The Cryptosporidium Parvum Transcriptome during In Vitro Development

Mauzy, Mary Jean; Enomoto, Shinichiro; Lancto, Cheryl A.; Abrahamsen, Mitchell S.; Rutherford, Mark S.

doi:10.1371/journal.pone.0031715

Cited by 106 publications

(130 citation statements)

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“…The original variant data set contained 622 variants not present in IbA10G2. Genes of interest that were broadly selected using the following criteria were as follows: (i) genes linked to oocyst formation and function in previous studies such as the COWP family genes (cgd7_1800, cgd6_200, and cgd6_210), which are believed to play a fundamental role in the durability of oocysts (25), and the CpCCP1 gene (cgd_1730), which is actively expressed at the sporozoite stage of development and yet may play a role in parasitophorous vacuole integrity (26,27), and (ii) genes with a Gene Ontology classification suggestive of being involved in cell homeostasis, development, or invasion, such as the TRAP-C2 gene (cgd5_3420), encoding a member of the thrombospondin-related anonymous protein family, linked to apicomplexan cell invasion (28). The full list of genes is shown in Table 4.…”

Section: Discussionmentioning

confidence: 99%

Genomic Variation in IbA10G2 and Other Patient-Derived Cryptosporidium hominis Subtypes

et al. 2017

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In order to improve genotyping and epidemiological analysis of Cryptosporidium spp., genomic data need to be generated directly from a broad range of clinical specimens. Utilizing a robust method that we developed for the purification and generation of amplified target DNA, we present its application for the successful isolation and whole-genome sequencing of 14 different Cryptosporidium hominis patient specimens. Six isolates of subtype IbA10G2 were analyzed together with a single representative each of 8 other subtypes: IaA20R3, IaA23R3, IbA9G3, IbA13G3, IdA14, IeA11G3T3, IfA12G1, and IkA18G1. Parasite burden was measured over a range of more than 2 orders of magnitude for all samples, while the genomes were sequenced to mean depths of between 17ϫ and 490ϫ coverage. Sequence homologybased functional annotation identified several genes of interest, including the gene encoding Cryptosporidium oocyst wall protein 9 (COWP9), which presented a predicted loss-of-function mutation in all the sequence subtypes, except for that seen with IbA10G2, which has a sequence identical to the Cryptosporidium parvum reference Iowa II sequence. Furthermore, phylogenetic analysis showed that all the IbA10G2 genomes form a monophyletic clade in the C. hominis tree as expected and yet display some heterogeneity within the IbA10G2 subtype. The current report validates the aforementioned method for isolating and sequencing Cryptosporidium directly from clinical stool samples. In addition, the analysis demonstrates the potential in mining data generated from sequencing multiple whole genomes of Cryptosporidium from human fecal samples, while alluding to the potential for a higher degree of genotyping within Cryptosporidium epidemiology.

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Section: Discussionmentioning

confidence: 99%

Genomic Variation in IbA10G2 and Other Patient-Derived Cryptosporidium hominis Subtypes

et al. 2017

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“…The transcript copies for each of the 4 loci generated using RT-PCR were calculated using RotorGene Q software. The amount of signal for COWP, cp15 and cp900 was normalized to 18S rRNA for each time period as previously described (Abrahamsen and Schroeder, 1999;Mauzy et al, 2012;Schroeder et al, 1998).…”

Section: Comparison Of Gene Expression In Hct-8 Versus Cell-free Cultmentioning

confidence: 99%

“…A recent study conducted a genome-wide transcriptome analysis of C. parvum infected HCT-8 cells over a 72 hr period (Mauzy et al, 2012). Quantitative-PCR (qPCR) for 3302 genes (87% of the protein coding genes) indicated that each gene has detectable transcription in at least one time point assessed (Mauzy et al, 2012).…”

Section: Article In Pressmentioning

confidence: 99%

“…Quantitative-PCR (qPCR) for 3302 genes (87% of the protein coding genes) indicated that each gene has detectable transcription in at least one time point assessed (Mauzy et al, 2012). Further studies, which involve a wider range of genes, should be conducted to better understand the expression of Cryptosporidium genes in cell-free culture.…”

Section: Article In Pressmentioning

confidence: 99%

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Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies

Yang

Elankumaran

Hijjawi

et al. 2015

Experimental Parasitology

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• Scanning electron microscopy (SEM) characterisation of life cycle stages in cell-free culture.• Comparison of gene expression in cell culture and cell-free culture.• Gene expression cell-free culture was delayed and was lower.• Data provide support for cell-free culture of Cryptosporidium. A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages. G R A P H I C A L A B S T R A C T

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“…Although intracellular development is asynchronous, at 48 h postinfection there is increased morphological complexity marked by reinvasion of epithelial cells by merozoites (37). A recent study of the transcriptome of the intracellular stages of C. parvum reported a spike in expression of 7 Cryptosporidium-specific genes, including 5 genes encoding mucin-like glycoproteins, at 48 h postinfection (37).…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of Cryptosporidium parvum Clec, a Novel C-Type Lectin Domain-Containing Mucin-Like Glycoprotein

et al. 2013

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dCryptosporidium species are waterborne apicomplexan parasites that cause diarrheal disease worldwide. Although the mechanisms underlying Cryptosporidium-host cell interactions are not well understood, mucin-like glycoproteins of the parasite are known to mediate attachment and invasion in vitro. We identified C. parvum Clec (CpClec), a novel mucin-like glycoprotein that contains a C-type lectin domain (CTLD) and has orthologs in C. hominis and C. muris. CTLD-containing proteins are ligand-binding proteins that function in adhesion and signaling and are present in a wide range of organisms, from humans to viruses. However, this is the first report of a CTLD-containing protein in protozoa and in Apicomplexa. CpClec is predicted to be a type 1 membrane protein, with a CTLD, an O-glycosylated mucin-like domain, a transmembrane domain, and a cytoplasmic tail containing a YXX sorting motif. The predicted structure of CpClec displays several characteristics of canonical CTLD-containing proteins, including a long loop region hydrophobic core associated with calcium-dependent glycan binding as well as predicted calcium-and glycan-binding sites. CpClec expression during C. parvum infection in vitro is maximal at 48 h postinfection, suggesting that it is developmentally regulated. The 120-kDa mass of native CpClec is greater than predicted, most likely due to O-glycosylation. CpClec is localized to the surface of the apical region and to dense granules of sporozoites and merozoites. Taken together, these findings, along with the known functions of C. parvum mucin-like glycoproteins and of CTLD-containing proteins, strongly implicate a significant role for CpClec in Cryptosporidium-host cell interactions.

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The Cryptosporidium Parvum Transcriptome during In Vitro Development

Cited by 106 publications

References 64 publications

Genomic Variation in IbA10G2 and Other Patient-Derived Cryptosporidium hominis Subtypes

Genomic Variation in IbA10G2 and Other Patient-Derived Cryptosporidium hominis Subtypes

Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies

Identification and Characterization of Cryptosporidium parvum Clec, a Novel C-Type Lectin Domain-Containing Mucin-Like Glycoprotein

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