Dietary environmental factors shape the immune defense against Cryptosporidium infection.

Cryptosporidium is a leading cause of diarrheal-related deaths in children, especially in resource-poor settings. It also targets the immunocompromised, chronically infecting people living with HIV and primary immunodeficiencies. There is no vaccine or effective treatment. Although it is known from human cases and animal models that CD4+ T cells play a role in curbing Cryptosporidium, the role of CD8+ T cells remains to be defined. Using a Cryptosporidium tyzzeri mouse model, we show that gut-resident CD8+ intraepithelial lymphocytes (IELs) confer resistance to parasite growth. CD8+ IELs express and depend on the ligand-dependent transcription factor aryl hydrocarbon receptor (AHR). AHR deficiency reduces CD8+ IELs, decreases their cytotoxicity, and worsens infection. Transfer of CD8+ IELs rescues severely immunodeficient mice from death following Cryptosporidium challenge. Finally, dietary supplementation of the AHR pro-ligand indole-3-carbinol in newborn mice promotes resistance to infection. Therefore, common dietary metabolites augment the host immune response to cryptosporidiosis, protecting against disease.

A genetic screen identifies a protective type III interferon response to Cryptosporidium that requires TLR3 dependent recognition.

Cryptosporidium is a leading cause of severe diarrhea and diarrheal-related death in children worldwide. As an obligate intracellular parasite, Cryptosporidium relies on intestinal epithelial cells to provide a niche for its growth and survival, but little is known about the contributions that the infected cell makes to this relationship. Here we conducted a genome wide CRISPR/Cas9 knockout screen to discover host genes that influence Cryptosporidium parvum infection and/or host cell survival. Gene enrichment analysis indicated that the host interferon response, glycosaminoglycan (GAG) and glycosylphosphatidylinositol (GPI) anchor biosynthesis are important determinants of susceptibility to C. parvum infection and impact on the viability of host cells in the context of parasite infection. Several of these pathways are linked to parasite attachment and invasion and C-type lectins on the surface of the parasite. Evaluation of transcript and protein induction of innate interferons revealed a pronounced type III interferon response to Cryptosporidium in human cells as well as in mice. Treatment of mice with IFNλ reduced infection burden and protected immunocompromised mice from severe outcomes including death, with effects that required STAT1 signaling in the enterocyte. Initiation of this type III interferon response was dependent on sustained intracellular growth and mediated by the pattern recognition receptor TLR3. We conclude that host cell intrinsic recognition of Cryptosporidium results in IFNλ production critical to early protection against this infection.

The Long and Short of Next Generation Sequencing for Cryptosporidium Research.

The intestinal parasite Cryptosporidium is a significant cause of severe diarrhoeal disease that can have long term effects. Therapeutic options remain limited despite a significant impact on public health, partly due to various challenges in the field of Cryptosporidium research, including the availability of genomic and transcriptomic data from environmental and clinical isolates. In this review we explore how long read DNA and RNA sequencing technologies have begun to provide novel insights into the biology of the parasite. The increased deployment of these technologies will help researchers address key gaps in the understanding of Cryptosporidium biology, and ultimately drive translational research and better parasite control.

Long-read assembly and comparative evidence-based reanalysis of Cryptosporidium genome sequences reveal expanded transporter repertoire and duplication of entire chromosome ends including subtelomeric regions.

Cryptosporidiosis is a leading cause of waterborne diarrheal disease globally and an important contributor to mortality in infants and the immunosuppressed. Despite its importance, the Cryptosporidium community has only had access to a good, but incomplete, Cryptosporidium parvum IOWA reference genome sequence. Incomplete reference sequences hamper annotation, experimental design, and interpretation. We have generated a new C. parvum IOWA genome assembly supported by Pacific Biosciences (PacBio) and Oxford Nanopore long-read technologies and a new comparative and consistent genome annotation for three closely related species: C. parvum, Cryptosporidium hominis, and Cryptosporidium tyzzeri We made 1926 C. parvum annotation updates based on experimental evidence. They include new transporters, ncRNAs, introns, and altered gene structures. The new assembly and annotation revealed a complete Dnmt2 methylase ortholog. Comparative annotation between C. parvum, C. hominis, and C. tyzzeri revealed that most "missing" orthologs are found, suggesting that the biological differences between the species must result from gene copy number variation, differences in gene regulation, and single-nucleotide variants (SNVs). Using the new assembly and annotation as reference, 190 genes are identified as evolving under positive selection, including many not detected previously. The new C. parvum IOWA reference genome assembly is larger, gap free, and lacks ambiguous bases. This chromosomal assembly recovers all 16 chromosome ends, 13 of which are contiguously assembled. The three remaining chromosome ends are provisionally placed. These ends represent duplication of entire chromosome ends including subtelomeric regions revealing a new level of genome plasticity that will both inform and impact future research.

Enterocyte-innate lymphoid cell crosstalk drives early IFN-γ-mediated control of Cryptosporidium.

The intestinal parasite, Cryptosporidium, is a major contributor to global child mortality and causes opportunistic infection in immune deficient individuals. Innate resistance to Cryptosporidium, which specifically invades enterocytes, is dependent on the production of IFN-γ, yet whether enterocytes contribute to parasite control is poorly understood. In this study, utilizing a mouse-adapted strain of C. parvum, we show that epithelial-derived IL-18 synergized with IL-12 to stimulate innate lymphoid cell (ILC) production of IFN-γ required for early parasite control. The loss of IFN-γ-mediated STAT1 signaling in enterocytes, but not dendritic cells or macrophages, antagonized early parasite control. Transcriptional profiling of enterocytes from infected mice identified an IFN-γ signature and enrichment of the anti-microbial effectors IDO, GBP, and IRG. Deletion experiments identified a role for Irgm1/m3 in parasite control. Thus, enterocytes promote ILC production of IFN-γ that acts on enterocytes to restrict the growth of Cryptosporidium.

The enteric pathogen Cryptosporidium parvum exports proteins into the cytosol of the infected host cell.

The parasite Cryptosporidium is responsible for diarrheal disease in young children causing death, malnutrition, and growth delay. Cryptosporidium invades enterocytes where it develops in a unique intracellular niche. Infected cells exhibit profound changes in morphology, physiology, and transcriptional activity. How the parasite effects these changes is poorly understood. We explored the localization of highly polymorphic proteins and found members of the Cryptosporidium parvum MEDLE protein family to be translocated into the cytosol of infected cells. All intracellular life stages engage in this export, which occurs after completion of invasion. Mutational studies defined an N-terminal host-targeting motif and demonstrated proteolytic processing at a specific leucine residue. Direct expression of MEDLE2 in mammalian cells triggered an ER stress response, which was also observed during infection. Taken together, our studies reveal the presence of a Cryptosporidium secretion system capable of delivering parasite proteins into the infected enterocyte.

Spontaneous Selection of Cryptosporidium Drug Resistance in a Calf Model of Infection.

The intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last ∼10 years, with numerous lead compounds identified, including several tRNA synthetase inhibitors. Here, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (Cryptosporidium parvum MetRS [CpMetRS]) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day 5. Parasites shed by each recrudescent calf had different amino acid-altering mutations in the gene encoding CpMetRS (CpMetRS), yielding either an aspartate 243-to-glutamate (D243E) or a threonine 246-to-isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant-expressing parasites, respectively, had 2093 half-maximal effective concentrations (EC50s) that were 613- and 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence.

The intestinal parasite Cryptosporidium is controlled by an enterocyte intrinsic inflammasome that depends on NLRP6.

The apicomplexan parasite Cryptosporidium infects the intestinal epithelium. While infection is widespread around the world, children in resource-poor settings suffer a disproportionate disease burden. Cryptosporidiosis is a leading cause of diarrheal disease, responsible for mortality and stunted growth in children. CD4 T cells are required to resolve this infection, but powerful innate mechanisms control the parasite prior to the onset of adaptive immunity. Here, we use the natural mouse pathogen Cryptosporidium tyzzeri to demonstrate that the inflammasome plays a critical role in initiating this early response. Mice lacking core inflammasome components, including caspase-1 and apoptosis-associated speck-like protein, show increased parasite burden and caspase 1 deletion solely in enterocytes phenocopies whole-body knockout (KO). This response was fully functional in germfree mice and sufficient to control Cryptosporidium infection. Inflammasome activation leads to the release of IL-18, and mice that lack IL-18 are more susceptible to infection. Treatment of infected caspase 1 KO mice with recombinant IL-18 is remarkably efficient in rescuing parasite control. Notably, NOD-like receptor family pyrin domain containing 6 (NLRP6) was the only NLR required for innate parasite control. Taken together, these data support a model of innate recognition of Cryptosporidium infection through an NLRP6-dependent and enterocyte-intrinsic inflammasome that leads to the release of IL-18 required for parasite control.

Crypto-Currency: Investing in New Models to Advance the Study of Cryptosporidium Infection and Immunity.

Cryptosporidiosis is a leading cause of diarrheal disease and an important contributor to global morbidity and mortality. Although the brunt of disease burden is felt by children in developing countries, Cryptosporidium is a ubiquitous intestinal parasite with frequent outbreaks around the world. There are no consistently effective treatments for cryptosporidiosis and the research to drive new developments has stagnated, largely due to a lack of efficient in vivo and in vitro models. Fortunately, these research barriers have started to fall. In this review, we highlight two recent advances aiding this process: A tractable mouse model for Cryptosporidium infection and stem cell-based in vitro culture systems that mimic the complexity of the host intestine. These models are paving the way for researchers to investigate Cryptosporidium infection and host immunity down to a molecular level. We believe that wise investments made to adopt and develop these new models will reap benefits not only for the Cryptosporidium community but also for the intestinal immunology field at large.

Update on Cryptosporidium spp.: highlights from the Seventh International Giardia and Cryptosporidium Conference.

While cryptosporidiosis is recognized as being among the most common causes of human parasitic diarrhea in the world, there is currently limited knowledge on Cryptosporidium infection mechanisms, incomplete codification of diagnostic methods, and a need for additional therapeutic options. In response, the Seventh International Giardia and Cryptosporidium Conference (IGCC 2019) was hosted from 23 to 26 June 2019, at the Rouen Normandy University, France. This trusted event brought together an international delegation of researchers to synthesize recent advances and identify key research questions and knowledge gaps. The program of the interdisciplinary conference included all aspects of host-parasite relationships from basic research to applications to human and veterinary medicine, and environmental issues associated with waterborne parasites and their epidemiological consequences. In relation to Cryptosporidium and cryptosporidiosis, the primary research areas for which novel findings and the most impressive communications were presented and discussed included: Cryptosporidium in environmental waters, seafood, and fresh produce; Animal epidemiology; Human cryptosporidiosis and epidemiology; Genomes and genomic evolution encompassing: Comparative genomics of Cryptosporidium spp., Genomic insights into biology, Acquiring and utilizing genome sequences, Genetic manipulation; Host-parasite interaction (immunology, microbiome); and Diagnosis and treatment. High quality presentations discussed at the conference reflected decisive progress and identified new opportunities that will engage investigators and funding agencies to spur future research in a "one health" approach to improve basic knowledge and the clinical and public health management of zoonotic cryptosporidiosis.

TITLE: Mise à jour sur Cryptosporidium spp.: Faits saillants de la Septième Conférence Internationale sur Giardia et Cryptosporidium. ABSTRACT: Bien que la cryptosporidiose soit reconnue comme l'une des premières causes de diarrhée parasitaire humaine dans le monde, la connaissance des mécanismes de l'infection par Cryptosporidium est limitée, la codification des méthodes diagnostiques est incomplète et des options thérapeutiques supplémentaires sont requises. En réponse à cette situation, la Septième Conférence Internationale sur Giardia and Cryptosporidium (IGCC 2019) s'est tenue du 23 au 26 juin 2019, à l'Université de Rouen-Normandie, France. Cet événement renommé a rassemblé une délégation internationale de chercheurs pour faire la synthèse des avancées récentes et identifier les principaux thèmes de recherche et les lacunes dans les connaissances. Le programme de cette conférence interdisciplinaire comprenait tous les aspects des relations hôte-parasite, de la recherche fondamentale aux applications à la médecine humaine et vétérinaire, ainsi que les questions environnementales liées aux parasites d'origine hydrique et leurs conséquences épidémiologiques. En ce qui concerne Cryptosporidium et la cryptosporidiose, les principaux domaines de recherche pour lesquels de nouvelles découvertes et les communications les plus impressionnantes ont été présentées et discutées comprenaient : Cryptosporidium dans les eaux environnementales, les fruits de mer et les produits frais ; Épidémiologie animale ; Cryptosporidiose et épidémiologie humaine ; Génomes et évolution génomique englobant : Génomique comparative des Cryptosporidium spp., Perspectives génomiques en biologie, Acquisition et utilisation des séquences du génome, Manipulation génétique ; Interaction hôte-parasite (immunologie, microbiome) ; Diagnostic et traitement. Les présentations de grande qualité discutées à la conférence ont fait état de progrès décisifs et ont permis de cerner de nouvelles possibilités qui inciteront les chercheurs et les organismes de financement à stimuler la recherche future dans une approche « une seule santé ¼ afin d'améliorer les connaissances de base et la gestion clinique et de santé publique de la cryptosporidiose zoonotique.

Analysis of Long Non-Coding RNA in Cryptosporidium parvum Reveals Significant Stage-Specific Antisense Transcription.

Cryptosporidium is a protist parasite that has been identified as the second leading cause of moderate to severe diarrhea in children younger than two and a significant cause of mortality worldwide. Cryptosporidium has a complex, obligate, intracellular but extra cytoplasmic lifecycle in a single host. How genes are regulated in this parasite remains largely unknown. Long non-coding RNAs (lncRNAs) play critical regulatory roles, including gene expression across a broad range of organisms. Cryptosporidium lncRNAs have been reported to enter the host cell nucleus and affect the host response. However, no systematic study of lncRNAs in Cryptosporidium has been conducted to identify additional lncRNAs. In this study, we analyzed a C. parvum in vitro strand-specific RNA-seq developmental time series covering both asexual and sexual stages to identify lncRNAs associated with parasite development. In total, we identified 396 novel lncRNAs, mostly antisense, with 86% being differentially expressed. Surprisingly, nearly 10% of annotated mRNAs have an antisense transcript. lncRNAs occur most often at the 3' end of their corresponding sense mRNA. Putative lncRNA regulatory regions were identified and many appear to encode bidirectional promoters. A positive correlation between lncRNA and upstream mRNA expression was observed. Evolutionary conservation and expression of lncRNA candidates was observed between C. parvum, C. hominis and C. baileyi. Ten C. parvum protein-encoding genes with antisense transcripts have P. falciparum orthologs that also have antisense transcripts. Three C. parvum lncRNAs with exceptional properties (e.g., intron splicing) were experimentally validated using RT-PCR and RT-qPCR. This initial characterization of the C. parvum non-coding transcriptome facilitates further investigations into the roles of lncRNAs in parasite development and host-pathogen interactions.

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.

Genetic ablation of purine salvage in Cryptosporidium parvum reveals nucleotide uptake from the host cell.

The apicomplexan parasite Cryptosporidium is a leading global cause of severe diarrheal disease and an important contributor to early-childhood mortality. Waterborne outbreaks occur frequently, even in countries with advanced water treatment capabilities, and there is currently no fully effective treatment. Nucleotide pathways are attractive targets for antimicrobial development, and several laboratories are designing inhibitors of these enzymes as potential treatment for Cryptosporidium infections. Here we take advantage of newly available molecular genetics for Cryptosporidium parvum to investigate nucleotide biosynthesis by directed gene ablation. Surprisingly, we found that the parasite tolerates the loss of classical targets including dihydrofolate reductase-thymidylate synthase (DHFR-TS) and inosine monophosphate dehydrogenase (IMPDH). We show that thymidine kinase provides a route to thymidine monophosphate in the absence of DHFR-TS. In contrast, only a single pathway has been identified for C. parvum purine nucleotide salvage. Nonetheless, multiple enzymes in the purine pathway, as well as the adenosine transporter, can be ablated. The resulting mutants are viable under normal conditions but are hypersensitive to inhibition of purine nucleotide synthesis in their host cell. Cryptosporidium might use as-yet undiscovered purine transporters and salvage enzymes; however, genetic and pharmacological experiments led us to conclude that Cryptosporidium imports purine nucleotides from the host cell. The potential for ATP uptake from the host has significant impact on our understanding of parasite energy metabolism given that Cryptosporidium lacks oxidative phosphorylation and glycolytic enzymes are not constitutively expressed throughout the parasite life cycle.

Life cycle progression and sexual development of the apicomplexan parasite Cryptosporidium parvum.

The apicomplexan parasite Cryptosporidium is a leading global cause of severe diarrhoeal disease and an important contributor to early childhood mortality. Currently, there are no fully effective treatments or vaccines available. Parasite transmission occurs through ingestion of oocysts, through either direct contact or consumption of contaminated water or food. Oocysts are meiotic spores and the product of parasite sex. Cryptosporidium has a single-host life cycle in which both asexual and sexual processes occur in the intestine of infected hosts. Here, we genetically engineered strains of Cryptosporidium to make life cycle progression and parasite sex tractable. We derive reporter strains to follow parasite development in culture and in infected mice and define the genes that orchestrate sex and oocyst formation through mRNA sequencing of sorted cells. After 2 d, parasites in cell culture show pronounced sexualization, but productive fertilization does not occur and infection falters. By contrast, in infected mice, male gametes successfully fertilize female parasites, which leads to meiotic division and sporulation. To rigorously test for fertilization, we devised a two-component genetic-crossing assay using a reporter that is activated by Cre recombinase. Our findings suggest obligate developmental progression towards sex in Cryptosporidium, which has important implications for the treatment and prevention of the infection.

A Genetically Tractable, Natural Mouse Model of Cryptosporidiosis Offers Insights into Host Protective Immunity.

Cryptosporidium is a leading cause of diarrheal disease and an important contributor to early childhood mortality, malnutrition, and growth faltering. Older children in high endemicity regions appear resistant to infection, while previously unexposed adults remain susceptible. Experimental studies in humans and animals support the development of disease resistance, but we do not understand the mechanisms that underlie protective immunity to Cryptosporidium. Here, we derive an in vivo model of Cryptosporidium infection in immunocompetent C57BL/6 mice by isolating parasites from naturally infected wild mice. Similar to human cryptosporidiosis, this infection causes intestinal pathology, and interferon-γ controls early infection while T cells are critical for clearance. Importantly, mice that controlled a live infection were resistant to secondary challenge and vaccination with attenuated parasites provided protection equal to live infection. Both parasite and host are genetically tractable and this in vivo model will facilitate mechanistic investigation and rational vaccine design.

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.

Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells.

Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment.