The Complete Mitochondrial Genome of the Foodborne Parasitic Pathogen Cyclospora cayetanensis

Abstract

Cyclospora cayetanensis is a human-specific coccidian parasite responsible for several food and water-related outbreaks around the world, including the most recent ones involving over 900 persons in 2013 and 2014 outbreaks in the USA. Multicopy organellar DNA such as mitochondrion genomes have been particularly informative for detection and genetic traceback analysis in other parasites. We sequenced the C. cayetanensis genomic DNA obtained from stool samples from patients infected with Cyclospora in Nepal using the Illumina MiSeq platform. By bioinformatically filtering out the metagenomic reads of non-coccidian origin sequences and concentrating the reads by targeted alignment, we were able to obtain contigs containing Eimeria-like mitochondrial, apicoplastic and some chromosomal genomic fragments. A mitochondrial genomic sequence was assembled and confirmed by cloning and sequencing targeted PCR products amplified from Cyclospora DNA using primers based on our draft assembly sequence. The results show that the C. cayetanensis mitochondrion genome is 6274 bp in length, with 33% GC content, and likely exists in concatemeric arrays as in Eimeria mitochondrial genomes. Phylogenetic analysis of the C. cayetanensis mitochondrial genome places this organism in a tight cluster with Eimeria species. The mitochondrial genome of C. cayetanensis contains three protein coding genes, cytochrome (cytb), cytochrome C oxidase subunit 1 (cox1), and cytochrome C oxidase subunit 3 (cox3), in addition to 14 large subunit (LSU) and nine small subunit (SSU) fragmented rRNA genes.

Fig 1. Confirmation of the C. Cayetanensis mt genome sequence.

A) Overlapping PCR fragments used to confirm the C. cayetanenesis mt genome sequence and concatemeric structure. PCR fragments are shown with dotted lines. Forward sequence reads are shown in blue or orange and reverse reads are shown in red or light blue. Terminal grey bands indicate identical sequence regions. B) A 659 bp contig constructed using overlapping junction PCR fragment sequences (see Table 2) is represented in gray. Diagonal red bars represent portions of the junction PCR product mapping to the ends of the complete C. cayetanensis mt genome. C) WGS reads spanning the junction region in the initial mt assembly. The vertical red lines mark the tail:head junction.

Fig 2. Gene arrangement of the C. cayetanensis mt genome.

Predicted genes on the Cyclospora mitochondrial genomes were derived based on ClustalW alignment with the Eimeria gallopavonis (KJ608417). Orange indicates coding genes, blue indicates fragments of LSU rRNA, and yellow indicates fragments of SSU rRNA. Transcriptional direction is indicated by arrowed end.

Fig 3. Phylogenetic relationships among Eimeria species and C. cayetanensis based on mitochondrial genome sequences.

NCBI accession numbers are included. The evolutionary relationship between the Eimeria (13), Cyclospora(1) and P.falciparum (1) mitochondrial genomes was inferred using the Maximum Likelihood method with 500 replications for bootstrap analysis. The confidence value for clustering is given as percentage above the branches. The scale bar points to the number of substitutions per site. After analysis, the outgroup branch was removed for clarity. Default parameters in MEGA6 were retained for phylogenetic analysis and tree building.