The Cryptosporidium signaling kinase CDPK5 plays an important role in male gametogenesis and parasite virulence.

The protozoan parasite Cryptosporidium is a leading cause of diarrhea in young children. The parasite's life cycle involves a coordinated and timely progression from asexual to sexual stages, leading to the formation of the transmissible oocyst. Underlying molecular signaling mechanisms orchestrating sexual development are not known. Here, we describe the function of a signaling kinase in Cryptosporidium male gametogenesis. We reveal the expression of Cryptosporidium parvum calcium-dependent protein kinase 5 (CDPK5) during male gamete development and its important role in the egress of mature gametes. Genetic ablation of this kinase results in viable parasites, indicating that this gene is dispensable for parasite survival. Interestingly, cdpk5 deletion decreases parasite virulence and impacts oocyst shedding in immunocompromised mice. Using phosphoproteomics, we identify possible CDPK5 substrates and biological processes regulated by this kinase. Collectively, these findings illuminate parasite cell biology by revealing a mechanism controlling male gamete production and a potential target to block disease transmission.

Tyrosine Kinase Inhibitors Display Potent Activity against Cryptosporidium parvum.

The protozoan parasite Cryptosporidium is a leading cause of diarrheal disease (cryptosporidiosis) and death in young children. Cryptosporidiosis can be life-threatening in individuals with weak immunity such as HIV/AIDS patients and organ transplant recipients. There is currently no effective drug to treat cryptosporidiosis in the pediatric and immunocompromised population. Therefore, there is an urgent need to expedite the drug discovery process in order to develop new and effective therapies to reduce the global disease burden of cryptosporidiosis. In this study, we employed a drug repurposing strategy to screen a library of 473 human kinase inhibitors to determine their activity against Cryptosporidium parvum. We have identified 67 new anti-cryptosporidial compounds using phenotypic screening based on a transgenic C. parvum strain expressing a luciferase reporter. Further, dose-response assays led to the identification of 11 hit compounds that showed potent inhibition of C. parvum at nanomolar concentration. Kinome profiling of these 11 prioritized hits identified compounds that displayed selectivity in targeting specific families of kinases, particularly tyrosine kinases. Overall, this study identified tyrosine kinase inhibitors that hold potential for future development as new drug candidates against cryptosporidiosis. IMPORTANCE The intestinal parasite Cryptosporidium parvum is a major cause of diarrhea-associated morbidity and mortality in children, immunocompromised people, and young ruminant animals. With no effective drug available to treat cryptosporidiosis in humans and animals, there is an urgent need to identify anti-parasitic compounds and new targets for drug development. To address this unmet need, we screened a GSK library of kinase inhibitors and identified several potent compounds, including tyrosine kinase inhibitors, that were highly effective in killing C. parvum. Overall, our study revealed several novel compounds and a new family of kinases that can be targeted for anti-cryptosporidial drug development.

Cryptosporidium: Host-Parasite Interactions and Pathogenesis.

PURPOSE OF REVIEW: Cryptosporidium spp. (C. hominis and C. parvum) are a major cause of diarrhea-associated morbidity and mortality in young children globally. While C. hominis only infects humans, C. parvum is a zoonotic parasite that can be transmitted from infected animals to humans. There are no treatment or control measures to fully treat cryptosporidiosis or prevent the infection in humans and animals. Our knowledge on the molecular mechanisms of Cryptosporidium-host interactions and the underlying factors that govern infectivity and disease pathogenesis is very limited. RECENT FINDINGS: Recent development of genetics and new animal models of infection, along with progress in cell culture platforms to complete the parasite lifecycle in vitro, is greatly advancing the Cryptosporidium field. SUMMARY: In this review, we will discuss our current knowledge of host-parasite interactions and how genetic manipulation of Cryptosporidium and promising infection models are opening the doors towards an improved understanding of parasite biology and disease pathogenesis.

Recent advances in genetic manipulation of Cryptosporidium.

Cryptosporidium is a leading cause of diarrhea-associated morbidity and mortality in young children. Currently, there is no fully effective drug to treat cryptosporidiosis and a complete lack of vaccine to prevent disease. For a long time, progress in the field of Cryptosporidium research has been hindered due to unavailability of methods to propagate the parasite, lack of efficient animal infection models and most importantly, the absence of technology to genetically manipulate the parasite. The recent advent of molecular genetics has been transformative for Cryptosporidium research, and is facilitating our fundamental understanding of parasite biology, and accelerating the pace of drug discovery. This review summarizes recent advancements in genetic manipulation and its applications for studying parasite gene function, host-parasite interactions and discovery of anti-cryptosporidial drugs.

Bicyclic azetidines kill the diarrheal pathogen Cryptosporidium in mice by inhibiting parasite phenylalanyl-tRNA synthetase.

Cryptosporidium is a protozoan parasite and a leading cause of diarrheal disease and mortality in young children. Currently, there are no fully effective treatments available to cure infection with this diarrheal pathogen. In this study, we report a broad drug repositioning effort that led to the identification of bicyclic azetidines as a new anticryptosporidial series. Members of this series blocked growth in in vitro culture of three Cryptosporidium parvum isolates with EC50 's in 1% serum of <0.4 to 96 nM, had comparable potencies against Cryptosporidium hominis and C. parvum, and was effective in three of four highly susceptible immunosuppressed mice with once-daily dosing administered for 4 days beginning 2 weeks after infection. Comprehensive genetic, biochemical, and chemical studies demonstrated inhibition of C. parvum phenylalanyl-tRNA synthetase (CpPheRS) as the mode of action of this new lead series. Introduction of mutations directly into the C. parvum pheRS gene by CRISPR-Cas9 genome editing resulted in parasites showing high degrees of compound resistance. In vitro, bicyclic azetidines potently inhibited the aminoacylation activity of recombinant ChPheRS. Medicinal chemistry optimization led to the identification of an optimal pharmacokinetic/pharmacodynamic profile for this series. Collectively, these data demonstrate that bicyclic azetidines are a promising series for anticryptosporidial drug development and establish a broad framework to enable target-based drug discovery for this infectious disease.

A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum.

Cryptosporidium spp., protozoan parasites, are a leading cause of global diarrhea-associated morbidity and mortality in young children and immunocompromised individuals. The limited efficacy of the only available drug and lack of vaccines make it challenging to treat and prevent cryptosporidiosis. Therefore, the identification of essential genes and understanding their biological functions are critical for the development of new therapies. Currently, there is no genetic tool available to investigate the function of essential genes in Cryptosporidium spp. Here, we describe the development of the first conditional system in Cryptosporidium parvum Our system utilizes the Escherichia coli dihydrofolate reductase degradation domain (DDD) and the stabilizing compound trimethoprim (TMP) for conditional regulation of protein levels in the parasite. We tested our system on the calcium-dependent protein kinase-1 (CDPK1), a leading drug target in C. parvum By direct knockout strategy, we establish that cdpk1 is refractory to gene deletion, indicating its essentiality for parasite survival. Using CRISPR/Cas9, we generated transgenic parasites expressing CDPK1 with an epitope tag, and localization studies indicate its expression during asexual parasite proliferation. We then genetically engineered C. parvum to express CDPK1 tagged with DDD. We demonstrate that TMP can regulate CDPK1 levels in this stable transgenic parasite line, thus revealing the critical role of this kinase in parasite proliferation. Further, these transgenic parasites show TMP-mediated regulation of CDPK1 levels in vitro and an increased sensitivity to kinase inhibitor upon conditional knockdown. Overall, this study reports the development of a powerful conditional system that can be used to study essential genes in CryptosporidiumIMPORTANCECryptosporidium parvum and Cryptosporidium hominis are leading pathogens responsible for diarrheal disease (cryptosporidiosis) and deaths in infants and children below 5 years of age. There are no effective treatment options and no vaccine for cryptosporidiosis. Therefore, there is an urgent need to identify essential gene targets and uncover their biological function to accelerate the development of new and effective anticryptosporidial drugs. Current genetic tool allows targeted disruption of gene function but leads to parasite lethality if the gene is essential for survival. In this study, we have developed a genetic tool for conditional degradation of proteins in Cryptosporidium spp., thus allowing us to study the function of essential genes. Our conditional system expands the molecular toolbox for Cryptosporidium, and it will help us to understand the biology of this important human diarrheal pathogen for the development of new drugs and vaccines.

Genetic Manipulation of Cryptosporidium parvum with CRISPR/Cas9.

Cryptosporidium parvum can be reliably genetically manipulated using CRISPR/Cas9-driven homologous repair coupled to in vivo propagation within immunodeficient mice. Recent modifications have simplified the initial protocol significantly. This chapter will guide through procedures for excystation, transfection, infection, collection, and purification of transgenic Cryptosporidium parvum.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

Generating and Maintaining Transgenic Cryptosporidium parvum Parasites.

The apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease and an important contributor to overall global child mortality. We currently lack effective treatment and immune prophylaxis. Recent advances now permit genetic modification of this important pathogen. We expect this to produce rapid advances in fundamental as well as translational research on cryptosporidiosis. Here we outline genetic engineering for Cryptosporidium in sufficient detail to establish transfection in any laboratory that requires access to this key technology. This chapter details the conceptual design consideration, as well as the experimental steps required to transfect, select, and isolate transgenic parasites. We also provide detail on key in vitro and in vivo assays to detect, validate, and quantify genetically modified Cryptosporidium parasites. © 2017 by John Wiley & Sons, Inc.

A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis.

Diarrhoeal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite Cryptosporidium to be a leading cause of paediatric diarrhoea, with particularly grave impact on infants and immunocompromised individuals. There is neither a vaccine nor an effective treatment. Here we establish a drug discovery process built on scalable phenotypic assays and mouse models that take advantage of transgenic parasites. Screening a library of compounds with anti-parasitic activity, we identify pyrazolopyridines as inhibitors of Cryptosporidium parvum and Cryptosporidium hominis. Oral treatment with the pyrazolopyridine KDU731 results in a potent reduction in intestinal infection of immunocompromised mice. Treatment also leads to rapid resolution of diarrhoea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection. Our results suggest that the Cryptosporidium lipid kinase PI(4)K (phosphatidylinositol-4-OH kinase) is a target for pyrazolopyridines and that KDU731 warrants further preclinical evaluation as a drug candidate for the treatment of cryptosporidiosis.

Necessity of Bumped Kinase Inhibitor Gastrointestinal Exposure in Treating Cryptosporidium Infection.

There is a substantial need for novel therapeutics to combat the widespread impact caused by Crytosporidium infection. However, there is a lack of knowledge as to which drug pharmacokinetic (PK) characteristics are key to generate an in vivo response, specifically whether systemic drug exposure is crucial for in vivo efficacy. To identify which PK properties are correlated with in vivo efficacy, we generated physiologically based PK models to simulate systemic and gastrointestinal drug concentrations for a series of bumped kinase inhibitors (BKIs) that have nearly identical in vitro potency against Cryptosporidium but display divergent PK properties. When BKI concentrations were used to predict in vivo efficacy with a neonatal model of Cryptosporidium infection, these concentrations in the large intestine were the sole predictors of the observed in vivo efficacy. The significance of large intestinal BKI exposure for predicting in vivo efficacy was further supported with an adult mouse model of Cryptosporidium infection. This study suggests that drug exposure in the large intestine is essential for generating a superior in vivo response, and that physiologically based PK models can assist in the prioritization of leading preclinical drug candidates for in vivo testing.

Bumped-Kinase Inhibitors for Cryptosporidiosis Therapy.

Bumped kinase inhibitors (BKIs) of Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) are leading candidates for treatment of cryptosporidiosis-associated diarrhea. Potential cardiotoxicity related to anti-human ether-à-go-go potassium channel (hERG) activity of the first-generation anti-Cryptosporidium BKIs triggered further testing for efficacy. A luminescence assay adapted for high-throughput screening was used to measure inhibitory activities of BKIs against C. parvum in vitro. Furthermore, neonatal and interferon γ knockout mouse models of C. parvum infection identified BKIs with in vivo activity. Additional iterative experiments for optimum dosing and selecting BKIs with minimum levels of hERG activity and frequencies of other safety liabilities included those that investigated mammalian cell cytotoxicity, C. parvum proliferation inhibition in vitro, anti-human Src inhibition, hERG activity, in vivo pharmacokinetic data, and efficacy in other mouse models. Findings of this study suggest that fecal concentrations greater than parasite inhibitory concentrations correlate best with effective therapy in the mouse model of cryptosporidiosis, but a more refined model for efficacy is needed.

Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum.

Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus. Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality. Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools. Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivo selection for aminoglycoside resistance. We derived reporter parasites suitable for in vitro and in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.

Genetic manipulation of the Toxoplasma gondii genome by fosmid recombineering.

UNLABELLED: Apicomplexa are obligate intracellular parasites that cause important diseases in humans and animals. Manipulating the pathogen genome is the most direct way to understand the functions of specific genes in parasite development and pathogenesis. In Toxoplasma gondii, nonhomologous recombination is typically highly favored over homologous recombination, a process required for precise gene targeting. Several approaches, including the use of targeting vectors that feature large flanks to drive site-specific recombination, have been developed to overcome this problem. We have generated a new large-insert repository of T. gondii genomic DNA that is arrayed and sequenced and covers 95% of all of the parasite's genes. Clones from this fosmid library are maintained at single copy, which provides a high level of stability and enhances our ability to modify the organism dramatically. We establish a robust recombineering pipeline and show that our fosmid clones can be easily converted into gene knockout constructs in a 4-day protocol that does not require plate-based cloning but can be performed in multiwell plates. We validated this approach to understand gene function in T. gondii and produced a conditional null mutant for a nucleolar protein belonging to the NOL1/NOP2/SUN family, and we show that this gene is essential for parasite growth. We also demonstrate a powerful complementation strategy in the context of chemical mutagenesis and whole-genome sequencing. This repository is an important new resource that will accelerate both forward and reverse genetic analysis of this important pathogen. IMPORTANCE: Toxoplasma gondii is an important genetic model to understand intracellular parasitism. We show here that large-insert genomic clones are effective tools that enhance homologous recombination and allow us to engineer conditional mutants to understand gene function. We have generated, arrayed, and sequenced a fosmid library of T. gondii genomic DNA in a copy control vector that provides excellent coverage of the genome. The fosmids are maintained in a single-copy state that dramatically improves their stability and allows modification by means of a simple and highly scalable protocol. We show here that modified and unmodified fosmid clones are powerful tools for forward and reverse genetics.

A nucleolar AAA-NTPase is required for parasite division.

Apicomplexa division involves several distinct phases shared with other eukaryote cell cycles including a gap period (G1) prior to chromosome synthesis, although how progression through the parasite cell cycle is controlled is not understood. Here we describe a cell cycle mutant that reversibly arrests in the G1 phase. The defect in this mutant was mapped by genetic complementation to a gene encoding a novel AAA-ATPase/CDC48 family member called TgNoAP1. TgNoAP1 is tightly regulated and expressed in the nucleolus during the G1/S phases. A tyrosine to a cysteine change upstream of the second AAA+ domain in the temperature sensitive TgNoAP1 allele leads to conditional protein instability, which is responsible for rapid cell cycle arrest and a primary defect in 28S rRNA processing as confirmed by knock-in of the mutation back into the parent genome. The interaction of TgNoAP1 with factors of the snoRNP and R2TP complexes indicates this protein has a role in pre-rRNA processing. This is a novel role for a cdc48-related chaperone protein and indicates that TgNoAP1 may be part of a dynamic mechanism that senses the health of the parasite protein machinery at the initial steps of ribosome biogenesis and conveys that information to the parasite cell cycle checkpoint controls.

Genetic variation in the Plasmodium falciparum circumsporozoite protein in India and its relevance to RTS,S malaria vaccine.

RTS,S is the most advanced malaria vaccine candidate, currently under phase-III clinical trials in Africa. This Plasmodium falciparum vaccine contains part of the central repeat region and the complete C-terminal T cell epitope region (Th2R and Th3R) of the circumsporozoite protein (CSP). Since naturally occurring polymorphisms at the vaccine candidate loci are critical determinants of the protective efficacy of the vaccines, it is imperative to investigate these polymorphisms in field isolates. In this study we have investigated the genetic diversity at the central repeat, C-terminal T cell epitope (Th2R and Th3R) and N-terminal T cell epitope regions of the CSP, in P. falciparum isolates from Madhya Pradesh state of India. These isolates were collected through a 5-year prospective study aimed to develop a well-characterized field-site for the future evaluation of malaria vaccine in India. Our results revealed that the central repeat (63 haplotypes, n = 161) and C-terminal Th2R/Th3R epitope (24 haplotypes, n = 179) regions were highly polymorphic, whereas N-terminal non-repeat region was less polymorphic (5 haplotypes, n = 161) in this population. We did not find any evidence of the role of positive natural selection in maintaining the genetic diversity at the Th2R/Th3R regions of CSP. Comparative analysis of the Th2R/Th3R sequences from this study to the global isolates (n = 1160) retrieved from the GenBank database revealed two important points. First, the majority of the sequences (~61%, n = 179) from this study were identical to the Dd2/Indochina type, which is also the predominant Th2R/Th3R haplotype in Asia (~59%, n = 974). Second, the Th2R/Th3R sequences in Asia, South America and Africa are geographically distinct with little allele sharing between continents. In conclusion, this study provides an insight on the existing polymorphisms in the CSP in a parasite population from India that could potentially influence the efficacy of RTS,S vaccine in this region.

Selective sweeps and genetic lineages of Plasmodium falciparum drug -resistant alleles in Ghana.

BACKGROUND: In 2005, Ghana adopted artemisinin-based combination therapy (ACT) for primary treatment of falciparum malaria. A comprehensive study of the drug-resistance-associated mutations and their genetic lineages will lead to a better understanding of the evolution of antimalarial drug resistance in this region. METHODS: The pfcrt, pfmdr1, dhps, and dhfr mutations associated with chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) resistance and the microsatellite loci flanking these genes were genotyped in Plasmodium falciparum isolates from Ghana. RESULTS: The prevalence of mutations associated with both CQ and SP resistance was high in Ghana. However, we observed a decrease in prevalence of the pfcrt K76T mutation in northern Ghana after the change in drug policy from CQ to ACT. Analysis of genetic diversity and differentiation at microsatellite loci flanking all 4 genes indicated that they have been under strong selection, because of CQ and SP use. The triple-mutant pfcrt and dhfr alleles in Ghana were derived from Southeast Asia, whereas the double-mutant dhfr, dhps, and pfmdr1 alleles were of African lineage. CONCLUSION: Because of the possible role of pfmdr1 in amodiaquine and mefloquine resistance, demonstrating selection on pfmdr1 and defining lineages of resistant alleles in an African population holds great importance.

Tracking origins and spread of sulfadoxine-resistant Plasmodium falciparum dhps alleles in Thailand.

The emergence and spread of drug-resistant Plasmodium falciparum have been a major impediment for the control of malaria worldwide. Earlier studies have shown that similar to chloroquine (CQ) resistance, high levels of pyrimethamine resistance in P. falciparum originated independently 4 to 5 times globally, including one origin at the Thailand-Cambodia border. In this study we describe the origins and spread of sulfadoxine-resistance-conferring dihydropteroate synthase (dhps) alleles in Thailand. The dhps mutations and flanking microsatellite loci were genotyped for P. falciparum isolates collected from 11 Thai provinces along the Burma, Cambodia, and Malaysia borders. Results indicated that resistant dhps alleles were fixed in Thailand, predominantly being the SGEGA, AGEAA, and SGNGA triple mutants and the AGKAA double mutant (mutated codons are underlined). These alleles had different geographical distributions. The SGEGA alleles were found mostly at the Burma border, while the SGNGA alleles occurred mainly at the Cambodia border and nearby provinces. Microsatellite data suggested that there were two major genetic lineages of the triple mutants in Thailand, one common for SGEGA/SGNGA alleles and another one independent for AGEAA. Importantly, the newly reported SGNGA alleles possibly originated at the Thailand-Cambodia border. All parasites in the Yala province (Malaysia border) had AGKAA alleles with almost identical flanking microsatellites haplotypes. They were also identical at putatively neutral loci on chromosomes 2 and 3, suggesting a clonal nature of the parasite population in Yala. In summary, this study suggests multiple and independent origins of resistant dhps alleles in Thailand.