Diversity of Cryptosporidium PKS gene and using metabolomics to identify a polyketide product

Abstract

The protozoan parasite Cryptosporidium causes cryptosporidiosis and it is a major cause of non-viral diarrhoeal illness. Currently, five Cryptosporidium species are considered as human infectious and over 90% of infections are caused by C. parvum and C. hominis. A polyketide synthase (PKS) gene identified in C. parvum has been speculated to be involved in toxin production. The PKS gene has been identified in the C. parvum, C. hominis, and C. muris but the PKS protein has only been identified in the human infectious intestinal species: C. parvum and C. hominis. In this study, the PKS gene was identified in C. cuniculus, C. fayeri, C. andersoni and C. bovis. The identification was based on degenerate primer sets targeting various domains within the PKS gene. The results revealed that significant variability exists between the PKS gene in intestinal and gastric species of Cryptosporidium. The PKS gene sequence of 30 Cryptosporidium isolates from 21 subtypes within the three most common and waterborne outbreak causing Cryptosporidium species: C. parvum, C. hominis, and C. cuniculus were characterised. The resulting PKS gene sequences were divided into two distinct groups where C. parvum isolates formed one cluster (II) and C. hominis and C. cuniculus isolates formed another cluster (I). The CpPKS1 in C. parvum was highly conserved, by comparison, there was much more variation observed at the species level in C. hominis and C. cuniculus. The feasibility of using mass spectroscopy (MS) for a metabolomics investigation into Cryptosporidium with an aim of identifying a potential polyketide was conducted. The results demonstrated the possibility of constructing a metabolomics profile of Cryptosporidium. While a polyketide was not identified, an unusual sphingoid base related molecule: 2-amino,3,4-dihydroxyl heptadecane was found. This molecule is potentially cell toxic and may be related to host cell apoptosis attributed to Cryptosporidium infections. Further works are required on sequencing more subtypes of C. parvum and C. hominis isolates to increase the confidence on the polymorphism identified between species. In the future, HPLC based separation should allow purification of the 2-amino, 3,4-dihydroxyl heptadecane from a crude extract, then the stereochemistry of the molecule can be determined by NMR and cell-based cell toxicity assays can be performed.