University of Georgia
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors

Tag: Cryptosporidium

Genomic and virulence analysis of in vitro cultured Cryptosporidium parvum

Donna Huber on February 28, 2024

Fig 1. Diagramatic section through the hollow fiber bioreactor.
Fig 1. Diagramatic section through the hollow fiber bioreactor.

 

Recent advances in the in vitro cultivation of Cryptosporidium parvum using hollow fiber bioreactor technology (HFB) have permitted continuous growth of parasites that complete all life cycle stages. The method provides access to all stages of the parasite and provides a method for non-animal production of oocysts for use in clinical trials. Here we examined the effect of long-term (>20 months) in vitro culture on virulence-factors, genome conservation, and in vivo pathogenicity of the host by in vitro cultured parasites. We find low-level sequence variation that is consistent with that observed in calf-passaged parasites. Further using a calf model infection, oocysts obtained from the HFB caused diarrhea of the same volume, duration and oocyst shedding intensity as in vivo passaged parasites.

Nigel Yarlett, Mary Morada, Deborah A Schaefer, Kevin Ackman, Elizabeth Carranza, Rodrigo de Paula Baptista, Michael W Riggs, Jessica Kissinger. PLoS Pathog. 2024 Feb 28;20(2):e1011992. doi: 10.1371/journal.ppat.1011992.

Genetic crosses within and between species of Cryptosporidium

Donna Huber on January 2, 2024

Figure 1 PheRS can be used as a selection marker for stable transgenesis.
PheRS can be used as a selection marker for stable transgenesis.

Parasites and their hosts are engaged in reciprocal coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium, genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach, multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing Cryptosporidium parvum, a parasite of cattle and humans, and Cryptosporidium tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity.

Sebastian Shaw, Ian S Cohn, Rodrigo P Baptista, Guoqin Xia, Bruno Melillo, Fiifi Agyabeng-Dadzie, Jessica C Kissinger, Boris Striepen. Proc Natl Acad Sci USA. 2024 Jan 2;121(1):e2313210120. doi: 10.1073/pnas.2313210120.

Small and intermediate size structural RNAs in the unicellular parasite Cryptosporidium parvum as revealed by sRNA-seq and comparative genomics

Donna Huber on May 10, 2022

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.

Yiran Li, Rodrigo P Baptista, Xiaohan Mei, Jessica C Kissinger. Microb Genom. 2022 May;8(5). doi: 10.1099/mgen.0.000821

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

Donna Huber on November 11, 2021

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.

Challenges for Cryptosporidium Population Studies

Donna Huber on June 10, 2021

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.

Baptista, Rodrigo P.; Cooper, Garrett W.; Kissinger, Jessica C. 2021. Genes 12, no. 6: 894. https://doi.org/10.3390/genes12060894

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

Donna Huber on January 14, 2021

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.

Yiran Li, Rodrigo P. Baptista, Adam Sateriale, Boris Striepen and Jessica C. Kissinger. Front Cell Infect Microbiol. 2021 Jan 14;10:608298. doi: 10.3389/fcimb.2020.608298. eCollection 2020.

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

Donna Huber on March 13, 2020

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.

Accessing Cryptosporidium Omic and Isolate Data via CryptoDB.org

Donna Huber on August 27, 2019

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.

Warrenfeltz S, Kissinger JC, EuPathDB Team. Methods Mol Biol. 2020;2052:139-192. doi: 10.1007/978-1-4939-9748-0_10.

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

Donna Huber on June 18, 2019

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.