as heme. The parasites heavily rely on their own heme production for intracellular survival. Notably, the antepenultimate step within this pathway is facilitated by coproporphyrinogen III oxidase (CPOX), which employs oxygen to convert coproporphyrinogen III to protoporphyrinogen IX through oxidative decarboxylation. Conversely, some bacteria can accomplish this conversion independently of oxygen through coproporphyrinogen dehydrogenase (CPDH). Genome analysis found a CPDH ortholog in Toxoplasma. The mutant Toxoplasma lacking CPOX displays significantly reduced growth, implying that T. gondii CPDH (TgCPDH) potentially functions as an alternative enzyme to perform the same reaction as CPOX under low-oxygen conditions. In this study, we demonstrated that TgCPDH exhibits CPDH activity by complementing it in a heme synthesis-deficient Salmonella mutant. Additionally, we observed an increase in TgCPDH expression in Toxoplasma when it grew under hypoxic conditions. However, deleting TgCPDH in both wild-type and heme-deficient parasites did not alter their intracellular growth under both ambient and low-oxygen conditions. This research marks the first report of a CPDH-like protein in eukaryotic cells. Although TgCPDH responds to hypoxic conditions and possesses enzymatic activity, our findings revealed that it does not directly affect acute Toxoplasma infections in vitro and in vivo. IMPORTANCE: Toxoplasma gondii is a ubiquitous parasite capable of infecting a wide range of warm-blooded hosts, including humans. During its life cycle, these parasites must adapt to varying environmental conditions, including situations with low-oxygen levels, such as intestine and spleen tissues. Our research, in conjunction with studies conducted by other laboratories, has revealed that Toxoplasma primarily relies on its own heme production during acute infections. Intriguingly, in addition to this classical heme biosynthetic pathway, the parasites encode a putative oxygen-independent coproporphyrinogen dehydrogenase (CPDH), suggesting its potential contribution to heme production under varying oxygen conditions, a feature typically observed in simpler organisms like bacteria. Notably, so far, CPDH has only been identified in some bacteria for heme biosynthesis. Our study discovered that Toxoplasma harbors a functional enzyme displaying CPDH activity, which alters its expression in the parasites when they face fluctuating oxygen levels in their surroundings.

Toxoplasma gondii harbors a hypoxia-responsive coproporphyrinogen dehydrogenase-like protein.

Toxoplasma gondii is an apicomplexan parasite that is the cause of toxoplasmosis, a potentially lethal disease for immunocompromised individuals. During in vivo infection, the parasites encounter various growth environments, such as hypoxia. Therefore, the metabolic enzymes in the parasites must adapt to such changes to fulfill their nutritional requirements. Toxoplasma can de novo biosynthesize some nutrients, such as heme. The parasites heavily rely on their own heme production for intracellular survival. Notably, the antepenultimate step within this pathway is facilitated by coproporphyrinogen III oxidase (CPOX), which employs oxygen to convert coproporphyrinogen III to protoporphyrinogen IX through oxidative decarboxylation. Conversely, some bacteria can accomplish this conversion independently of oxygen through coproporphyrinogen dehydrogenase (CPDH). Genome analysis found a CPDH ortholog in Toxoplasma. The mutant Toxoplasma lacking CPOX displays significantly reduced growth, implying that TgCPDH potentially functions as an alternative enzyme to perform the same reaction as CPOX under low oxygen conditions. In this study, we demonstrated that TgCPDH exhibits coproporphyrinogen dehydrogenase activity by complementing it in a heme synthesis-deficient Salmonella mutant. Additionally, we observed an increase in TgCPDH expression in Toxoplasma when it grew under hypoxic conditions. However, deleting TgCPDH in both wildtype and heme-deficient parasites did not alter their intracellular growth under both ambient and low oxygen conditions. This research marks the first report of a coproporphyrinogen dehydrogenase-like protein in eukaryotic cells. Although TgCPDH responds to hypoxic conditions and possesses enzymatic activity, our findings suggest that it does not directly affect intracellular infection or the pathogenesis of Toxoplasma parasites.

The vacuolar iron transporter mediates iron detoxification in Toxoplasma gondii.

Iron is essential to cells as a cofactor in enzymes of respiration and replication, however without correct storage, iron leads to the formation of dangerous oxygen radicals. In yeast and plants, iron is transported into a membrane-bound vacuole by the vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including in Toxoplasma gondii. Here, we assess the role of VIT and iron storage in T. gondii. By deleting VIT, we find a slight growth defect in vitro, and iron hypersensitivity, confirming its essential role in parasite iron detoxification, which can be rescued by scavenging of oxygen radicals. We show VIT expression is regulated by iron at transcript and protein levels, and by altering VIT localization. In the absence of VIT, T. gondii responds by altering expression of iron metabolism genes and by increasing antioxidant protein catalase activity. We also show that iron detoxification has an important role both in parasite survival within macrophages and in virulence in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification in T. gondii, we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.

A cathepsin C-like protease mediates the post-translation modification of Toxoplasma gondii secretory proteins for optimal invasion and egress.

Microbial pathogens use proteases for their infections, such as digestion of proteins for nutrients and activation of their virulence factors. As an obligate intracellular parasite, Toxoplasma gondii must invade host cells to establish its intracellular propagation. To facilitate invasion, the parasites secrete invasion effectors from microneme and rhoptry, two unique organelles in apicomplexans. Previous work has shown that some micronemal invasion effectors experience a series of proteolytic cleavages within the parasite's secretion pathway for maturation, such as the aspartyl protease (TgASP3) and the cathepsin L-like protease (TgCPL), localized within the post-Golgi compartment and the endolysosomal system, respectively. Furthermore, it has been shown that the precise maturation of micronemal effectors is critical for Toxoplasma invasion and egress. Here, we show that an endosome-like compartment (ELC)-residing cathepsin C-like protease (TgCPC1) mediates the final trimming of some micronemal effectors, and its loss further results in defects in the steps of invasion, egress, and migration throughout the parasite's lytic cycle. Notably, the deletion of TgCPC1 completely blocks the activation of subtilisin-like protease 1 (TgSUB1) in the parasites, which globally impairs the surface-trimming of many key micronemal invasion and egress effectors. Additionally, we found that Toxoplasma is not efficiently inhibited by the chemical inhibitor targeting the malarial CPC ortholog, suggesting that these cathepsin C-like orthologs are structurally different within the apicomplexan phylum. Collectively, our findings identify a novel function of TgCPC1 in processing micronemal proteins within the Toxoplasma parasite's secretory pathway and expand the understanding of the roles of cathepsin C protease. IMPORTANCE Toxoplasma gondii is a microbial pathogen that is well adapted for disseminating infections. It can infect virtually all warm-blooded animals. Approximately one-third of the human population carries toxoplasmosis. During infection, the parasites sequentially secrete protein effectors from the microneme, rhoptry, and dense granule, three organelles exclusively found in apicomplexan parasites, to help establish their lytic cycle. Proteolytic cleavage of these secretory proteins is required for the parasite's optimal function. Previous work has revealed that two proteases residing within the parasite's secretory pathway cleave micronemal and rhoptry proteins, which mediate parasite invasion and egress. Here, we demonstrate that a cathepsin C-like protease (TgCPC1) is involved in processing several invasion and egress effectors. The genetic deletion of TgCPC1 prevented the complete maturation of some effectors in the parasites. Strikingly, the deletion led to a full inactivation of one surface-anchored protease, which globally impaired the trimming of some key micronemal proteins before secretion. Therefore, this finding represents a novel post-translational mechanism for the processing of virulence factors within microbial pathogens.

A cathepsin C-like protease post-translationally modifies Toxoplasma gondii secretory proteins for optimal invasion and egress.

Microbial pathogens use proteases for their infections, such as digestion of proteins for nutrients and activation of their virulence factors. As an obligate intracellular parasite, Toxoplasma gondii must invade host cells to establish its intracellular propagation. To facilitate invasion, the parasites secrete invasion effectors from microneme and rhoptry, two unique organelles in apicomplexans. Previous work has shown that some micronemal invasion effectors experience a series of proteolytic cleavages within the parasite's secretion pathway for maturation, such as the aspartyl protease (TgASP3) and the cathepsin L-like protease (TgCPL), localized within the post-Golgi compartment (1) and the endolysosomal system (2), respectively. Furthermore, it has been shown that the precise maturation of micronemal effectors is critical for Toxoplasma invasion and egress (1). Here, we show that an endosome-like compartment (ELC)-residing cathepsin C-like protease (TgCPC1) mediates the final trimming of some micronemal effectors, and its loss further results in defects in the steps of invasion, egress, and migration throughout the parasite's lytic cycle. Notably, the deletion of TgCPC1 completely blocks the activation of subtilisin-like protease 1 (TgSUB1) in the parasites, which globally impairs the surface-trimming of many key micronemal invasion and egress effectors. Additionally, we found that TgCPC1 was not efficiently inhibited by the chemical inhibitor targeting its malarial ortholog, suggesting that these cathepsin C-like orthologs are structurally different within the apicomplexan phylum. Taken together, our findings identify a novel function of TgCPC1 in the processing of micronemal proteins within the secretory pathway of Toxoplasma parasites and expand the understanding of the roles of cathepsin C protease. IMPORTANCE: Toxoplasma gondii is a microbial pathogen that is well adapted for disseminating infections. It can infect virtually all warm-blooded animals. Approximately one-third of the human population carries toxoplasmosis. During infection, the parasites sequentially secrete protein effectors from the microneme, rhoptry, and dense granule, three organelles exclusively found in apicomplexan parasites, to help establish their lytic cycle. Proteolytic cleavage of these secretory proteins is required for the parasite's optimal function. Previous work has revealed that two proteases residing within the parasite's secretory pathway cleave micronemal and rhoptry proteins, which mediate parasite invasion and egress. Here, we demonstrate that a cathepsin C-like protease (TgCPC1) is involved in processing several invasion and egress effectors. The genetic deletion of TgCPC1 prevented the complete maturation of some effectors in the parasites. Strikingly, the deletion led to a full inactivation of one surface-anchored protease, which globally impaired the trimming of some key micronemal proteins before secretion. Therefore, this finding represents a novel post-translational mechanism for the processing of virulence factors within microbial pathogens.

Safe, Effective, and Inexpensive Clearance of Mycoplasma Contamination from Cultures of Apicomplexan Parasites with Sparfloxacin.

Most commercial products cannot be used for clearance of Mycoplasma contamination from cultures of apicomplexan parasites due to the parasites' dependence on the apicoplast, an essential organelle with DNA replication and translation machinery of cyanobacterial origin. The lone exception, mycoplasma removal agent (MRA), is relatively expensive, and some mycoplasma strains have shown resistance to clearance with MRA. Here, we report that the fluoroquinolone antibiotic sparfloxacin is a safe, effective, and inexpensive alternative for treatment of mycoplasma contamination in cultures of apicomplexan parasites. Sparfloxacin cleared both MRA-sensitive and MRA-resistant mycoplasma species from P. falciparum cultures at 1 and 4 µg/mL, respectively. We show that cultures of three different apicomplexan parasites can be maintained at concentrations of sparfloxacin required to clear mycoplasma without resulting in substantial deleterious effects on parasite growth. We also describe an alternative low-cost, in-house PCR assay for detecting mycoplasma. These findings will be useful to laboratories maintaining apicomplexan parasites in vitro, especially in low-resource environments, where the high cost of commercial products creates an economic barrier for detecting and eliminating mycoplasma from culture. IMPORTANCE These findings will be useful to laboratories maintaining apicomplexan parasites in vitro, especially in low-resource environments, where the high cost of commercial products creates an economic barrier for detecting and eliminating Mycoplasma from culture.

The Toxoplasma plant-like vacuolar compartment (PLVAC).

Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. T. gondii shares several characteristics with plants including a nonphotosynthetic plastid termed apicoplast and a multivesicular organelle that was named the plant-like vacuole (PLV) or vacuolar compartment (VAC). The name plant-like vacuole was selected based on its resemblance in composition and function to plant vacuoles. The name VAC represents its general vacuolar characteristics. We will refer to the organelle as PLVAC in this review. New findings in recent years have revealed that the PLVAC represents the lysosomal compartment of T. gondii which has adapted peculiarities to fulfill specific Toxoplasma needs. In this review, we discuss the composition and functions of the PLVAC highlighting its roles in ion storage and homeostasis, endocytosis, exocytosis, and autophagy.

Oxadiazon Derivatives Elicit Potent Intracellular Growth Inhibition against Toxoplasma gondii by Disrupting Heme Biosynthesis.

Infections of Toxoplasma gondii can cause severe and sometimes fatal diseases in immunocompromised individuals. The de novo heme biosynthesis pathway is required for intracellular growth and pathogenesis, making it an appealing therapeutic target. We synthesized a small library of derivatives of the herbicide oxadiazon, a known inhibitor of the penultimate reaction within the heme biosynthesis pathway in plants, catalyzed by protoporphyrinogen oxidase (PPO). Seven of the 18 analogs exhibit potent intracellular growth inhibition of wild-type T. gondii (IC50 = 1 to 2.4 µM). An assay of the compounds against Toxoplasma PPO knockout and complementation strains confirmed the mode of action to be due to the potent inhibition of PPO. The most potent compounds have no detectable cytotoxicity against human foreskin fibroblast cells up to 100 µM. This study suggests that oxadiazon derivatives may represent a new molecular scaffold for the effective treatment of T. gondii infections.

An Uninvited Seat at the Dinner Table: How Apicomplexan Parasites Scavenge Nutrients from the Host.

Obligate intracellular parasites have evolved a remarkable assortment of strategies to scavenge nutrients from the host cells they parasitize. Most apicomplexans form a parasitophorous vacuole (PV) within the invaded cell, a replicative niche within which they survive and multiply. As well as providing a physical barrier against host cell defense mechanisms, the PV membrane (PVM) is also an important site of nutrient uptake that is essential for the parasites to sustain their metabolism. This means nutrients in the extracellular milieu are separated from parasite metabolic machinery by three different membranes, the host plasma membrane, the PVM, and the parasite plasma membrane (PPM). In order to facilitate nutrient transport from the extracellular environment into the parasite itself, transporters on the host cell membrane of invaded cells can be modified by secreted and exported parasite proteins to maximize uptake of key substrates to meet their metabolic demand. To overcome the second barrier, the PVM, apicomplexan parasites secrete proteins contained in the dense granules that remodel the vacuole and make the membrane permissive to important nutrients. This bulk flow of host nutrients is followed by a more selective uptake of substrates at the PPM that is operated by specific transporters of this third barrier. In this review, we recapitulate and compare the strategies developed by Apicomplexa to scavenge nutrients from their hosts, with particular emphasis on transporters at the parasite plasma membrane and vacuolar solute transporters on the parasite intracellular digestive organelle.

Fluorescence-based Heme Quantitation in Toxoplasma Gondii.

Toxoplasma gondii is a highly prevalent protozoan pathogen throughout the world. As a eukaryotic intracellular pathogen, Toxoplasma ingests nutrients from host cells to support its intracellular growth. The parasites also encode full or partial metabolic pathways for the biosynthesis of certain nutrients, such as heme. Heme is an essential nutrient in virtually all living organisms, acting as a co-factor for mitochondrial respiration complexes. Free heme is toxic to cells; therefore, it gets conjugated to proteins or other metabolites to form a "labile heme pool," which is readily available for the biosynthesis of hemoproteins. Previous literature has shown that Toxoplasma gondii carries a fully functional de novo heme biosynthesis pathway and principally depends on this pathway for intracellular survival. Our recent findings also showed that the parasite's intracellular replication is proportional to the total abundance of heme within the cells. Moreover, heme abundance is linked to mitochondrial oxygen consumption for ATP production in these parasites; thus, they may need to regulate their cellular heme levels for optimal infection when present in different environments. Therefore, quantitative measurement of heme abundance within Toxoplasma will help us to understand the roles of heme in subcellular activities such as mitochondrial respiration and other events related to energy metabolism.

Toxoplasma gondii requires its plant-like heme biosynthesis pathway for infection.

Heme, an iron-containing organic ring, is essential for virtually all living organisms by serving as a prosthetic group in proteins that function in diverse cellular activities ranging from diatomic gas transport and sensing, to mitochondrial respiration, to detoxification. Cellular heme levels in microbial pathogens can be a composite of endogenous de novo synthesis or exogenous uptake of heme or heme synthesis intermediates. Intracellular pathogenic microbes switch routes for heme supply when heme availability fluctuates in their replicative environment throughout infection. Here, we show that Toxoplasma gondii, an obligate intracellular human pathogen, encodes a functional heme biosynthesis pathway. A chloroplast-derived organelle, termed apicoplast, is involved in heme production. Genetic and chemical manipulation revealed that de novo heme production is essential for T. gondii intracellular growth and pathogenesis. Surprisingly, the herbicide oxadiazon significantly impaired Toxoplasma growth, consistent with phylogenetic analyses that show T. gondii protoporphyrinogen oxidase is more closely related to plants than mammals. This inhibition can be enhanced by 15- to 25-fold with two oxadiazon derivatives, lending therapeutic proof that Toxoplasma heme biosynthesis is a druggable target. As T. gondii has been used to model other apicomplexan parasites, our study underscores the utility of targeting heme biosynthesis in other pathogenic apicomplexans, such as Plasmodium spp., Cystoisospora, Eimeria, Neospora, and Sarcocystis.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay.

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than Δcpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Toxoplasma Cathepsin Protease B and Aspartyl Protease 1 Are Dispensable for Endolysosomal Protein Digestion.

The lysosome-like vacuolar compartment (VAC) is a major site of proteolysis in the intracellular parasite Toxoplasma gondii Previous studies have shown that genetic ablation of a VAC-residing cysteine protease, cathepsin protease L (CPL), resulted in the accumulation of undigested protein in the VAC and loss of parasite viability during the chronic stage of infection. However, since the maturation of another VAC localizing protease, cathepsin protease B (CPB), is dependent on CPL, it remained unknown whether these defects result directly from ablation of CPL or indirectly from a lack of CPB maturation. Likewise, although a previously described cathepsin D-like aspartyl protease 1 (ASP1) could also play a role in proteolysis, its definitive residence and function in the Toxoplasma endolysosomal system were not well defined. Here, we demonstrate that CPB is not necessary for protein turnover in the VAC and that CPB-deficient parasites have normal growth and viability in both the acute and chronic stages of infection. We also show that ASP1 depends on CPL for correct maturation, and it resides in the T. gondii VAC, where, similar to CPB, it plays a dispensable role in protein digestion. Taken together with previous work, our findings suggest that CPL is the dominant protease in a hierarchy of proteolytic enzymes within the VAC. This unusual lack of redundancy for CPL in T. gondii makes it a single exploitable target for disrupting chronic toxoplasmosis.IMPORTANCE Roughly one-third of the human population is chronically infected with the intracellular single-celled parasite Toxoplasma gondii, but little is known about how this organism persists inside people. Previous research suggested that a parasite proteolytic enzyme, termed cathepsin protease L, is important for Toxoplasma persistence; however, it remained possible that other associated proteolytic enzymes could also be involved in the long-term survival of the parasite during infection. Here, we show that two proteolytic enzymes associated with cathepsin protease L play dispensable roles and are dependent on cathepsin L to reach maturity, which differs from the corresponding enzymes in humans. These findings establish a divergent hierarchy of proteases and help focus attention principally on cathepsin protease L as a potential target for interrupting Toxoplasma chronic infection.

An ortholog of Plasmodium falciparum chloroquine resistance transporter (PfCRT) plays a key role in maintaining the integrity of the endolysosomal system in Toxoplasma gondii to facilitate host invasion.

Toxoplasma gondii is an apicomplexan parasite with the ability to use foodborne, zoonotic, and congenital routes of transmission that causes severe disease in immunocompromised patients. The parasites harbor a lysosome-like organelle, termed the "Vacuolar Compartment/Plant-Like Vacuole" (VAC/PLV), which plays an important role in maintaining the lytic cycle and virulence of T. gondii. The VAC supplies proteolytic enzymes that contribute to the maturation of invasion effectors and that digest autophagosomes and endocytosed host proteins. Previous work identified a T. gondii ortholog of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) that localized to the VAC. Here, we show that TgCRT is a membrane transporter that is functionally similar to PfCRT. We also genetically ablate TgCRT and reveal that the TgCRT protein plays a key role in maintaining the integrity of the parasite's endolysosomal system by controlling morphology of the VAC. When TgCRT is absent, the VAC dramatically increases in volume by ~15-fold and overlaps with adjacent endosome-like compartments. Presumably to reduce aberrant swelling, transcription and translation of endolysosomal proteases are decreased in ΔTgCRT parasites. Expression of subtilisin protease 1 is significantly reduced, which impedes trimming of microneme proteins, and significantly decreases parasite invasion. Chemical or genetic inhibition of proteolysis within the VAC reverses these effects, reducing VAC size and partially restoring integrity of the endolysosomal system, microneme protein trimming, and invasion. Taken together, these findings reveal for the first time a physiological role of TgCRT in substrate transport that impacts VAC volume and the integrity of the endolysosomal system in T. gondii.

Toxoplasma depends on lysosomal consumption of autophagosomes for persistent infection.

Globally, nearly 2 billion people are infected with the intracellular protozoan Toxoplasma gondii1. This persistent infection can cause severe disease in immunocompromised people and is epidemiologically linked to major mental illnesses2 and cognitive impairment3. There are currently no options for curing this infection. The lack of effective therapeutics is due partly to a poor understanding of the essential pathways that maintain long-term infection. Although it is known that Toxoplasma replicates slowly within intracellular cysts demarcated with a cyst wall, precisely how it sustains itself and remodels organelles in this niche is unknown. Here, we identify a key role for proteolysis within the parasite lysosomal organelle (the vacuolar compartment or VAC) in turnover of autophagosomes and persistence during neural infection. We found that disrupting a VAC-localized cysteine protease compromised VAC digestive function and markedly reduced chronic infection. Death of parasites lacking the VAC protease was preceded by accumulation of undigested autophagosomes in the parasite cytoplasm. These findings suggest an unanticipated function for parasite lysosomal degradation in chronic infection, and identify an intrinsic role for autophagy in the T. gondii parasite and its close relatives. This work also identifies a key element of Toxoplasma persistence and suggests that VAC proteolysis is a prospective target for pharmacological development.

Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component.

The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concentrating-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and ß, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins. In contrast to the ß-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystallization. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for ß-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

Toxoplasma gondii ingests and digests host cytosolic proteins.

The protozoan parasite Toxoplasma gondii resides within a nonfusogenic vacuole during intracellular replication. Although the limiting membrane of this vacuole provides a protective barrier to acidification and degradation by lysosomal hydrolases, it also physically segregates the parasite from the host cytosol. Accordingly, it has been suggested that T. gondii acquires material from the host via membrane channels or transporters. The ability of the parasite to internalize macromolecules via endocytosis during intracellular replication has not been tested. Here, we show that Toxoplasma ingests host cytosolic proteins and digests them using cathepsin L and other proteases within its endolysosomal system. Ingestion was reduced in mutant parasites lacking an intravacuolar network of tubular membranes, implicating this apparatus as a possible conduit for trafficking to the parasite. Genetic ablation of proteins involved in the pathway is associated with diminished parasite replication and virulence attenuation. We show that both virulent type I and avirulent type II strain parasites ingest and digest host-derived protein, indicating that the pathway is not restricted to highly virulent strains. The findings provide the first definitive evidence that T. gondii internalizes proteins from the host during intracellular residence and suggest that protein digestion within the endolysosomal system of the parasite contributes to toxoplasmosis. Importance: Toxoplasma gondii causes significant disease in individuals with weak immune systems. Treatment options for this infection have drawbacks, creating a need to understand how this parasite survives within the cells it infects as a prelude to interrupting its survival strategies. This study reveals that T. gondii internalizes proteins from the cytoplasm of the cells it infects and degrades such proteins within a digestive compartment within the parasite. Disruption of proteins involved in the pathway reduced parasite replication and lessened disease severity. The identification of a novel parasite ingestion pathway opens opportunities to interfere with this process and improve the outcome of infection.

A vacuolar-H(+) -pyrophosphatase (TgVP1) is required for microneme secretion, host cell invasion, and extracellular survival of Toxoplasma gondii.

The vacuolar proton pyrophosphatase (H(+) -PPase) of Toxoplasma gondii (TgVP1), a membrane proton pump, localizes to acidocalcisomes and a novel lysosome-like compartment termed plant-like vacuole (PLV) or vacuolar compartment (VAC). We report the characterization of a T. gondii null mutant for the TgVP1 gene. Propagation of these mutants decreased significantly because of deficient attachment and invasion of host cells, which correlated with deficient microneme secretion. Processing of cathepsin L (CPL) in these mutants was deficient only when the parasites were incubated in the presence of low concentrations of the vacuolar H(+) -ATPase (V-H(+) -ATPase) inhibitor bafilomycin A1 , suggesting that either TgVP1 or the T. gondii V-H(+) -ATPase (TgVATPase) are sufficient to support CPL processing. The lack of TgVP1 did not affect processing of micronemal proteins, indicating that it does not contribute to proMIC maturations. The TgVP1 null mutants were more sensitive to extracellular conditions and were less virulent in mice. We demonstrate that T. gondii tachyzoites possess regulatory volume decrease capability during hypo-osmotic stress and this ability is impaired in TgVP1 null mutants implicating TgVP1 in osmoregulation. We hypothesize that osmoregulation is needed for host cell invasion and that TgVP1 plays a role during the normal lytic cycle of T. gondii.

Characterization of the chloroquine resistance transporter homologue in Toxoplasma gondii.

Mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein confer resistance to the antimalarial drug chloroquine. PfCRT localizes to the parasite digestive vacuole, the site of chloroquine action, where it mediates resistance by transporting chloroquine out of the digestive vacuole. PfCRT belongs to a family of transporter proteins called the chloroquine resistance transporter family. CRT family proteins are found throughout the Apicomplexa, in some protists, and in plants. Despite the importance of PfCRT in drug resistance, little is known about the evolution or native function of CRT proteins. The apicomplexan parasite Toxoplasma gondii contains one CRT family protein. We demonstrate that T. gondii CRT (TgCRT) colocalizes with markers for the vacuolar (VAC) compartment in these parasites. The TgCRT-containing VAC is a highly dynamic organelle, changing its morphology and protein composition between intracellular and extracellular forms of the parasite. Regulated knockdown of TgCRT expression resulted in modest reduction in parasite fitness and swelling of the VAC, indicating that TgCRT contributes to parasite growth and VAC physiology. Together, our findings provide new information on the role of CRT family proteins in apicomplexan parasites.