Mendelian segregation and high recombination rates facilitate genetic analyses in Cryptosporidium parvum.

Very little is known about the process of meiosis in the apicomplexan parasite Cryptosporidium despite the essentiality of sex in its life cycle. Most cell lines only support asexual growth of Cryptosporidium parvum (C. parvum), but stem cell derived intestinal epithelial cells grown under air-liquid interface (ALI) conditions support the sexual cycle. To examine chromosomal dynamics during meiosis in C. parvum, we generated two transgenic lines of parasites that were fluorescently tagged with mCherry or GFP on chromosomes 1 or 5, respectively. Infection of ALI cultures or Ifngr1-/- mice with mCherry and GFP parasites resulted in cross-fertilization and the formation of "yellow" oocysts, which contain 4 haploid sporozoites that are the product of meiosis. Recombinant oocysts from the F1 generation were purified and used to infect HCT-8 cultures, and phenotypes of the progeny were observed by microscopy. All possible phenotypes predicted by independent segregation were represented equally (~25%) in the population, indicating that C. parvum chromosomes exhibit a Mendelian inheritance pattern. The most common pattern observed from the outgrowth of single oocysts included all possible parental and recombinant phenotypes derived from a single meiotic event, suggesting a high rate of crossover. To estimate the frequency of crossover, additional loci on chromosomes 1 and 5 were tagged and used to monitor intrachromosomal crosses in Ifngr1-/- mice. Both chromosomes showed a high frequency of crossover compared to other apicomplexans with map distances (i.e., 1% recombination) of 3-12 kb. Overall, a high recombination rate may explain many unique characteristics observed in Cryptosporidium spp. such as high rates of speciation, wide variation in host range, and rapid evolution of host-specific virulence factors.

Combination of inhibitors for two glycolytic enzymes portrays high synergistic efficacy against Cryptosporidium parvum.

Cryptosporidium is an intracellular protozoan parasite that causes serious enteric disease in humans and in a wide range of animals worldwide. Despite its high prevalence, no effective therapeutic drugs are available against life-threatening cryptosporidiosis in at-risk populations including malnourished children, immunocompromised patients, and neonatal calves. Thus, new efficacious drugs are urgently needed to treat all susceptible populations with cryptosporidiosis. Unlike other apicomplexans, Cryptosporidium parvum lacks the tricarboxylic acid cycle and the oxidative phosphorylation steps, making it solely dependent on glycolysis for metabolic energy production. We have previously reported that individual inhibitors of two unique glycolytic enzymes, the plant-like pyruvate kinase (CpPyK) and the bacterial-type lactate dehydrogenase (CpLDH), are effective against C. parvum, both in vitro and in vivo. Herein, we have derived combinations of CpPyK and CpLDH inhibitors with strong synergistic effects against the growth and survival of C. parvum, both in vitro and in an infection mouse model. In infected immunocompromised mice, compound combinations of NSC303244 + NSC158011 and NSC252172 + NSC158011 depicted enhanced efficacy against C. parvum reproduction and ameliorated intestinal lesions of cryptosporidiosis at doses fourfold lower than the total effective doses of individual compounds. Importantly, unlike individual compounds, NSC303244 + NSC158011 combination was effective in clearing the infection completely without relapse in immunocompromised mice. Collectively, our study has unveiled compound combinations that simultaneously block two essential catalytic steps for metabolic energy production in C. parvum to achieve improved efficacy against the parasite. These combinations are, therefore, lead compounds for the development of a new generation of efficacious anti-cryptosporidial drugs.

Broad-Spectrum Inhibitors for Conserved Unique Phosphoethanolamine Methyltransferases in Parasitic Nematodes Possess Anthelmintic Efficacy.

In humans, nematode infections are prevalent in developing countries, causing long-term ill health, particularly in children. Worldwide, nematode infections are prevalent in livestock and pets, affecting productivity and health. Anthelmintic drugs are the primary means of controlling nematodes, but there is now high prevalence of anthelmintic resistance, requiring urgent identification of new molecular targets for anthelmintics with novel mechanisms of action. Here, we identified orthologous genes for phosphoethanolamine methyltransferases (PMTs) in nematodes within the families Trichostrongylidae, Dictyocaulidae, Chabertiidae, Ancylostomatoidea, and Ascarididae. We characterized these putative PMTs and found that they possess bona fide PMT catalytic activities. By complementing a mutant yeast strain lacking the ability to synthesize phosphatidylcholine, the PMTs were validated to catalyze the biosynthesis of phosphatidylcholine. Using an in vitro phosphoethanolamine methyltransferase assay with PMTs as enzymes, we identified compounds with cross-inhibitory effects against the PMTs. Corroboratively, treatment of PMT-complemented yeast with the PMT inhibitors blocked growth of the yeast, underscoring the essential role of the PMTs in phosphatidylcholine synthesis. Fifteen of the inhibitors with the highest activity against complemented yeast were tested against Haemonchus contortus using larval development and motility assays. Among them, four were found to possess potent anthelmintic activity against both multiple drug-resistant and susceptible isolates of H. contortus, with IC50 values (95% confidence interval) of 4.30 µM (2.15-8.28), 4.46 µM (3.22-6.16), 28.7 µM (17.3-49.5), and 0.65 µM (0.21-1.88). Taken together, we have validated a molecular target conserved in a broad range of nematodes and identified its inhibitors that possess potent in vitro anthelmintic activity.

Novel lactate dehydrogenase inhibitors with in vivo efficacy against Cryptosporidium parvum.

Cryptosporidium parvum is a highly prevalent zoonotic and anthroponotic protozoan parasite that causes a diarrheal syndrome in children and neonatal livestock, culminating in growth retardation and mortalities. Despite the high prevalence of C. parvum, there are no fully effective and safe drugs for treating infections, and there is no vaccine. We have previously reported that the bacterial-like C. parvum lactate dehydrogenase (CpLDH) enzyme is essential for survival, virulence and growth of C. parvum in vitro and in vivo. In the present study, we screened compound libraries and identified inhibitors against the enzymatic activity of recombinant CpLDH protein in vitro. We tested the inhibitors for anti-Cryptosporidium effect using in vitro infection assays of HCT-8 cells monolayers and identified compounds NSC158011 and NSC10447 that inhibited the proliferation of intracellular C. parvum in vitro, with IC50 values of 14.88 and 72.65 µM, respectively. At doses tolerable in mice, we found that both NSC158011 and NSC10447 consistently significantly reduced the shedding of C. parvum oocysts in infected immunocompromised mice's feces, and prevented intestinal villous atrophy as well as mucosal erosion due to C. parvum. Together, our findings have unveiled promising anti-Cryptosporidium drug candidates that can be explored further for the development of the much needed novel therapeutic agents against C. parvum infections.

Small GTPase Immunity-Associated Proteins Mediate Resistance to Toxoplasma gondii Infection in Lewis Rat.

Rats vary in their susceptibilities to Toxoplasma gondii infection depending on the rat strain. Compared to the T. gondii-susceptible Brown Norway (BN) rat, the Lewis (LEW) rat is extremely resistant to T. gondii Thus, these two rat strains are ideal models for elucidating host mechanisms that are important for host resistance to T. gondii infection. Therefore, in our efforts to unravel molecular factors directing the protective early innate immune response in the LEW rat, we performed RNA sequencing analysis of the LEW versus BN rat with or without T. gondii infection. We identified three candidate small GTPase immunity-associated proteins (GIMAPs) that were upregulated (false discovery rate, 0.05) in the LEW rat in response to T. gondii infection. Subsequently, we engineered T. gondii-susceptible NR8383 rat macrophage cells for overexpression of LEW rat-derived candidate GIMAP 4, 5, and 6. By immunofluorescence analysis we observed that GIMAP 4, 5, and 6 in T. gondii-infected NR8383 cells each colocalized with GRA5, a parasite parasitophorous vacuole membrane (PVM) marker protein, suggesting their translocation to the PVM. Interestingly, overexpression of each candidate GIMAP in T. gondii-infected NR8383 cells induced translocation of LAMP1, a lysosome marker protein, to the T. gondii surface membrane. Importantly, overexpression of GIMAP 4, 5, or 6 individually inhibited intracellular T. gondii growth, with GIMAP 4 having the highest inhibitory effect. Together, our findings indicate that upregulation of GIMAP 4, 5, and 6 contributes to the robust refractoriness of the LEW rat to T. gondii through induction of lysosomal fusion to the otherwise nonfusogenic PVM.

Inherent Oxidative Stress in the Lewis Rat Is Associated with Resistance to Toxoplasmosis.

The course of Toxoplasma gondii infection in rats closely resembles that in humans. However, compared to the Brown Norway (BN) rat, the Lewis (LEW) rat is extremely resistant to T. gondii infection. Thus, we performed RNA sequencing analysis of the LEW rat versus the BN rat, with or without T. gondii infection, in order to unravel molecular factors directing robust and rapid early T. gondii-killing mechanisms in the LEW rat. We found that compared to the uninfected BN rat, the uninfected LEW rat has inherently higher transcript levels of cytochrome enzymes (Cyp2d3, Cyp2d5, and Cybrd1, which catalyze generation of reactive oxygen species [ROS]), with concomitant higher levels of ROS. Interestingly, despite having higher levels of ROS, the LEW rat had lower transcript levels for antioxidant enzymes (lactoperoxidase, microsomal glutathione S-transferase 2 and 3, glutathione S-transferase peroxidase kappa 1, and glutathione peroxidase) than the BN rat, suggesting that the LEW rat maintains cellular oxidative stress that it tolerates. Corroboratively, we found that scavenging of superoxide anion by Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) decreased the refractoriness of LEW rat peritoneal cells to T. gondii infection, resulting in proliferation of parasites in LEW rat peritoneal cells which, in turn, led to augmented cell death in the infected cells. Together, our results indicate that the LEW rat maintains inherent cellular oxidative stress that contributes to resistance to invading T. gondii, and they thus unveil new avenues for developing therapeutic agents targeting induction of host cell oxidative stress as a mechanism for killing T. gondii.

In vitro antagonistic and indifferent activity of combination of 3-deoxyanthocyanidins against Toxoplasma gondii.

Toxoplasma gondii is a ubiquitous intracellular zoonotic parasite estimated to affect about 30-90% of the world's human population. The most affected are immunocompromised individuals such as HIV-AIDS and cancer patients, organ and tissue transplant recipients, and congenitally infected children. No effective and safe drugs and vaccines are available against all forms of the parasite. We report here the antagonistic and indifferent activity of the combination of five different formulations of pure synthetic 3-deoxyanthocyaninidin (3-DA) chloride compounds against T. gondii tachyzoites and the synergistic and additive interaction against a human foreskin fibroblast (HFF) cell line in vitro using fluorescence microscopy, trypan blue assay, and fractional inhibitory concentration index. The individual and the combined pure 3-DA compounds were observed to have effective inhibition against T. gondii parasites with less cytotoxic effect in a ratio of 1:1. The IC50 values for parasite inhibition ranged from 1.88 µg/mL (1.51-2.32 µg/mL) for luteolinindin plus 7-methoxyapigeninindin (LU/7-MAP) and 2.23 µg/mL (1.66-2.97 µg/mL) for apigeninindin plus 7-methoxyapigeninindin (AP/7-MAP) combinations at 95% confidence interval (CI) after 48 h of culture. We found LU/7-MAP to be antagonistic and AP/7-MAP to be indifferent in interaction against T. gondii growth. Both individual and combination 3-DA compounds not only depicted very strong inhibitory activity against T. gondii, but also had synergistic and additive cytotoxic effects against HFF cells. These synthetic 3-DAs have potential as antiparasitic agents for the treatment of human toxoplasmosis.

In vitro activity of Sorghum bicolor extracts, 3-deoxyanthocyanidins, against Toxoplasma gondii.

We investigated dried red leaf extracts of Sorghum bicolor for activity against Toxoplasma gondii tachyzoites. S. bicolor red leaf extracts were obtained by bioassay-guided fractionation using ethanol and ethyl acetate as solvents. Analysis of the crude and fractionated extracts from S. bicolor using electrospray ionization mass spectrometry (ESI-MS) showed that they contained significant amounts of apigeninidin, luteolinidin, 7-methoxyapigeninidin, 5-methoxyapigeninidin, 5-methoxyluteolinidin, 7-methoxyluteolinidin 5,7-dimethoxyapigeninidin or 5,7-dimethoxyluteolinidin, based on mass per charge (m/z). When tested in vitro, the IC50s for inhibitory activity against T. gondii tachyzoites' growth of the ethanol and ethyl acetate extracts were 2.3- and 4-fold, respectively, lower than their cytotoxic IC50s in mammalian cells. Ethyl acetate extracts fractionated in chloroform-methanol and chloroform had IC50s against T. gondii that were 56.1- and 3-fold lower than their respective cytotoxic IC50s in mammalian cells. These antiparasitic activities were found to be consistent with those of the respective pure 3-deoxyanthocyanidin compounds identified to be contained in the fractions in significant amounts. Further, we observed that, the position and number of methoxy groups possessed by the 3-deoyanthocyanidins influenced their antiparasitic activity. Together, our findings indicate that S. bicolor red-leaf 3-deoxyanthocyanidins-rich extracts have potent in vitro inhibitory activity against the proliferative stage of T. gondii parasites.

Plasmodium falciparum phosphoethanolamine methyltransferase is essential for malaria transmission.

Efficient transmission of Plasmodium species between humans and Anopheles mosquitoes is a major contributor to the global burden of malaria. Gametocytogenesis, the process by which parasites switch from asexual replication within human erythrocytes to produce male and female gametocytes, is a critical step in malaria transmission and Plasmodium genetic diversity. Nothing is known about the pathways that regulate gametocytogenesis and only few of the current drugs that inhibit asexual replication are also capable of inhibiting gametocyte development and blocking malaria transmission. Here we provide genetic and pharmacological evidence indicating that the pathway for synthesis of phosphatidylcholine in Plasmodium falciparum membranes from host serine is essential for parasite gametocytogenesis and malaria transmission. Parasites lacking the phosphoethanolamine N-methyltransferase enzyme, which catalyzes the limiting step in this pathway, are severely altered in gametocyte development, are incapable of producing mature-stage gametocytes, and are not transmitted to mosquitoes. Chemical screening identified 11 inhibitors of phosphoethanolamine N-methyltransferase that block parasite intraerythrocytic asexual replication and gametocyte differentiation in the low micromolar range. Kinetic studies in vitro as well as functional complementation assays and lipid metabolic analyses in vivo on the most promising inhibitor NSC-158011 further demonstrated the specificity of inhibition. These studies set the stage for further optimization of NSC-158011 for development of a class of dual activity antimalarials to block both intraerythrocytic asexual replication and gametocytogenesis.

Molecular target validation, antimicrobial delivery, and potential treatment of Toxoplasma gondii infections.

Toxoplasma gondii persistently infects over two billion people worldwide. It can cause substantial morbidity and mortality. Existing treatments have associated toxicities and hypersensitivity and do not eliminate encysted bradyzoites that recrudesce. New, improved medicines are needed. Transductive peptides carry small molecule cargos across multiple membranes to enter intracellular tachyzoites and encysted bradyzoites. They also carry cargos into retina when applied topically to eyes, and cross blood brain barrier when administered intravenously. Phosphorodiamidate morpholino oligomers (PMO) inhibit gene expression in a sequence-specific manner. Herein, effect of transductive peptide conjugated PMO (PPMO) on tachyzoite protein expression and replication in vitro and in vivo was studied. Initially, sequence-specific PPMO successfully reduced transfected T. gondii's fluorescence and luminescence. PPMO directed against T. gondii's dihydrofolate reductase (DHFR), an enzyme necessary for folate synthesis, limited tachyzoite replication. Rescue with exogenous folate demonstrated DHFR PPMO's specificity. PPMO directed against enoyl-ACP reductase (ENR), an enzyme of type II fatty acid synthesis that is structurally distinct in T. gondii from ENR in mammalian cells was investigated. PPMO directed against plant-like Apetela 2 (AP2) domain transcription factor XI-3 (AP2XI-3), not present in human cells, was characterized. ENR and AP2XI-3 PPMO each restricted intracellular parasite replication validating these molecular targets in tachyzoites. DHFR-specific PPMO administered to infected mice diminished parasite burden. Thus, these antisense oligomers are a versatile approach to validate T. gondii molecular targets, reduce essential T. gondii proteins in vitro and in vivo, and have potential for development as curative medicines.

ALOX12 in human toxoplasmosis.

ALOX12 is a gene encoding arachidonate 12-lipoxygenase (12-LOX), a member of a nonheme lipoxygenase family of dioxygenases. ALOX12 catalyzes the addition of oxygen to arachidonic acid, producing 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which can be reduced to the eicosanoid 12-HETE (12-hydroxyeicosatetraenoic acid). 12-HETE acts in diverse cellular processes, including catecholamine synthesis, vasoconstriction, neuronal function, and inflammation. Consistent with effects on these fundamental mechanisms, allelic variants of ALOX12 are associated with diseases including schizophrenia, atherosclerosis, and cancers, but the mechanisms have not been defined. Toxoplasma gondii is an apicomplexan parasite that causes morbidity and mortality and stimulates an innate and adaptive immune inflammatory reaction. Recently, it has been shown that a gene region known as Toxo1 is critical for susceptibility or resistance to T. gondii infection in rats. An orthologous gene region with ALOX12 centromeric is also present in humans. Here we report that the human ALOX12 gene has susceptibility alleles for human congenital toxoplasmosis (rs6502997 [P, <0.000309], rs312462 [P, <0.028499], rs6502998 [P, <0.029794], and rs434473 [P, <0.038516]). A human monocytic cell line was genetically engineered using lentivirus RNA interference to knock down ALOX12. In ALOX12 knockdown cells, ALOX12 RNA expression decreased and levels of the ALOX12 substrate, arachidonic acid, increased. ALOX12 knockdown attenuated the progression of T. gondii infection and resulted in greater parasite burdens but decreased consequent late cell death of the human monocytic cell line. These findings suggest that ALOX12 influences host responses to T. gondii infection in human cells. ALOX12 has been shown in other studies to be important in numerous diseases. Here we demonstrate the critical role ALOX12 plays in T. gondii infection in humans.

Multiple pathways for glucose phosphate transport and utilization support growth of Cryptosporidium parvum.

Cryptosporidium parvum is an obligate intracellular parasite with a highly reduced mitochondrion that lacks the tricarboxylic acid cycle and the ability to generate ATP, making the parasite reliant on glycolysis. Genetic ablation experiments demonstrated that neither of the two putative glucose transporters CpGT1 and CpGT2 were essential for growth. Surprisingly, hexokinase was also dispensable for parasite growth while the downstream enzyme aldolase was required, suggesting the parasite has an alternative way of obtaining phosphorylated hexose. Complementation studies in E. coli support a role for direct transport of glucose-6-phosphate from the host cell by the parasite transporters CpGT1 and CpGT2, thus bypassing a requirement for hexokinase. Additionally, the parasite obtains phosphorylated glucose from amylopectin stores that are released by the action of the essential enzyme glycogen phosphorylase. Collectively, these findings reveal that C. parvum relies on multiple pathways to obtain phosphorylated glucose both for glycolysis and to restore carbohydrate reserves.

Mendelian segregation and high recombination rates facilitate genetic analyses in Cryptosporidium parvum.

Very little is known about the process of meiosis in the apicomplexan parasite Cryptosporidium despite the essentiality of sex in its life cycle. Most cell lines only support asexual growth of Cryptosporidium parvum (C. parvum), but stem cell derived intestinal epithelial cells grown under air-liquid interface (ALI) conditions support the sexual cycle. To examine chromosomal dynamics during meiosis in C. parvum, we generated two transgenic lines of parasites that were fluorescently tagged with mCherry or GFP on chromosomes 1 or 5, respectively. Infection of ALI cultures or Ifngr1-/- mice with mCherry and GFP parasites produced "yellow" oocysts generated by cross-fertilization. Outcrossed oocysts from the F1 generation were purified and used to infect HCT-8 cultures, and phenotypes of the progeny were observed by microscopy. All possible phenotypes predicted by independent segregation were represented equally (~25%) in the population, indicating that C. parvum chromosomes exhibit a Mendelian inheritance pattern. Unexpectedly, the most common pattern observed from the outgrowth of single oocysts included all possible parental and recombinant phenotypes derived from a single meiotic event, suggesting a high rate of crossover. To estimate the frequency of crossover, additional loci on chromosomes 1 and 5 were tagged and used to monitor intrachromosomal crosses in Ifngr1-/- mice. Both chromosomes showed a high frequency of crossover compared to other apicomplexans with map distances (i.e., 1% recombination) of 3-12 kb. Overall, a high recombination rate may explain many unique characteristics observed in Cryptosporidium spp. such as high rates of speciation, wide variation in host range, and rapid evolution of host-specific virulence factors.

Cryptosporidium infection of human small intestinal epithelial cells induces type III interferon and impairs infectivity of Rotavirus.

Cryptosporidiosis is a major cause of severe diarrheal disease in infants from resource poor settings. The majority of infections are caused by the human-specific pathogen C. hominis and absence of in vitro growth platforms has limited our understanding of host-pathogen interactions and development of effective treatments. To address this problem, we developed a stem cell-derived culture system for C. hominis using human enterocytes differentiated under air-liquid interface (ALI) conditions. Human ALI cultures supported robust growth and complete development of C. hominis in vitro including all life cycle stages. Cryptosporidium infection induced a strong interferon response from enterocytes, possibly driven, in part, by an endogenous dsRNA virus in the parasite. Prior infection with Cryptosporidium induced type III IFN secretion and consequently blunted infection with Rotavirus, including live attenuated vaccine strains. The development of hALI provides a platform for further studies on human-specific pathogens, including clinically important coinfections that may alter vaccine efficacy.

Isolation, cloning, and pathologic analysis of Trypanosoma evansi field isolates.

In recent years, the emergence of highly pathogenic Trypanosoma evansi strains in the Philippines has resulted in substantial losses in livestock production. In this study, we isolated T. evansi from infected-water buffaloes in the Philippines and analyzed their virulence using mice and cattle. A total of 10 strains of T. evansi were isolated. Evaluation of the virulence of each strain using mice depicted significant differences among the strains in the prepatent period, the level of parasitemia, and the survival time of the infected animals. In mice infected with the highly pathogenic T. evansi, signs of excessive inflammation such as marked splenomegaly and increase more than 6-fold in the number of leukocytes were observed at 8 days post-infection. To study the virulence of the parasite strains in cattle (which are the common T. evansi hosts in Philippines), cattle were infected with the T. evansi isolates that showed high and low virulence in mice. The rate of parasite growth and the length of the prepatent periods were found to be similar to those observed in mice for the respective strains. The cattle infected with the highly pathogenic strain developed anemia and a marked decrease in leukocyte counts. To determine the cause of the pathological changes, we analyzed the expression levels of inflammatory cytokines and observed up-regulation of tumor necrosis factor-α in anemic infected cattle. Our findings suggest that the epidemic of T. evansi in the Philippines is characterized by T. evansi strains with varying virulences from low to very high pathogenicity in cattle.

Past, current, and potential treatments for cryptosporidiosis in humans and farm animals: A comprehensive review.

The intracellular protozoan parasite of the genus Cryptosporidium is among the leading causes of waterborne diarrheal disease outbreaks throughout the world. The parasite is transmitted by ingestion of infective oocysts that are highly stable in the environment and resistant to almost all conventional disinfection methods and water treatments. Control of the parasite infection is exceedingly difficult due to the excretion of large numbers of oocysts in the feces of infected individuals that contaminate the environment and serve as a source of infection for susceptible hosts including humans and animals. Drug development against the parasite is challenging owing to its limited genetic tractability, absence of conventional drug targets, unique intracellular location within the host, and the paucity of robust cell culture platforms for continuous parasite propagation. Despite the high prevalence of the parasite, the only US Food and Drug Administration (FDA)-approved treatment of Cryptosporidium infections is nitazoxanide, which has shown moderate efficacy in immunocompetent patients. More importantly, no effective therapeutic drugs are available for treating severe, potentially life-threatening cryptosporidiosis in immunodeficient patients, young children, and neonatal livestock. Thus, safe, inexpensive, and efficacious drugs are urgently required to reduce the ever-increasing global cryptosporidiosis burden especially in low-resource countries. Several compounds have been tested for both in vitro and in vivo efficacy against the disease. However, to date, only a few experimental compounds have been subjected to clinical trials in natural hosts, and among those none have proven efficacious. This review provides an overview of the past and present anti-Cryptosporidium pharmacotherapy in humans and agricultural animals. Herein, we also highlight the progress made in the field over the last few years and discuss the different strategies employed for discovery and development of effective prospective treatments for cryptosporidiosis.

Multiple pathways for glucose phosphate transport and utilization support growth of Cryptosporidium parvum.

Cryptosporidium parvum is an obligate intracellular parasite with a highly reduced mitochondrion that lacks the TCA cycle and the ability to generate ATP, making the parasite reliant on glycolysis. Genetic ablation experiments demonstrated that neither of the two putative glucose transporters CpGT1 and CpGT2 were essential for growth. Surprisingly, hexokinase was also dispensable for parasite growth while the downstream enzyme aldolase was required, suggesting the parasite has an alternative way of obtaining phosphorylated hexose. Complementation studies in E. coli support a role for direct transport of glucose-6-phosphate from the host cell by the parasite transporters CpGT1 and CpGT2, thus bypassing a requirement for hexokinase. Additionally, the parasite obtains phosphorylated glucose from amylopectin stores that are released by the action of the essential enzyme glycogen phosphorylase. Collectively, these findings reveal that C. parvum relies on multiple pathways to obtain phosphorylated glucose both for glycolysis and to restore carbohydrate reserves.

Cryptosporidium infection of human small intestinal epithelial cells induces type III interferon and impairs infectivity of Rotavirus.

Cryptosporidiosis is a major cause of severe diarrheal disease in infants from resource poor settings. The majority of infections are caused by the human-specific pathogen C. hominis and absence of in vitro growth platforms has limited our understanding of host-pathogen interactions and development of effective treatments. To address this problem, we developed a stem cell-derived culture system for C. hominis using human enterocytes differentiated under air-liquid interface (ALI) conditions. Human ALI cultures supported robust growth and complete development of C. hominis in vitro including all life cycle stages. C. hominis infection induced a strong interferon response from enterocytes, likely driven by an endogenous dsRNA virus in the parasite. Prior infection with Cryptosporidium induced type III IFN secretion and consequently blunted infection with Rotavirus, including live attenuated vaccine strains. The development of hALI provides a platform for further studies on human-specific pathogens, including clinically important coinfections that may alter vaccine efficacy.

Novel N-benzoyl-2-hydroxybenzamide disrupts unique parasite secretory pathway.

Toxoplasma gondii is a protozoan parasite that can damage the human brain and eyes. There are no curative medicines. Herein, we describe our discovery of N-benzoyl-2-hydroxybenzamides as a class of compounds effective in the low nanomolar range against T. gondii in vitro and in vivo. Our lead compound, QQ-437, displays robust activity against the parasite and could be useful as a new scaffold for development of novel and improved inhibitors of T. gondii. Our genome-wide investigations reveal a specific mechanism of resistance to N-benzoyl-2-hydroxybenzamides mediated by adaptin-3ß, a large protein from the secretory protein complex. N-Benzoyl-2-hydroxybenzamide-resistant clones have alterations of their secretory pathway, which traffics proteins to micronemes, rhoptries, dense granules, and acidocalcisomes/plant-like vacuole (PLVs). N-Benzoyl-2-hydroxybenzamide treatment also alters micronemes, rhoptries, the contents of dense granules, and, most markedly, acidocalcisomes/PLVs. Furthermore, QQ-437 is active against chloroquine-resistant Plasmodium falciparum. Our studies reveal a novel class of compounds that disrupts a unique secretory pathway of T. gondii, with the potential to be used as scaffolds in the search for improved compounds to treat the devastating diseases caused by apicomplexan parasites.

P2X7 receptor-mediated killing of an intracellular parasite, Toxoplasma gondii, by human and murine macrophages.

The P2X7R is highly expressed on the macrophage cell surface, and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this study, we show that macrophages from people with the 1513C (rs3751143, NM_002562.4:c.1487A>C) loss-of-function P2X7R single nucleotide polymorphism are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X7R-specific effect on T. gondii, macrophages from P2X7R knockout mice (P2X7R-/-) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X7R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production.