Small and intermediate-size noncoding RNAs (sRNAs and is-ncRNAs) have been shown to play important regulatory roles in the development of several eukaryotic organisms. However, they have not been thoroughly explored in Cryptosporidium parvum, an obligate zoonotic protist parasite responsible for the diarrhoeal disease cryptosporidiosis. Using Illumina sequencing of a small RNA library, a systematic identification of novel small and is-ncRNAs was performed in C. parvum excysted sporozoites. A total of 79 novel is-ncRNA candidates, including antisense, intergenic and intronic is-ncRNAs, were identified, including 7 new small nucleolar RNAs (snoRNAs). Expression of select novel is-ncRNAs was confirmed by RT-PCR. Phylogenetic conservation was analysed using covariance models (CMs) in related Cryptosporidium and apicomplexan parasite genome sequences. A potential new type of small ncRNA derived from tRNA fragments was observed. Overall, a deep profiling analysis of novel is-ncRNAs in C. parvum and related species revealed structural features and conservation of these novel is-ncRNAs. Covariance models can be used to detect is-ncRNA genes in other closely related parasites. These findings provide important new sequences for additional functional characterization of novel is-ncRNAs in the protist pathogen C. parvum.
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 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 1,926 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.
Rodrigo P Baptista, Yiran Li, Adam Sateriale, Karen L Brooks, Alan Tracey, Mandy J Sanders, Brendan R E Ansell, Aaron R Jex, Garrett W Cooper, Ethan D Smith, Rui Xiao, Jennifer E Dumaine, Peter Georgeson, Bernard Pope, Matthew Berriman, Boris Striepen, James A Cotton, Jessica C Kissinger. Genome Res. 2021 Nov 11;gr.275325.121. doi: 10.1101/gr.275325.121.
Cryptosporidiosis is ranked sixth in the list of the most important food-borne parasites globally, and it is an important contributor to mortality in infants and the immunosuppressed. Recently, the number of genome sequences available for this parasite has increased drastically. The majority of the sequences are derived from population studies of Cryptosporidium parvum and Cryptosporidium hominis, the most important species causing disease in humans. Work with this parasite is challenging since it lacks an optimal, prolonged, in vitro culture system, which accurately reproduces the in vivo life cycle. This obstacle makes the cloning of isolates nearly impossible. Thus, patient isolates that are sequenced represent a population or, at times, mixed infections. Oocysts, the lifecycle stage currently used for sequencing, must be considered a population even if the sequence is derived from single-cell sequencing of a single oocyst because each oocyst contains four haploid meiotic progeny (sporozoites). Additionally, the community does not yet have a set of universal markers for strain typing that are distributed across all chromosomes. These variables pose challenges for population studies and require careful analyses to avoid biased interpretation. This review presents an overview of existing population studies, challenges, and potential solutions to facilitate future population analyses.
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.
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.
Giovanni Widmer, David Carmena, Martin Kváč, Rachel M. Chalmers, Jessica C. Kissinger, Lihua Xiao, Adam Sateriale, Boris Striepen, Fabrice Laurent, Sonia Lacroix-Lamandé, Gilles Gargala and Loïc Favennec. Parasite. 2020;27:14. doi: 10.1051/parasite/2020011.
Cryptosporidium has historically been a difficult organism to work with, and molecular genomic data for this important pathogen have typically lagged behind other prominent protist pathogens. CryptoDB ( http://cryptodb.org/ ) was launched in 2004 following the appearance of draft genome sequences for both C. parvum and C. hominis. CryptoDB merged with the EuPathDB Bioinformatics Resource Center family of databases ( https://eupathdb.org ) and has been maintained and updated regularly since its establishment. These resources are freely available, are web-based, and permit users to analyze their own sequence data in the context of reference genome sequences in our user workspaces. Advances in technology have greatly facilitated Cryptosporidium research in the last several years greatly enhancing and extending the data and types of data available for this genus. Currently, 13 genome sequences are available for 9 species of Cryptosporidium as well as the distantly related Gregarina niphandrodes and two free-living alveolate outgroups of the Apicomplexa, Chromera velia and Vitrella brassicaformis. Recent years have seen several new genome sequences for both existing and new Cryptosporidium species as well as transcriptomics, proteomics, SNP, and isolate population surveys. This chapter introduces the extensive data mining and visualization capabilities of the EuPathDB software platform and introduces the data types and tools that are currently available for Cryptosporidium. Key features are demonstrated with Cryptosporidium-relevant examples and explanations.
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.
Adam Sateriale, Jan Šlapeta, Rodrigo Baptista, Julie B. Engiles, Jodi A. Gullicksrud, Gillian T. Herbert, Carrie F. Brooks, Emily M. Kugler, Jessica C. Kissinger, Christopher A. Hunter, Boris Striepen. Cell Host Microbe. 2019 Jun 18. pii: S1931-3128 (19) 30251-3. doi: 10.1016/j.chom.2019.05.006.
Athens, Ga. – Infectious disease scientists from research institutions including the University of Georgia have reported the discovery and early validation of a drug that shows promise for treating cryptosporidiosis, a diarrheal disease that is a major cause of child mortality and for which there is no vaccine or effective treatment.
“Cryptosporidiosis is largely a disease of poverty,” said Boris Striepen, Distinguished Research Professor of Cellular Biology in UGA’s Franklin College of Arts and Sciences and a member of the Center for Tropical and Emerging Global Diseases. “Globally, it primarily affects infants in developing countries, but there are patients in the U.S.-those with weakened immune systems, such as HIV/AIDS or transplant patients-that would benefit greatly from new therapeutics.”
Striepen began studying crypto, as researchers often call the parasite that causes cryptosporidiosis, more than a decade ago. Now he and Sumiti Vinayak, assistant research scientist at UGA’s Center for Tropical and Emerging Global Diseases, along with scientists at Novartis and Washington State University, have reported the discovery of KDU731, a potent inhibitor of cryptosporidium, in the journal Nature.
Identifying KDU731 as a potential drug for the treatment of cryptosporidiosis began with the screening of a selection of 6,200 compounds that showed strong activity against the related malaria parasite. The Novartis team led by Ujjini H. Manjunatha and Thierry T. Diagana identified compounds with activity against crypto and found KDU731 particularly promising based on preclinical data.
Using a new mouse model, UGA’s Striepen and Vinayak showed that oral treatment with the drug dramatically reduced intestinal infection of immunocompromised mice. Additional research, led by Jennifer A. Zambriski at Washington State University, showed that treatment with KDU731 also leads to rapid resolution of diarrhea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection.
Crypto is most commonly spread through tainted drinking or recreational water. When a person drinks contaminated water, parasites emerge from spores and invade the cells that line the small intestine, causing severe diarrhea that can last for up to three weeks.
In 1993, more than 400,000 people living in the Milwaukee, Wisconsin, area were infected and became ill when one of the city’s water treatment systems malfunctioned. More than 100 people, mostly AIDS patients, died during the outbreak.
Outbreaks have also been linked to swimming pools and water parks. Crypto is the most common cause of diarrheal illness and outbreaks linked to recreational water because it is not easily killed by chlorine and can survive up to 10 days in properly treated water.
The Centers for Disease Control and Prevention reported at least 32 outbreaks in U.S. facilities during 2016-twice as many as in 2014, according to preliminary data in the agency’s May 18 Morbidity and Mortality Weekly Report.
Recent global studies have shown crypto to be one of the most important causes of life-threatening diarrhea in infants and toddlers, especially in areas that lack access to clean water. There is no vaccine and only one drug, nitazoxanide, approved by the U.S. Food and Drug Administration, but it provides no benefit for those in gravest danger-malnourished infants and immunocompromised patients.
Crypto is notoriously difficult to work with in a laboratory setting, but Striepen has developed new genetic techniques that make it easier to detect and follow the parasite. One technique involves manipulating crypto so that it emits light and is easier to detect and measure. For this study, Striepen’s team engineered a new “reporter” parasite that is amenable to whole-animal imaging, allowing the researchers to non-invasively track and record dissipation of the infection during treatment.
Striepen’s genetically modified organisms have been made available to researchers across the world in the hope that more scientists will be drawn to studying crypto.
“This is an important problem,” he said. “No one institution can solve it alone. It needs significant investment, and it needs a lot of people with good ideas.”
“The discovery of this compound represents an important step toward urgently needed treatment for gravely ill children around the world,” said Thierry Diagana, head of the Novartis Institutes for Tropical Diseases.
An online version of the study is available at http://dx.doi.org/10.1038/nature22337.
Writer: Allyson Mann
Contact:Boris Striepen Sumiti Vinayak