A pyruvate transporter in the apicoplast of apicomplexan parasites.

Pyruvate lies at a pivotal node of carbon metabolism in eukaryotes. It is involved in diverse metabolic pathways in multiple organelles, and its interorganelle shuttling is crucial for cell fitness. Many apicomplexan parasites harbor a unique organelle called the apicoplast that houses metabolic pathways like fatty acid and isoprenoid precursor biosyntheses, requiring pyruvate as a substrate. However, how pyruvate is supplied in the apicoplast remains enigmatic. Here, deploying the zoonotic parasite Toxoplasma gondii as a model apicomplexan, we identified two proteins residing in the apicoplast membranes that together constitute a functional apicoplast pyruvate carrier (APC) to mediate the import of cytosolic pyruvate. Depletion of APC results in reduced activities of metabolic pathways in the apicoplast and impaired integrity of this organelle, leading to parasite growth arrest. APC is a pyruvate transporter in diverse apicomplexan parasites, suggesting a common strategy for pyruvate acquisition by the apicoplast in these clinically relevant intracellular pathogens.

Two enzymes contribute to citrate production in the mitochondrion of Toxoplasma gondii.

Citrate synthase catalyzes the first and the rate-limiting reaction of the tricarboxylic acid (TCA) cycle, producing citrate from the condensation of oxaloacetate and acetyl-coenzyme A. The parasitic protozoan Toxoplasma gondii has full TCA cycle activity but its physiological roles remain poorly understood. In this study, we identified three proteins with predicted citrate synthase (CS) activities and two of which were localized in the mitochondrion, including the 2-methylcitrate synthase (PrpC) that was thought to be involved in the 2-methylcitrate cycle, an alternative pathway for propionyl-CoA detoxification. Further analyses on the two mitochondrial enzymes showed that both had citrate synthase activity, but the catalytic efficiency of CS1 was much higher than that of PrpC. Consistently, deletion of CS1 resulted in significantly reduced flux of glucose derived carbons into TCA cycle intermediates, leading to decreased parasite growth. In contrast, disruption of PrpC had little effect. On the other hand, simultaneous disruption of both CS1 and PrpC resulted in more severe metabolic changes and growth defects than single deletion of either gene, suggesting that PrpC does contribute to citrate production under physiological conditions. Interestingly, deleting Δcs1 and Δprpc individually or in combination only mildly or negligibly affected the virulence of parasites in mice, suggesting that both enzymes are dispensable in vivo. The dispensability of CS1 and PrpC suggests that either the TCA cycle is not essential for the asexual reproduction of tachyzoites, or there are other routes of citrate supply in the parasite mitochondrion.

Essential role of pyrophosphate homeostasis mediated by the pyrophosphate-dependent phosphofructokinase in Toxoplasma gondii.

Many biosynthetic pathways produce pyrophosphate (PPi) as a by-product, which is cytotoxic if accumulated at high levels. Pyrophosphatases play pivotal roles in PPi detoxification by converting PPi to inorganic phosphate. A number of apicomplexan parasites, including Toxoplasma gondii and Cryptosporidium parvum, express a PPi-dependent phosphofructokinase (PPi-PFK) that consumes PPi to power the phosphorylation of fructose-6-phosphate. However, the physiological roles of PPi-PFKs in these organisms are not known. Here, we report that Toxoplasma expresses both ATP- and PPi-dependent phosphofructokinases in the cytoplasm. Nonetheless, only PPi-PFK was indispensable for parasite growth, whereas the deletion of ATP-PFK did not affect parasite proliferation or virulence. The conditional depletion of PPi-PFK completely arrested parasite growth, but it did not affect the ATP level and only modestly reduced the flux of central carbon metabolism. However, PPi-PFK depletion caused a significant increase in cellular PPi and decreased the rates of nascent protein synthesis. The expression of a cytosolic pyrophosphatase in the PPi-PFK depletion mutant reduced its PPi level and increased the protein synthesis rate, therefore partially rescuing its growth. These results suggest that PPi-PFK has a major role in maintaining pyrophosphate homeostasis in T. gondii. This role may allow PPi-PFK to fine-tune the balance of catabolism and anabolism and maximize the utilization efficiency for carbon nutrients derived from host cells, increasing the success of parasitism. Moreover, PPi-PFK is essential for parasite propagation and virulence in vivo but it is not present in human hosts, making it a potential drug target to combat toxoplasmosis.

Two apicoplast dwelling glycolytic enzymes provide key substrates for metabolic pathways in the apicoplast and are critical for Toxoplasma growth.

Many apicomplexan parasites harbor a non-photosynthetic plastid called the apicoplast, which hosts important metabolic pathways like the methylerythritol 4-phosphate (MEP) pathway that synthesizes isoprenoid precursors. Yet many details in apicoplast metabolism are not well understood. In this study, we examined the physiological roles of four glycolytic enzymes in the apicoplast of Toxoplasma gondii. Many glycolytic enzymes in T. gondii have two or more isoforms. Endogenous tagging each of these enzymes found that four of them were localized to the apicoplast, including pyruvate kinase2 (PYK2), phosphoglycerate kinase 2 (PGK2), triosephosphate isomerase 2 (TPI2) and phosphoglyceraldehyde dehydrogenase 2 (GAPDH2). The ATP generating enzymes PYK2 and PGK2 were thought to be the main energy source of the apicoplast. Surprisingly, deleting PYK2 and PGK2 individually or simultaneously did not cause major defects on parasite growth or virulence. In contrast, TPI2 and GAPDH2 are critical for tachyzoite proliferation. Conditional depletion of TPI2 caused significant reduction in the levels of MEP pathway intermediates and led to parasite growth arrest. Reconstitution of another isoprenoid precursor synthesis pathway called the mevalonate pathway in the TPI2 depletion mutant partially rescued its growth defects. Similarly, knocking down the GAPDH2 enzyme that produces NADPH also reduced isoprenoid precursor synthesis through the MEP pathway and inhibited parasite proliferation. In addition, it reduced de novo fatty acid synthesis in the apicoplast. Together, these data suggest a model that the apicoplast dwelling TPI2 provides carbon source for the synthesis of isoprenoid precursor, whereas GAPDH2 supplies reducing power for pathways like MEP, fatty acid synthesis and ferredoxin redox system in T. gondii. As such, both enzymes are critical for parasite growth and serve as potential targets for anti-toxoplasmic intervention designs. On the other hand, the dispensability of PYK2 and PGK2 suggest additional sources for energy in the apicoplast, which deserves further investigation.

Metabolic flexibilities and vulnerabilities in the pentose phosphate pathway of the zoonotic pathogen Toxoplasma gondii.

Metabolic pathways underpin the growth and virulence of intracellular parasites and are therefore promising antiparasitic targets. The pentose phosphate pathway (PPP) is vital in most organisms, providing a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) and ribose sugar for nucleotide synthesis; however, it has not yet been studied in Toxoplasma gondii, a widespread intracellular pathogen and a model protozoan organism. Herein, we show that T. gondii has a functional PPP distributed in the cytoplasm and nucleus of its acutely-infectious tachyzoite stage. We produced eight parasite mutants disrupting seven enzymes of the PPP in T. gondii. Our data show that of the seven PPP proteins, the two glucose-6-phosphate dehydrogenases (TgG6PDH1, TgG6PDH2), one of the two 6-phosphogluconate dehydrogenases (Tg6PGDH1), ribulose-5-phosphate epimerase (TgRuPE) and transaldolase (TgTAL) are dispensable in vitro as well as in vivo, disclosing substantial metabolic plasticity in T. gondii. Among these, TgG6PDH2 plays a vital role in defense against oxidative stress by the pathogen. Further, we show that Tg6PGDH2 and ribulose-5-phosphate isomerase (TgRPI) are critical for tachyzoite growth. The depletion of TgRPI impairs the flux of glucose in central carbon pathways, and causes decreased expression of ribosomal, microneme and rhoptry proteins. In summary, our results demonstrate the physiological need of the PPP in T. gondii while unraveling metabolic flexibility and antiparasitic targets.

The beta subunit of AMP-activated protein kinase is critical for cell cycle progression and parasite development in Toxoplasma gondii.

Toxoplasma gondii is a widespread eukaryotic pathogen that causes life-threatening diseases in humans and diverse animals. It has a complex life cycle with multiple developmental stages, which are timely adjusted according to growth conditions. But the regulatory mechanisms are largely unknown. Here we show that the AMP-activated protein kinase (AMPK), a key regulator of energy homeostasis in eukaryotes, plays crucial roles in controlling the cell cycle progression and bradyzoite development in Toxoplasma. Deleting the ß regulatory subunit of AMPK in the type II strain ME49 caused massive DNA damage and increased spontaneous conversion to bradyzoites (parasites at chronic infection stage), leading to severe growth arrest and reduced virulence of the parasites. Under alkaline stress, all Δampkß mutants converted to a bradyzoite-like state but the cell division pattern was significantly impaired, resulting in compromised parasite viability. Moreover, we found that phosphorylation of the catalytic subunit AMPKα was greatly increased in alkaline stressed parasites, whereas AMPKß deletion mutants failed to do so. Phosphoproteomics found that many proteins with predicted roles in cell cycle and cell division regulation were differentially phosphorylated after AMPKß deletion, under both normal and alkaline stress conditions. Together, these results suggest that the parasite AMPK has critical roles in safeguarding cell cycle progression, and guiding the proper exist of the cell cycle to form mature bradyzoites when the parasites are stressed. Consistent with this model, growth of parasites was not significantly altered when AMPKß was deleted in a strain that was naturally reluctant to bradyzoite development.

Seroprevalence and risk factors of Toxoplasma gondii in urban cats from China.

BACKGROUND: Toxoplasma gondii infects almost all warm-blooded animals, and cats play a crucial role in the epidemiology of T. gondii as the definitive host. Despite sporadic reports on the seroprevalence of T. gondii in domestic cats, systematic surveys are lacking and some regions remain in China uninvestigated. METHODS: A total of 1,521 serum samples were collected from 10 regions of China and analyzed by antibodies against T. gondii by ELISA with the purpose of identifying risk factors of T. gondii infection in cats across China and obtaining seroprevalence data from some previously uninvestigated areas. RESULTS: Antibodies to T. gondii were detected in 62 of 1,478 (4.2%) urban pet cats and in 9 of 43 (20.9%) stray cats. Among the regions examined, the prevalence was 13% in Sichuan, 12.8% in Chongqing, 6.4% in Hunan, 2.5% in Hubei and 0.9% in Guangdong. Additionally, this is the first report on the seroprevalence of T. gondii in urban pet cats from Qinghai (6.2%), Anhui (3.1%), Jiangxi (2.5%), Shaanxi (2.4%) and Ningxia (1.6%). The age and lifestyle (stray or pet) of cats were identified as the risk factors for seropositivity by multivariate analysis of the data. CONCLUSIONS: Our findings improve our understanding of seroprevalence and risk factors of T. gondii infection in cats across China, and provide useful information for the formulating of preventive and control measures against this widespread zoonotic parasite.

Evaluation of immunoprotective effects of recombinant proteins and DNA vaccines derived from Eimeria tenella surface antigen 6 and 15 in vivo.

Coccidiosis is an intestinal parasitic disease that causes huge economic losses to the poultry industry globally. Eimeria tenella belonging to protozoon is the causative agent of cecal coccidiosis in chicken, and it causes enormous damage to poultry industry. The surface antigens (SAGs) of apicomplexan parasites have functions of attachment and invasion in host-parasite interaction. As a result of parasitic invasion, host immune response is triggered. However, the immunogenicity and potency of E. tenella surface antigen 6 and 15 (EtSAG 6 and 15), as vaccinal candidate antigen, remain largely unknown. Therefore, gene fragments of E. tenella EtSAG 6 and 15 were amplified and transformed to pET28a prokaryotic vector for recombinant protein expression. The pEGFP-N1 eukaryotic vectors with EtSAG 6 and 15 amplification fragments (pEGFP-N1-EtSAG 5 and 16) were transformed into 293 T cell line. The results of reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis revealed successful expressions of EtSAG 6 and 15 in Escherichia coli and 293 T cells. Subsequently, animal experiments of 49 cobb broilers were performed to evaluate immunoprotection of recombinant proteins and DNA vaccines derived from E. tenella EtSAG 5 and 16 with an immunizing dose of 100 µg, respectively. Chickens vaccinated with rEtSAG 6 protein, rEtSAG 15 protein, pEGFP-N1-EtSAG 6 plasmid, or pEGFP-N1-EtSAG 15 plasmid showed no significant increase in IFN-γor interleukin-4 (IL-4) level compared with control groups. Chickens vaccinated with protein rEtSAG 6, protein rEtSAG 15, pEGFP-N1-EtSAG 6 plasmid, or pEGFP-N1-EtSAG 15 exhibited higher weight gains, lower oocyst output, and lower mean lesion scores, compared with infection control group. Among the four immunized groups, plasmid EGFP-N1-EtSAG 6 (100 µg) group exhibited the highest anticoccidial index (ACI) value (150.20). Overall, plasmids EGFP-N1-EtSAG 6 and 15, as DNA vaccines, provided a more effective immunoprotection for chickens against E. tenella than protein rEtSAG 6 and protein rEtSAG 15 as subunit vaccines. EtSAG 6 and 15 are promising candidate antigen genes for developing coccidiosis vaccine.

Acquisition of exogenous fatty acids renders apicoplast-based biosynthesis dispensable in tachyzoites of Toxoplasma.

Toxoplasma gondii is a common protozoan parasite that infects a wide range of hosts, including livestock and humans. Previous studies have suggested that the type 2 fatty acid synthesis (FAS2) pathway, located in the apicoplast (a nonphotosynthetic plastid relict), is crucial for the parasite's survival. Here we examined the physiological relevance of fatty acid synthesis in T. gondii by focusing on the pyruvate dehydrogenase complex and malonyl-CoA-[acyl carrier protein] transacylase (FabD), which are located in the apicoplast to drive de novo fatty acid biosynthesis. Our results disclosed unexpected metabolic resilience of T. gondii tachyzoites, revealing that they can tolerate CRISPR/Cas9-assisted genetic deletions of three pyruvate dehydrogenase subunits or FabD. All mutants were fully viable in prolonged cultures, albeit with impaired growth and concurrent loss of the apicoplast. Even more surprisingly, these mutants displayed normal virulence in mice, suggesting an expendable role of the FAS2 pathway in vivo Metabolic labeling of the Δpdh-e1α mutant showed reduced incorporation of glucose-derived carbon into fatty acids with medium chain lengths (C14:0 and C16:0), revealing that FAS2 activity was indeed compromised. Moreover, supplementation of exogenous C14:0 or C16:0 significantly reversed the growth defect in the Δpdh-e1α mutant, indicating salvage of these fatty acids. Together, these results demonstrate that the FAS2 pathway is dispensable during the lytic cycle of Toxoplasma because of its remarkable flexibility in acquiring fatty acids. Our findings question the long-held assumption that targeting this pathway has significant therapeutic potential for managing Toxoplasma infections.

The Sec1/Munc18-like proteins TgSec1 and TgVps45 play pivotal roles in assembly of the pellicle and sub-pellicle network in Toxoplasma gondii.

Post-Golgi vesicle trafficking is indispensable for precise movement of proteins to the pellicle, the sub-pellicle network and apical secretory organelles in Apicomplexa. However, only a small number of molecular complexes involved in trafficking, tethering and fusion of vesicles have been identified in Toxoplasma gondii. Consequently, it is unclear how complicated vesicle trafficking is accomplished in this parasite. Sec1/Munc18-like (SM) proteins are essential components of protein complexes involved in vesicle fusion. Here, we found that depletion of the SM protein TgSec1 using an auxin-inducible degron-based conditional knockout strategy led to mislocalization of plasma membrane proteins. By contrast, conditional depletion of the SM protein TgVps45 led to morphological changes, asymmetrical loss of the inner membrane complex and defects in nucleation of sub-pellicular microtubules, polarization and symmetrical assembly of daughter parasites during repeated endodyogeny. TgVps45 interacts with the SNARE protein TgStx16 and TgVAMP4-1. Conditional ablation of TgStx16 causes the similar growth defect like TgVps45 deficiency suggested they work together for the vesicle fusion at TGN. These findings indicate that these two SM proteins are crucial for assembly of pellicle and sub-pellicle network in T. gondii respectively.

Antitumor effects of a Toxoplasma mutant lacking lactate dehydrogenases.

"Bug as drug" is a concept recognized over a century ago and has gained significant research attention recently for fighting diseases such as immune disorders and others. Bacteria and viruses are constantly studied for this purpose, but the use of parasitic organisms is still rare. Recently, we found that Toxoplasma gondii mutants lacking two lactate dehydrogenases (ME49 Δldh1-Δldh2) were avirulent in mice but able to stimulate high levels of Th1 immunity. This outcome prompted us to determine whether Δldh mutants also displayed antitumor activities. Using a mouse melanoma model, we showed that intratumoral administration of Δldh1-Δldh2 repressed the growth of established tumors and helped to inhibit lethal tumor development in the mice. The sera of parasite-treated mice had high levels of TNF-α and INF-γ, which likely contributed to the tumor-repressing activity. We also found that chronic Toxoplasma infection, which is common in animals and humans, also led to antitumor activity. In addition, pre-existing chronic infections did not affect the antitumor efficiency of the Δldh1-Δldh2 mutant. Together, these results suggest that the attenuated T. gondii mutant Δldh1-Δldh2 has the potential to be a good antitumor therapy and provide new insights into the development of novel tumor therapeutics.

Prevalence of Eimeria parasites in the Hubei and Henan provinces of China.

Coccidiosis is an intestinal parasitic disease that causes huge economic losses in the poultry industry globally. Henan and Hubei, as important poultry production provinces in China, have great pressure for the prevention and control of chicken coccidiosis. In order to obtain information on the local prevalence of Eimeria species, we used an internal transcribed spacer 1 (ITS1) sequence of ribosomal DNA to identify the species from 318 fresh fecal samples. The fecal samples and the data relating to farm information were collected from 137 farms in Hubei and Henan provinces. As shown by genus-specific PCR results, the positivity rate of Eimeria was 97.17% (309/318), and the most common species were Eimeria mitis (66.67%), E. tenella (46.86%), and E. necatrix (41.51%). Then, we analyzed the correlation between the background information of each sample and the PCR identification results, which showed that indigenous farms in Henan province were at the greatest risk of harboring highly pathogenic Eimeria species and a larger proportion of such farms were positive for E. necatrix, the most pathogenic species. The results of this study showed that chicken coccidia was widespread, which provides important insights into the control of chicken coccidiosis in this region.

Transcriptomic analyses reveal distinct response of porcine macrophages to Toxoplasma gondii infection.

Toxoplasma gondii is an obligate protozoan parasite infecting diverse hosts. Studies have demonstrated that different hosts respond differently to Toxoplasma infection. Pigs are among the most susceptible hosts of T. gondii, but the host-pathogen interactions that shape the outcome of infection in pigs are completely unknown. Here, we used dual RNA-seq to profile the transcriptomic changes of porcine alveolar macrophages (PAMs) upon Toxoplasma infection. Our results indicated that PAMs initiated different responses to Toxoplasma infection compared with mouse macrophages. First, although infected PAMs upregulated numerous pro-inflammatory factors, IL-12, which plays critical roles in IL-12~IFN-γ-mediated immunity against Toxoplasma infection in mice, was found unchanged during PAM infection. Second, the gene encoding iNOS that is responsible for nitric oxide (NO) production was also not induced in infected PAMs. Consistently, there was no NO level change in PAMs after infection. Third, it seems like Toxoplasma infection inhibited apoptosis in PAMs. On the parasite side, the most obvious change is the upregulation of genes involved in metabolism and macromolecule synthesis, such as the type II fatty acid synthesis in the apicoplast. Together, these results revealed distinct responses of PAMs to Toxoplasma infection and provide novel insights into Toxoplasma-pig interactions.

Identification of Novel Dense-Granule Proteins in Toxoplasma gondii by Two Proximity-Based Biotinylation Approaches.

Toxoplasma gondii is an opportunistic pathogen infecting humans and a variety of vertebrate animals. Secretory dense-granule proteins (GRAs) play diverse roles in the mediation of host-parasite interactions and facilitate parasitism, but many of them still remain to be identified. Here, we used two proximity-based protein labeling techniques to identify novel GRA proteins. Taking GRA1 as bait, transgenic strains expressing GRA1-BirA* or GRA1-APEX were constructed to biotinylate GRAs. Using these methods, a total of 46 proteins were identified, 20 of which were known GRA proteins. Among these 46, 17 were identified by both strategies, and 14 out of the 17 were known GRAs. The other three were all confirmed to localize to dense granules. Nonetheless a significant portion of the proteins were only identified by either APEX or BirA*, indicating that there are differences between these methods. Of the 26 novel GRAs, 5 were validated as bona fide GRAs by localization studies. The majority of these novel GRAs are only present in coccidian parasites and are likely dispensable for parasite growth in vitro; they may play roles during animal infections. The identification of novel GRAs laid the foundation for further studies investigating the mechanisms underlying parasite-host interactions.

Functional analysis of Toxoplasma lactate dehydrogenases suggests critical roles of lactate fermentation for parasite growth in vivo.

Glycolysis was thought to be the major pathway of energy supply in both fast-replicating tachyzoites and slowly growing bradyzoites of Toxoplasma gondii. However, its biological significance has not been clearly verified. The genome of T. gondii encodes two lactate dehydrogenases (LDHs), which are differentially expressed in tachyzoites and bradyzoites. In this study, we knocked out the two LDH genes individually and in combination and found that neither gene was required for tachyzoite growth in vitro under standard growth conditions. However, during infection in mice, Δldh1 and Δldh1 Δldh2 mutants were unable to propagate and displayed significant virulence attenuation and cyst formation defects. LDH2 only played minor roles in these processes. To further elucidate the mechanisms underlying the critical requirement of LDH in vivo, we found that Δldh1 Δldh2 mutants replicated significantly more slowly than wild-type parasites when cultured under conditions with physiological levels of oxygen (3%). In addition, Δldh1 Δldh2 mutants were more susceptible to the oxidative phosphorylation inhibitor oligomycin A. Together these results suggest that lactate fermentation is critical for parasite growth under physiological conditions, likely because energy production from oxidative phosphorylation is insufficient when oxygen is limited and lactate fermentation becomes a key supplementation.

Synthesis and in vitro and in vivo anti-inflammatory activity of novel 4-ferrocenylchroman-2-one derivatives.

A series of novel 4-ferrocenylchroman-2-one derivatives were designed and synthesised to discover potent anti-inflammatory agents for treatment of arthritis. All the target compounds had been screened for their anti-inflammatory activity by evaluating the inhibition effect of LPS-induced NO production in RAW 264.7 macrophages. Among them, 4-ferrocenyl-3,4-dihydro-2H-benzo[g]chromen-2-one (3h) was found to be the most potent compound in inhibiting the productions of NO with low toxicity. This compound also exhibited significant inhibition of the productions of IL-6 and TNF-α in RAW 264.7 macrophages. Preliminary mechanism studies indicated that compound 3h could inhibit the activation of LPS-induced NF-κB and MAPKs signalling pathways. The in vivo anti-inflammatory effect of this compound was determined in the rat adjuvant-induced arthritis model.

Seroprevalence of Toxoplasma gondii in one-humped camels (Camelus dromedarius) of Thal and Cholistan deserts, Punjab, Pakistan.

Toxoplasma (T.) gondii is an important zoonotic protozoan infecting humans and a wide range of animals. In this study, we determine the seroprevalence and risk factors associated with the seroprevalence of T. gondii in one-humped camels (Camelus dromedarius) in Pakistan. Camels are still an important mean of transportation in some desert areas in Pakistan. In addition, they are the main source of meat and milk for people in those regions; therefore, they have the potential to transmit T. gondii to humans. In order to estimate the seroprevalence of T. gondii, a total of 897 sera samples were collected from camels in the Thal (n = 359) and Cholistan (n = 440) deserts, along with other districts of Chakwal (n = 44) and Faisalabad (n = 54) Punjab, Pakistan, through convenient and snowball sampling techniques. These samples were then analyzed by an indirect enzyme-linked immune-sorbent assay (ELISA) for the presence of T. gondii-specific antibodies, using purified recombinant micronemal protein 3 (MIC3) as an antibody-catching antigen. Our results showed an overall seroprevalence of T. gondii as 40.1% (Thal = 45%; Cholistan = 35.9%; other districts = 33.7%). Risk factor analysis suggested that infection rate was higher in older animals (70.6%). In addition, female camels carried frequent infection (48.8%) than males (22.4%). What's more, female animals having abortion history showed even higher infection rate (75%) compared to pregnant (68.4%) and non-pregnant (42.4%) animals. Our results reported high seroprevelance of T. gondii in camels in Pakistan which provided important information with respect to public health and disease controls.

Micronemal protein 13 contributes to the optimal growth of Toxoplasma gondii under stress conditions.

Toxoplasma gondii is a ubiquitous parasitic protozoan infecting humans and a wide variety of animals. Fast-replicating tachyzoites during acute infection and slowly growing bradyzoites during chronic infection are the two basic forms of T. gondii in intermediate hosts. Interconversion between the two contributes to the transmission and pathogenesis of this parasite. Secretory micronemal proteins are thought to mediate interactions with host cells and facilitate parasite invasion, therefore the majority of them are highly expressed in tachyzoites. Micronemal protein 13 (MIC13) is unique in that its expression is low in tachyzoites and is upregulated under bradyzoite-inducing conditions. Previous attempts to disrupt this gene were not successful, implying that it may play critical roles during parasite growth. However, in this study, MIC13 was successfully disrupted in type 1 strain RH and type 2 strain ME49 using CRISPR/Cas9-mediated gene disruption techniques. Consistent with its low expression in tachyzoites and increased expression under stress or bradyzoite-inducing conditions, MIC13-inactivated mutants displayed normal growth, host cell invasion, intracellular replication, and egress, as well as acute virulence at the tachyzoite stage. However, under stress conditions, such as high pH or oxygen limitation, MIC13-disrupted parasites showed significantly slower growth rates compared to the parental strains, suggesting that it is required for optimal parasite growth under bradyzoite-inducing or stress conditions. This is the first micronemal protein reported to have such expression pattern and function modes, which expands our understanding of the diverse functions of micronemal proteins.

Toxoplasma aldolase is required for metabolism but dispensable for host-cell invasion.

Gliding motility and host-cell invasion by apicomplexan parasites depend on cell-surface adhesins that are translocated via an actin-myosin motor beneath the membrane. The current model posits that fructose-1,6-bisphosphate aldolase (ALD) provides a critical link between the cytoplasmic tails of transmembrane adhesins and the actin-myosin motor. Here we tested this model using the Toxoplasma gondii apical membrane protein 1 (TgAMA1), which binds to aldolase in vitro. TgAMA1 cytoplasmic tail mutations that disrupt ALD binding in vitro showed no correlation with host-cell invasion, indicating this interaction is not essential. Furthermore, ALD-depleted parasites were impaired when grown in glucose, yet they showed normal gliding and invasion in glucose-free medium. Depletion of ALD in the presence of glucose led to accumulation of fructose-1,6-bisphosphate, which has been associated with toxicity in other systems. Finally, TgALD knockout parasites and an ALD mutant that specifically disrupts adhesin binding in vitro also supported normal invasion when cultured in glucose-free medium. Taken together, these results suggest that ALD is primarily important for energy metabolism rather than interacting with microneme adhesins, challenging the current model for apicomplexan motility and invasion.

Identification of host proteins, Spata3 and Dkk2, interacting with Toxoplasma gondii micronemal protein MIC3.

As an obligate intracellular protozoan, Toxoplasma gondii is a successful pathogen infecting a variety of animals, including humans. As an adhesin involving in host invasion, the micronemal protein MIC3 plays important roles in host cell attachment, as well as modulation of host EGFR signaling cascade. However, the specific host proteins that interact with MIC3 are unknown and the identification of such proteins will increase our understanding of how MIC3 exerts its functions. This study was designed to identify host proteins interacting with MIC3 by yeast two-hybrid screens. Using MIC3 as bait, a library expressing mouse proteins was screened, uncovering eight mouse proteins that showed positive interactions with MIC3. Two of which, spermatogenesis-associated protein 3 (Spata3) and dickkopf-related protein 2 (Dkk2), were further confirmed to interact with MIC3 by additional protein-protein interaction tests. The results also revealed that the tandem repeat EGF domains of MIC3 were critical in mediating the interactions with the identified host proteins. This is the first study to show that MIC3 interacts with host proteins that are involved in reproduction, growth, and development. The results will provide a clearer understanding of the functions of adhesion-associated micronemal proteins in T. gondii.