Reassociation kinetics-based approach for partial genome sequencing of the cattle tick, Rhipicephalus (Boophilus) microplus

Background: The size and repetitive nature of the Rhipicephalus microplus genome makes obtaining a full genome sequence fiscally and technically problematic. To selectively obtain gene-enriched regions of this tick's genome, Cot filtration was performed, and Cot-filtered DNA was sequenced via 454 FLX pyrosequencing.Results: The sequenced Cot-filtered genomic DNA was assembled with an EST-based gene index of 14,586 unique entries where each EST served as a potential "seed" for scaffold formation. The new sequence assembly extended the lengths of 3,913 of the 14,586 gene index entries. Over half of the extensions corresponded to extensions of over 30 amino acids. To survey the repetitive elements in the tick genome, the complete sequences of five BAC clones were determined. Both Class I and II transposable elements were found. Comparison of the BAC and Cot filtration data indicates that Cot filtration was highly successful in filtering repetitive DNA out of the genomic DNA used in 454 sequencing.Conclusion: Cot filtration is a very useful strategy to incorporate into genome sequencing projects on organisms with large genome sizes and which contain high percentages of repetitive, difficult to assemble, genomic DNA. Combining the Cot selection approach with 454 sequencing and assembly with a pre-existing EST database as seeds resulted in extensions of 27% of the members of the EST database

Genomic analysis of Campylobacter fetus subspecies: identification of candidate virulence determinants and diagnostic assay targets

Background: Campylobacter fetus subspecies venerealis is the causative agent of bovine genital campylobacteriosis, asymptomatic in bulls the disease is spread to female cattle causing extensive reproductive loss. The microbiological and molecular differentiation of C. fetus subsp. venerealis from C. fetus subsp. fetus is extremely difficult. This study describes the analysis of the available C. fetus subsp. venerealis AZUL-94 strain genome (~75–80%) to identify elements exclusively found in C. fetus subsp. venerealis strains as potential diagnostic targets and the characterisation of subspecies virulence genes. Results: Eighty Kb of genomic sequence (22 contigs) was identified as unique to C. fetus subsp. venerealis AZUL-94 and consisted of type IV secretory pathway components, putative plasmid genes and hypothetical proteins. Of the 9 PCR assays developed to target C. fetus subsp. venerealis type IV secretion system genes, 4 of these were specific for C. fetus subsp. venerealis biovar venerealis and did not detect C. fetus subsp. venerealis biovar intermedius. Two assays were specific for C. fetus subsp. venerealis AZUL-94 strain, with a further single assay specific for the AZUL-94 strain and C. fetus subsp. venerealis biovar intermedius (and not the remaining C. fetus subsp. venerealis biovar venerealis strains tested). C. fetus subsp. fetus and C. fetus subsp. venerealis were found to share most common Campylobacter virulence factors such as SAP, chemotaxis, flagellar biosynthesis, 2-component systems and cytolethal distending toxin subunits (A, B, C). We did not however, identify in C. fetus the full complement of bacterial adherence candidates commonly found in other Campylobacter spp. Conclusion: The comparison of the available C. fetus subsp. venerealis genome sequence with the C. fetus subsp. fetus genome identified 80 kb of unique C. fetus subsp. venerealis AZUL94 sequence, with subsequent PCR confirmation demonstrating inconsistent amplification of these targets in all other C. fetus subsp. venerealis strains and biovars tested. The assays developed here highlight the complexity of targeting strain specific virulence genes for field studies for the molecular identification and epidemiology of C. fetus

The complexity of Rhipicephalus (Boophilus) microplus genome characterised through detailed analysis of two BAC clones

<p>Abstract</p> <p>Background</p> <p><it>Rhipicephalus (Boophilus) microplus (Rmi) </it>a major cattle ectoparasite and tick borne disease vector, impacts on animal welfare and industry productivity. In arthropod research there is an absence of a complete Chelicerate genome, which includes ticks, mites, spiders, scorpions and crustaceans. Model arthropod genomes such as <it>Drosophila </it>and <it>Anopheles </it>are too taxonomically distant for a reference in tick genomic sequence analysis. This study focuses on the <it>de-novo </it>assembly of two <it>R. microplus </it>BAC sequences from the understudied <it>R microplus </it>genome. Based on available <it>R. microplus </it>sequenced resources and comparative analysis, tick genomic structure and functional predictions identify complex gene structures and genomic targets expressed during tick-cattle interaction.</p> <p>Results</p> <p>In our BAC analyses we have assembled, using the correct positioning of BAC end sequences and transcript sequences, two challenging genomic regions. Cot DNA fractions compared to the BAC sequences confirmed a highly repetitive BAC sequence BM-012-E08 and a low repetitive BAC sequence BM-005-G14 which was gene rich and contained short interspersed elements (SINEs). Based directly on the BAC and Cot data comparisons, the genome wide frequency of the SINE Ruka element was estimated. Using a conservative approach to the assembly of the highly repetitive BM-012-E08, the sequence was de-convoluted into three repeat units, each unit containing an 18S, 5.8S and 28S ribosomal RNA (rRNA) encoding gene sequence (rDNA), related internal transcribed spacer and complex intergenic region.</p> <p>In the low repetitive BM-005-G14, a novel gene complex was found between to 2 genes on the same strand. Nested in the second intron of a large 9 Kb <it>papilin </it>gene was a <it>helicase </it>gene. This <it>helicase </it>overlapped in two exonic regions with the <it>papilin</it>. Both these genes were shown expressed in different tick life stage important in ectoparasite interaction with the host. Tick specific sequence differences were also determined for the <it>papilin </it>gene and the protein binding sites of the 18S subunit in a comparison to <it>Bos taurus</it>.</p> <p>Conclusion</p> <p>In the absence of a sequenced reference genome we have assembled two complex BAC sequences, characterised novel gene structure that was confirmed by gene expression and sequencing analyses. This is the first report to provide evidence for 2 eukaryotic genes with exon regions that overlap on the same strand, the first to describe <it>Rhipicephalinae papilin</it>, and the first to report the complete ribosomal DNA repeated unit sequence structure for ticks. The Cot data estimation of genome wide sequence frequency means this research will underpin future efforts for genome sequencing and assembly of the <it>R. microplus </it>genome.</p

De novo assembly of Euphorbia fischeriana root transcriptome identifies prostratin pathway related genes

Background Euphorbia fischeriana is an important medicinal plant found in Northeast China. The plant roots contain many medicinal compounds including 12-deoxyphorbol-13-acetate, commonly known as prostratin that is a phorbol ester from the tigliane diterpene series. Prostratin is a protein kinase C activator and is effective in the treatment of Human Immunodeficiency Virus (HIV) by acting as a latent HIV activator. Latent HIV is currently the biggest limitation for viral eradication. The aim of this study was to sequence, assemble and annotate the E. fischeriana transcriptome to better understand the potential biochemical pathways leading to the synthesis of prostratin and other related diterpene compounds. Results In this study we conducted a high throughput RNA-seq approach to sequence the root transcriptome of E. fischeriana. We assembled 18,180 transcripts, of these the majority encoded protein-coding genes and only 17 transcripts corresponded to known RNA genes. Interestingly, we identified 5,956 protein-coding transcripts with high similarity (>=75%) to Ricinus communis, a close relative to E. fischeriana. We also evaluated the conservation of E. fischeriana genes against EST datasets from the Euphorbeacea family, which included R. communis, Hevea brasiliensis and Euphorbia esula. We identified a core set of 1,145 gene clusters conserved in all four species and 1,487 E. fischeriana paralogous genes. Furthermore, we screened E. fischeriana transcripts against an in-house reference database for genes implicated in the biosynthesis of upstream precursors to prostratin. This identified 24 and 9 candidate transcripts involved in the terpenoid and diterpenoid biosyntehsis pathways, respectively. The majority of the candidate genes in these pathways presented relatively low expression levels except for 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS) and isopentenyl diphosphate/dimethylallyl diphosphate synthase (IDS), which are required for multiple downstream pathways including synthesis of casbene, a proposed precursor to prostratin. Conclusion The resources generated in this study provide new insights into the upstream pathways to the synthesis of prostratin and will likely facilitate functional studies aiming to produce larger quantities of this compound for HIV research and/or treatment of patients

The Complete Genome Sequence of the Pathogenic Intestinal Spirochete Brachyspira pilosicoli and Comparison with Other Brachyspira Genomes

Background: The anaerobic spirochete Brachyspira pilosicoli colonizes the large intestine of various species of birds and mammals, including humans. It causes ''intestinal spirochetosis'', a condition characterized by mild colitis, diarrhea and reduced growth. This study aimed to sequence and analyse the bacterial genome to investigate the genetic basis of its specialized ecology and virulence. Methodology/Principal Findings: The genome of B. pilosicoli 95/1000 was sequenced, assembled and compared with that of the pathogenic Brachyspira hyodysenteriae and a near-complete sequence of Brachyspira murdochii. The B. pilosicoli genome was circular, composed of 2,586,443 bp with a 27.9 mol% G+C content, and encoded 2,338 genes. The three Brachyspira species shared 1,087 genes and showed evidence of extensive genome rearrangements. Despite minor differences in predicted protein functional groups, the species had many similar features including core metabolic pathways. Genes distinguishing B. pilosicoli from B. hyodysenteriae included those for a previously undescribed bacteriophage that may be useful for genetic manipulation, for a glycine reductase complex allowing use of glycine whilst protecting from oxidative stress, and for aconitase and related enzymes in the incomplete TCA cycle, allowing glutamate synthesis and function of the cycle during oxidative stress. B. pilosicoli had substantially fewer methyl-accepting chemotaxis genes than B. hyodysenteriae and hence these species are likely to have different chemotactic responses that may help to explain their different host range and colonization sites. B. pilosicoli lacked the gene for a new putative hemolysin identified in B. hyodysenteriae WA1. Both B. pilosicoli and B. murdochii lacked the rfbBADC gene cluster found on the B. hyodysenteriae plasmid, and hence were predicted to have different lipooligosaccharide structures. Overall, B. pilosicoli 95/1000 had a variety of genes potentially contributing to virulence. Conclusions/Significance: The availability of the complete genome sequence of B. pilosicoli 95/1000 will facilitate functional genomics studies aimed at elucidating host-pathogen interactions and virulence

Proteases of haematophagous arthropod vectors are involved in blood-feeding, yolk formation and immunity : a review

Ticks, triatomines, mosquitoes and sand flies comprise a large number of haematophagous arthropods considered vectors of human infectious diseases. While consuming blood to obtain the nutrients necessary to carry on life functions, these insects can transmit pathogenic microorganisms to the vertebrate host. Among the molecules related to the blood-feeding habit, proteases play an essential role. In this review, we provide a panorama of proteases from arthropod vectors involved in haematophagy, in digestion, in egg development and in immunity. As these molecules act in central biological processes, proteases from haematophagous vectors of infectious diseases may influence vector competence to transmit pathogens to their prey, and thus could be valuable targets for vectorial control

Wheat grain proteomics for the food industry

The analysis of protein in wheat grain is now a high priority because this provides the major source of protein utilized in the food industry. The novel aspects in this area that are of particular interest are the advances in wheat and barley genomics which now provide a basis for a whole-of-proteome approach to the analysis of the wheat grain. Studies reviewing food proteomics related to aspects of wheat and covering plant proteomics for cereal research, as well as the potential in new methodologies are provided. Although rice proteomics is the most advanced database available for analysis it is now clear that when this is taken together with the rapidly developing dataset of information on wheat and barley grain protein new insights are generated to the many different attributes of these cereal proteins. It is now feasible to identify many wheat and barley proteins in 2DE and mass spectroscopy analysis based on homology between wheat/barley expressed genes and rice/Arabidopsis genes. Case studies are described where this fundamental information has provided new insights and faster diagnostics for attributes associated with wheat protein

Differential recognition by tick-resistant cattle of the recombinantly expressed Rhipicephalus microplus serine protease inhibitor-3 (RMS-3)

Rhipicephalus microplus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host-parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZaA vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2mgl-1. qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host-parasite interaction to overcome the immune responses in resistant cattle

Rhipicephalus microplus lipocalins (LRMs): Genomic identification and analysis of the bovine immune response using in silico predicted B and T cell epitopes

The attachment to host skin by Rhipicephalus microplus larvae induces a series of physiological events at the attachment site. The host-parasite interaction might induce a rejection of the larvae, as is frequently observed in Bos taurus indicus cattle, and under certain conditions in Bos taurus taurus cattle. Ticks deactivate the host rejection response by secreting specific proteins and lipids that play an essential role in manipulation of the host immune response. The available genomic information on the R. microplus tick was mined using bioinformatics approaches to identify R. microplus lipocalins (LRMs). This in silico examination revealed a total of 12 different putative R. microplus LRMs (LRM1-LRM12). The identity of the LRM family showed high sequence variability: from 6% between LRM7 and LRM8 to 55.9% between LRM2 and LRM6. However, the three-dimensional structure of the lipocalin family was conserved in the LRMs. The B and T cell epitopes in these lipocalins were then predicted, and six of the LRMs (5, 6, 9, 10, 11 and 12) were used to examine the host immune interactions with sera and peripheral blood mononuclear cells (PBMCs) collected from tick-susceptible and tick-resistant cattle challenged with R. microplus. On days 28-60 after tick infestation, the anti-LRM titres were higher in the resistant group compared with the susceptible cattle. After 60. day, the anti-LRM titres (except LRM9 and LRM11) decreased to zero in the sera of both the tick-resistant and tick-susceptible cattle. Using cell proliferation assays, the PBMCs challenged with some of the predicted T cell epitopes (LRM1_T1, T2; LRM_T1, T2 and LRM12_T) exhibited a significantly higher number of IFN-?-secreting cells (Th1) in tick-susceptible Holstein-Friesians compared with tick-resistant Brahman cattle. In contrast, expression of the Th2 cytokine (IL-4) was lower in Holstein-Friesians cattle compared with Brahman cattle. Moreover, this study found that LRM6, LRM9 and LRM11 play important roles in the mechanism by which R. microplus interferes with the host's haemostasis mechanisms. © 2013 Australian Society for Parasitology Inc