Ebook Cryptosporidium - Parasite and disease: Part 2

Part 2 book "Cryptosporidium - Parasite and disease" includes content: Cryptosporidium - current state of genomics and systems biological research; from genome to proteome - transcriptional and proteomic analysis of cryptosporidium parasites; cryptosporidium metabolism, human cryptosporidiosis - a clinical perspective; immunology of cryptosporidiosis; treatment of cryptosporidiosis,... and other contents.

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Ebook Cryptosporidium - Parasite and disease: Part 2 Part II Molecular Biology Chapter 6 Cryptosporidium: Current State of Genomics and Systems Biological Research Aaron R. Jex and Robin B. Gasser Abstract Recent years have seen an unprecedented expansion in our knowledge of Cryptosporidium and cryptosporidiosis through the emergence of the genomics and systems biological age. High-quality draft genome sequences are now published for C. parvum and C. hominis, and the draft assembly of C. muris has been made publicly available. These genome sequences reveal a highly stream-lined parasite with limited metabolic and biosynthetic pathways and a heavy reliance on the host- cell. Bottlenecks in these pathways may be exploited for new drugs, which remain stubbornly illusive for these parasites. As more genomic information becomes available, fundamental research into gene regulation, genomic evolution and genome-wide variation becomes possible. This research will provide new insights into the transmission dynamics of these parasites and markers associated with host-specificity, virulence and pathogenicity, and will allow the identification of novel loci for use as molecular-diagnostic markers and genes under heavy immunoselection, potentially providing a basis for vaccine development. With the accelerating reduction in costs associated with ‘omic’ research, improved accessibility to analytical tools and in vitro culture of Cryptospordium, this field has tremendous potential to shape our understanding of their biology in the coming years. A.R. Jex (*) The University of Melbourne, Werribee, Victoria, Australia e-mail: ajex@unimelb.edu.au R.B. Gasser The University of Melbourne, Parkville, Victoria 3010, Australia e-mail: robinbg@unimelb.edu.au ` S.M. Caccio and G. Widmer (eds.), Cryptosporidium: parasite and disease, 327 DOI 10.1007/978-3-7091-1562-6_6, © Springer-Verlag Wien 2014 328 A.R. Jex and R.B. Gasser 6.1 Genomics of Cryptosporidium Research of Cryptosporidium in the genomics age is landmarked by the publication of the complete genome sequence of C. parvum Iowa strain (Abrahamsen et al. 2004). This genome was sequenced to ~13-fold coverage using a shotgun Sanger sequencing approach (Table 6.1). Briefly, this process involved isolating total genomic DNA from purified C. parvum oocysts, randomly shearing the DNA (2–5 kb) and constructing plasmid libraries, from which 120,000 clones were sequenced. In addition, to facilitate scaffolding and the resolution of repeat regions, the authors sequenced the genome to ~0.5-fold depth using data generated from large-insert (~15 kb) λ-phage libraries constructed from genomic DNA of the same Iowa isolate. The assembly of the sequence data from both libraries was guided by a physical map, which had been generated previously for this C. parvum isolate using ~200 oligonucleotide primers to screen haploid amounts (i.e., a “HAPPY” map) of C. parvum DNA by the polymerase chain reaction (PCR) for marker regions spaced ~50 kp apart (Piper et al. 1998; Abrahamsen et al. 2004) and a draft sequence for chromosome 6 (Bankier et al. 2003). The combination of the long sequence reads, large-insert libraries and the HAPPY map, combined with selective PCR-based sequencing for the purpose of gap closure, allowed the assembly of the C. parvum genome into its eight chromosomes, leaving five physical gaps. The final assembly revealed an AT-rich (70 %) and highly compact genome of ~9.1 Mb, which is relatively small in comparison with related apicomplexans, such as Eimeria tenella (~60 Mb) (Shirley 1994, 2000) and Plasmodium falciparum (~23 Mb) (Gardner et al. 2002a) and, unlike many apicomplexans, includes no mitochondrial or apicoplast genomes. The annotation of the C. parvum genome was achieved primarily through the prediction of open reading frames (ORFs) of at least 67 amino acids in length from the assembled genome sequence. Although gene prediction methods based on the use of hidden Markov and machine learning models for the analysis of expressed sequence tag (EST) data, and, more recently, deep RNA-Sequencing data (Wang et al. 2009) are considered standard approaches for current genomic sequencing projects (Jex et al. 2011a; Young et al. 2012), extensive EST data are not yet available for Cryptosporidium species and are challenging to generate from the endogenous developmental stages. Noting this, the C. parvum genome appears to have few intronic sequences and, thus, the straight-forward prediction of ORFs was sufficient to allow the identification of 3,952 (3,807 protein-coding) genes in C. parvum. These genes were fewer in number than predicted to be encoded by the genomes of related apicomplexans, such species of Plasmodium (~5,300 genes) (Gardner et al. 2002b), Toxoplasma (~7,700) (Radke et al. 2005) and Theileria (~4,000) (Gardner et al. 2005), which, coupled to a lack of introns and limited non-coding sequence (~25 % of the entire genome), explains the smaller size of the C. parvum genome (see also Bankier et al. 2003). 6 Cryptosporidium: Current State of Genomics and Systems Biological Research 329 Table 6.1 Comparison of characteristics and metrics of each Cryptosporidium genome sequenced and available in CryptoDB (www.cryptodb.org) C. parvum C. hominis C. parvum C. muris Feature Iowa II TU502 TU114 RN66 Project accession AAEE01000000 AAEL000000 not available PRJNA19553 Assembly size (Mb) 9.1 8.7 not available 9.2 Assembly metrics 8: 1.10 Mb 1,422: 14.5 kb not available 45: 0.72 Mb (Contigs: N50) Number of genes 3,952 3,994 ~3,952 3,986 GC richness 30.3 31.7 ...