Recent studies into the global causes of severe diarrhea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrheal pathogen after rotavirus1–3. Diarrheal disease is estimated to be responsible for 10.5% of overall child mortality4. Cryptosporidium is also an opportunistic pathogen in the context of HIV-AIDS and organ transplantation5,6. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger, malnourished children and immunocompromised patients7,8. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes lack of continuous culture, facile animal models, and molecular genetic tools3,9. Here we describe an experimental framework to genetically modify this important human pathogen. We establish and optimize transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we develop a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium CRISPR/Cas9 system, and in vivo selection for aminoglycoside resistance. We derive reporter parasites suitable for in vitro and in vivo drug screening, and we evaluate the basis of drug susceptibility by gene knock out. We anticipate the ability to genetically engineer the 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.

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions.

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

The combination of sulfadoxine-pyrimethamine (SP) is used as a second line of therapy for the treatment of uncomplicated chloroquine-resistant Plasmodium falciparum malaria. Resistance to SP arises due to certain point mutations in the genes for the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) enzymes of the parasite. We have analyzed these mutations in 312 field isolates of P. falciparum collected from different parts of India to assess the effects of drug pressure. The rate of mutation in the gene for DHFR was found to be higher than that in the gene for DHPS, although the latter had mutations in more alleles. There was a temporal rise in the number of isolates with double dhfr mutations and single dhps mutations, resulting in an increased total number of mutations in the loci for DHFR and DHPS combined over a 5-year period. During these 5 years, the number of isolates with drug-sensitive genotypes decreased and the number of isolates with drug-resistant genotypes (double DHFR mutations and a single DHPS mutation) increased significantly. The number of isolates with the triple mutations in each of the genes for the two enzymes (for a total of six mutations), however, remained very low, coinciding with the very low rate of SP treatment failure in the country. There was a regional bias in the mutation rate, as isolates from the northeastern region (the state of Assam) showed higher rates of mutation and more complex genotypes than isolates from the other regions. It was concluded that even though SP is prescribed as a second line of treatment in India, the mutations associated with SP resistance continue to be progressively increasing.Plasmodium falciparum is the most lethal of all human malaria parasites. This parasite causes epidemics in countries where malaria is endemic, resulting in large numbers of deaths. Widespread chloroquine resistance has forced many countries to use alternate drugs for the treatment of falciparum malaria, such as the combination of sulfadoxine and pyrimethamine (SP). However, the parasite can develop resistance to this drug combination as well through mutations in the genes for the enzymes involved in the folate biosynthesis pathway. Such mutations lead to the lowering of the drug binding affinity of the parasite enzymes (18,26,34,36,41). Resistance to pyrimethamine is attributed to mutations in the gene for the parasite enzyme dihydrofolate reductase (DHFR), whereas sulfadoxine resistance is associated with mutations in the gene for the parasite enzyme dihydropteroate synthetase (DHPS). The increased level of resistance has been found to be associated with increased numbers of mutations in the genes for these two enzymes. Multiple mutations in the genes for both enzymes result in SP treatment failure (39). Detection of these mutations in field isolates has been proposed as an alternate strategy for rapid screening for antifolate drug resistance (9,12,16,17,27,38).In India, chloroquine-resistant malaria was first reported in 1973, and since then resistance to ...

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.

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-stagePlasmodium falciparumandCryptosporidium parvumin cell-culture studies. Target deconvolution inP. falciparumhas 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 bothPfKRS1 andC. parvumKRS (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 betweenPfKRS1 andCpKRS. This series of compounds inhibitCpKRS andC. parvumandCryptosporidium hominisin 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 forPfKRS1 andCpKRS vs. (human)HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

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.

Background The emergence of artesunate-mefloquine (AS+MQ)–resistant Plasmodium falciparum in the Thailand-Cambodia region is a major concern for malaria control. Studies indicate that copy number increase and key alleles in the pfmdr1 gene are associated with AS+MQ resistance. In the present study, we investigated evidence for a selective sweep around pfmdr1 because of the spread of adaptive mutation and/or multiple copies of this gene in the P. falciparum population in Cambodia. Methods We characterized 13 microsatellite loci flanking (± 99 kb) pfmdr1 in 93 single-clone P. falciparum infections, of which 31 had multiple copies and 62 had a single copy of the pfmdr1 gene. Results Genetic analysis revealed no difference in the mean (± standard deviation) expected heterozygosity (He) at loci around single (0.75 ± 0.03) and multiple (0.76 ± 0.04) copies of pfmdr1. Evidence of genetic hitchhiking with the selective sweep of certain haplotypes was seen around mutant (184F) pfmdr1 allele, irrespective of the copy number. There was an overall reduction of 28% in mean He (± SD) around mutant allele (0.56 ± 0.05), compared with wild-type allele (0.84 ± 0.02). Significant linkage disequilibrium was also observed between the loci flanking mutant pfmdr1 allele. Conclusion The 184F mutant allele is under selection, whereas amplification of pfmdr1 gene in this population occurs on multiple genetic backgrounds.