Babesia microti, a tick-transmitted, intraerythrocytic protozoan parasite circulating mainly among small mammals, is the primary cause of human babesiosis. While most cases are transmitted by Ixodes ticks, the disease may also be transmitted through blood transfusion and perinatally. A comprehensive analysis of genome composition, genetic diversity, and gene expression profiling of seven B. microti isolates revealed that genetic variation in isolates from the Northeast United States is almost exclusively associated with genes encoding the surface proteome and secretome of the parasite. Furthermore, we found that polymorphism is restricted to a small number of genes, which are highly expressed during infection. In order to identify pathogen-encoded factors involved in host-parasite interactions, we screened a proteome array comprised of 174 B. microti proteins, including several predicted members of the parasite secretome. Using this immuno-proteomic approach we identified several novel antigens that trigger strong host immune responses during the onset of infection. The genomic and immunological data presented herein provide the first insights into the determinants of B. microti interaction with its mammalian hosts and their relevance for understanding the selective pressures acting on parasite evolution.
Human cryptosporidiosis is caused primarily by Cryptosporidium hominis, C. parvum and C. meleagridis. To accelerate research on parasites in the genus Cryptosporidium, we generated annotated, draft genome sequences of human C. hominis isolates TU502_2012 and UKH1, C. meleagridis UKMEL1, also isolated from a human patient, and the avian parasite C. baileyi TAMU-09Q1. The annotation of the genome sequences relied in part on RNAseq data generated from the oocyst stage of both C. hominis and C. baileyi. The genome assembly of C. hominis is significantly more complete and less fragmented than that available previously, which enabled the generation of a much-improved gene set for this species, with an increase in average gene length of 500 bp relative to the protein-encoding genes in the 2004 C. hominis annotation. Our results reveal that the genomes of C. hominis and C. parvum are very similar in both gene density and average gene length. These data should prove a valuable resource for the Cryptosporidium research community.
Babesia bovis is a hemoprotozoan parasite of cattle that has a complex life cycle within vertebrate and invertebrate hosts. In the mammalian host, B. bovis undergoes asexual reproduction while in the tick midgut, gametes are induced, fuse, and form zygotes. The zygote infects tick gut epithelial cells and transform into kinetes that are released into the hemolymph and invade other tick tissues such as the ovaries, resulting in transovarial transmission to tick offspring. To compare gene regulation between different B. bovis life stages, we collected parasites infecting bovine erythrocytes and tick hemolymph. Total RNA samples were isolated, and multiplexed libraries sequenced using paired-end 100 cycle reads of a HiSeq 2500. The data was normalized using the TMM method and analysed for significant differential expression using the generalized linear model likelihood ratio test (GLM LRT) in edgeR. To validate our datasets, ten genes were selected using NormFinder. Genes that had no significant fold change between the blood and tick stages in the RNA-Seq datasets were tested by quantitative PCR to determine their suitability as “housekeeping” genes. The normalized RNA-Seq data revealed genes upregulated during infection of the mammalian host or tick vector and six upregulated genes were validated by quantitative PCR. These datasets can help identify useful targets for controlling bovine babesiosis.
Supplemental Figure S1. Architecture of the Theileria parva Muguga genome and associated new structural annotation and RNAseq expression data.Data are shown for each of the 4 T. parva nuclear and one mitochondrial chromosomes. The five concentric rings, from outer-to innermost, represent (i) the boundaries of each contig (blue), with the last and most AT-rich contig representing the mitochondrion, (ii) the deviation from the average GC percentage (red), (iii) the genes on the forward strand (green = SVSP family genes, red = Tpr genes, purple = TpHN genes), (iv) the genes on the reverse strand (same colors as forward), and (v) RNAseq coverage depth (blue, on light purple background). The figure was generated using the Circleator software. The apicoplast genome was not re-annotated since only three of its 70 currently annotated genes had representative RNAseq coverage, and their existing annotation was consistent with the RNAseq data. Supplemental Figure S2. Updated gene annotation efforts in theTheileria parva genome reveal the existence of many genes that overlap adjacent genes at either UTR or CDS sequences. (A) Boxplots of the distribution of the total overlap length by genes that overlap by only the untranslated regions (UTR), or where the overlap includes a protein coding sequence (CDS) on either the same or opposite strands. (B) Shown are boxplots for the distribution of CDS length overlap in adjacent genes. Distributions were compared with a one-way ANOVA using alpha = 0.05. Significance is shown as follows: * 0.05>p≥0.01; ** 0.01>p≥0.001; *** p<0.001. Bar plot lines represent the mean and 5-95% percentiles from the mean. Supplemental Figure S3. RNA-seq reads that map to introns in the Theileria parva Muguga genome support the existence of genes where a subset of introns are not spliced. Read coverage per intron and protein coding sequence (CDSs) was calculated using HT-seq and their ratio plotted as a frequency histogram. While most introns had a coverage of zero (hence an intron_coverage/CDS_coverage ratio of zero), 1,610 introns had some read coverage, 701 of which had an intron_coverage/CDS_coverage ratio above or equal to 1.0. These 1,610 introns correspond to 744 genes. Supplemental Figure S4. Histogram of mRNA length across all annotated transcripts in the current T. parva Muguga annotation.All mRNAs were used to make this graph, including those for which no untranslated region was annotated. SupplementaryFigure S5. Types of transcription initiated in Theileria parva. Transcription in T. parva Muguga results from potential bidirectional (A) and cryptic promoters (B). Antisense transcription can be spliced (C), and may result in run-through transcription overlapping another gene (D). The model of transcription that emerges from these data is one of ubiquitous sense transcription of most genes in the schizont stage, but with a wide range of expression levels. Transcription can arise from potential bidirectional and cryptic promoters with highly prevalent antisense transcription. Blue boxes indicate exo...
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