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Miller, C. N.

Publications and source records attributed to Miller, C. N..

2 recordsLinked to original sources

Metabolic changes of the host-pathogen environment in a Cryptosporidium infection

Cryptosporidium is an important gut microbe whose contributions towards infant and immunocompromise patient mortality rates are steadily increasing. Current techniques for diagnosing, curing or simply understanding the biology of the parasite are few and far between, relying on a combination of in-silico predictions modelled on a varied and unique group of organisms and medical reports. The development of an in-vitro culture system, using COLO-680N cells, has provided the Cryptosporidium community with the opportunity to expand its toolkit for investigating this disease. One area in particular that is sorely overlooked is the metabolic alterations upon infection. Existing research is extremely limited and has already shown that significant variation can be found between the metabolome of different infected host species. Using a 1H Nuclear Magnetic Resonance approach to metabolomics, we have explored the nature of the mouse gut metabolome as well as providing the first insight into the metabolome of an infected cell line. Through a combination of Partial Least Squares Discriminant Analysis and predictive modelling, we exhibit new and potentially game changing insights into the effects of a Cryptosporidium parvum infection, while verifying the presence of known metabolic changes. Of particular note is the potential contribution of host derived taurine to the diuretic aspects of the disease previously attributed to a solely parasite based alteration of the gut environment. This practical and informative approach can spearhead our understanding of the Cryptosporidium-host metabolic exchange and thus provide novel targets for tackling this deadly parasite.

microbiology

A Cell Culture Platform For Cryptosporidium That Enables Long-Term Cultivation And New Tools For The Systematic Investigation Of Its Biology

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.

microbiology