A cell culture platform forcryptosporidiumthat enables long-term cultivation and new tools for the systematic investigation of its biology

Abstract

Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking. Currently, Cryptosporidium oocysts for research must be freshly produced in animals and cannot be long-term stored. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains (Moredun, Iowa) produce sufficient infectious oocysts to infect subsequent cultures. Oocyst identity was confirmed by specific staining (Crypt-a-glo, Vicia Villosa lectin, Sporo-glo), PCR-based amplification of Cryptosporidium-specific genes, lipidomics fingerprinting, and atomic force microscopy (AFM). Antibody-stained oocysts produced unstained oocysts confirming production of novel oocysts. Infected cultures could be cryoconserved and continued to produce infectious oocysts after resuscitation. Transmission electron microscopy identified all key Cryptosporidium life cycle stages. Infected cultures produced thick-walled (primarily involved in Cryptosporidium transmission between organisms) and thin-walled oocysts (important for Cryptosporidium propagation within a host/tissue) as indicated by DAPI staining (only thin-walled oocysts are permeable to DAPI staining, thus allowing visualisation of sporozoites) and AFM. In conclusion, we present a novel, easy-to-handle cell culture system that enables the propagation, cryopreservation and detailed investigation of Cryptosporidium at a laboratory scale. Its availability will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/134270 doi: bioRxiv preprint first posted online May. 4, 2017; 3 Cryptosporidiosis causes a significant number of deaths in children and immunocompromised individuals [1][2][3][4] . It is caused by species of the genus Cryptosporidium, in humans typically by C. parvum and C. hominis. The Cryptosporidium species belong to the phylum Apicomplexa and it has recently been proposed for the species to be reclassified as a member of the subclass of gregarina 5,6 . They are waterborne pathogens, and cryptosporidiosis has commonly been associated with disease in developing countries [7][8][9] . However, more recent molecular epidemiology studies suggested that the disease is also an increasing health concern in developed countries and may have reached epidemic levels 1, 2, 10-14 . Only one moderately effective drug (nitazoxanide) is available for the treatment of cryptosporidiosis.More effective drugs are urgently needed 10,15,16 .Cryptosporidium is a parasite that invades host cells, within the boundaries of the host cell membrane, residing intracellularly yet extra-cytoplasmic sometimes referred to simply as epicellular 17 . Cryptosporidium typically infects e...

NMR metabolomics reveals effects of Cryptosporidium infections on host cell metabolome

“…Previous studies recently demonstrated the successful long-term propagation of C. parvum in COLO-680N cell culture [22]. The ability of the cell culture to maintain the parasite for up to 8 weeks [22,23] along with the presence of organelles around the parasite (e.g.…”

“…We validated our methodology by comparing faecal profile results to the previously published studies, which used different methodologies, namely GC-MS [27,28]. Further experimentation utilised a recently published infectible culture, COLO-680N [22], to determine if any similarities or differences in response to infection could be determined.…”

“…Recent papers have attempted to rectify this by proposing improved or entirely novel techniques for culturing the parasite ex vivo in cell cultures, using the cultured cancer cells as host cells [20,21]. A recent study identified that infection of COLO-680N cell cultures produced a longer term and higher production volume culture of the parasite compared to previously existing in vitro cultures [22,23]. These advances have allowed higher in depth microscopy-based studies and even promise to provide a solution to developing a genetic engineering platform for the parasite.…”

The Symbiotic Spectrum: Where Do the Gregarines Fit?

“…have mutualistic, commensalistic, and parasitic effects on this host (Table S1). A long-term goal would be the development of an in vitro and axenic (animal-and tissue-free) culturing system [63,64] that would further permit the exploration of the basic cell and developmental biology of gregarines. Despite their importance, research on gregarines is progressing very slowly, and it will require researchers from various disciplines to come together and provide their expertise in expanding our knowledge on the evolution of parasitism in these microbes.…”

“…The debate about the relationship between Cryptosporidium and the gregarines has not been settled yet, and the current phylogenetic placement of the Cryptosporidia has two important implications: (i) parasitism of vertebrates in the apicomplexans might have evolved twice (Figure 1), and (ii) if this proves to be true, Cryptosporidium could be used as a model system to elucidate the biology of gregarines. There have been rapid advancements in the methodological approaches [3] for the study of the parasites, including the development of an in vitro cell culturing system in cell lines [63] and organoids [70,71] along with the development of genetic tools such as CRISPR/Cas9 [72] and small interfering RNA (siRNA) [73]. In addition, there are several 'omics' methods that have been introduced including in vitro transcriptomics [74,75], proteomics [63,76], metabolomics [60], and state-of-the art microscopy techniques [63], including immunomicroscopy [63,77], to understand the invasion strategies and host-parasite interactions.…”