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Lowell S Kappmeyer,
Mathangi Thiagarajan,
David R Herndon,
Joshua D Ramsay,
Elisabet Caler,
Appolinaire Djikeng,
Joseph J Gillespie,
Audrey Ot Lau,
Eric H Roalson,
Joana C Silva,
Marta G Silva,
Carlos E Suarez,
Massaro W Ueti, Vishvanath M Nene,
Robert H Mealey,
Donald P Knowles,
Kelly A Brayton
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ABSTRACT: BACKGROUND: Transmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an uncertain taxonomy. Understanding mechanisms by which apicomplexan parasites evade immune or chemotherapeutic elimination is required for development of effective vaccines or chemotherapeutics. The continued risk of transmission of T. equi from clinically silent, persistently infected equids impedes the goal of returning the U. S. to non-endemic status. Therefore comparative genomic analysis of T. equi was undertaken to: 1) identify genes contributing to immune evasion and persistence in equid hosts, 2) identify genes involved in PBMC infection biology and 3) define the phylogenetic position of T. equi relative to sequenced apicomplexan parasites. RESULTS: The known immunodominant proteins, EMA1, 2 and 3 were discovered to belong to a ten member gene family with a mean amino acid identity, in pairwise comparisons, of 39%. Importantly, the amino acid diversity of EMAs is distributed throughout the length of the proteins. Eight of the EMA genes were simultaneously transcribed. As the agents that cause bovine theileriosis infect and transform host cell PBMCs, we confirmed that T. equi infects equine PBMCs, however, there is no evidence of host cell transformation. Indeed, a number of genes identified as potential manipulators of the host cell phenotype are absent from the T. equi genome. Comparative genomic analysis of T. equi revealed the phylogenetic positioning relative to seven apicomplexan parasites using deduced amino acid sequences from 150 genes placed it as a sister taxon to Theileria spp. CONCLUSIONS: The EMA family does not fit the paradigm for classical antigenic variation, and we propose a novel model describing the role of the EMA family in persistence. T. equi has lost the putative genes for host cell transformation, or the genes were acquired by T. parva and T. annulata after divergence from T. equi. Our analysis identified 50 genes that will be useful for definitive phylogenetic classification of T. equi and closely related organisms.
BMC Genomics 11/2012; 13(1):603. · 4.07 Impact Factor
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Joseph J Gillespie,
Vinita Joardar,
Kelly P Williams,
Timothy Driscoll,
Jessica B Hostetler,
Eric Nordberg,
Maulik Shukla,
Brian Walenz,
Catherine A Hill, Vishvanath M Nene,
Abdu F Azad,
Bruno W Sobral,
Elisabet Caler
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ABSTRACT: We present the draft genome for the Rickettsia endosymbiont of Ixodes scapularis (REIS), a symbiont of the deer tick vector of Lyme disease in North America. Among Rickettsia species (Alphaproteobacteria: Rickettsiales), REIS has the largest genome sequenced to date (>2 Mb) and contains 2,309 genes across the chromosome and four plasmids (pREIS1 to pREIS4). The most remarkable finding within the REIS genome is the extraordinary proliferation of mobile genetic elements (MGEs), which contributes to a limited synteny with other Rickettsia genomes. In particular, an integrative conjugative element named RAGE (for Rickettsiales amplified genetic element), previously identified in scrub typhus rickettsiae (Orientia tsutsugamushi) genomes, is present on both the REIS chromosome and plasmids. Unlike the pseudogene-laden RAGEs of O. tsutsugamushi, REIS encodes nine conserved RAGEs that include F-like type IV secretion systems similar to that of the tra genes encoded in the Rickettsia bellii and R. massiliae genomes. An unparalleled abundance of encoded transposases (>650) relative to genome size, together with the RAGEs and other MGEs, comprise ~35% of the total genome, making REIS one of the most plastic and repetitive bacterial genomes sequenced to date. We present evidence that conserved rickettsial genes associated with an intracellular lifestyle were acquired via MGEs, especially the RAGE, through a continuum of genomic invasions. Robust phylogeny estimation suggests REIS is ancestral to the virulent spotted fever group of rickettsiae. As REIS is not known to invade vertebrate cells and has no known pathogenic effects on I. scapularis, its genome sequence provides insight on the origin of mechanisms of rickettsial pathogenicity.
Journal of bacteriology 11/2011; 194(2):376-94. · 3.94 Impact Factor
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ABSTRACT: Completion of the Babesia bovis (T2Bo strain) genome provides detailed data concerning the predicted proteome of this parasite, and allows for a bioinformatics approach to gene discovery. Comparative genomics of the hemoprotozoan parasites B. bovis and Theileria parva revealed a highly conserved syntenic block of genes flanking the p67 gene of T. parva, a sporozoite stage-specific vaccine candidate against East Coast fever (ECF). The syntenic gene in B. bovis, designated bov57, encodes a protein of limited amino acid sequence identity (11.8%) to p67. Monoclonal antibodies were produced against recombinant BOV57 and were used to demonstrate expression of BOV57 in merozoite and kinete stages of the T2Bo strain of B. bovis. Transcript levels of bov57 in kinetes were increased 100-fold in comparison to msa-1, a previously identified gene encoding an erythrocyte stage surface protein. Amino acid sequence comparisons between the T2Bo strain and two attenuated and virulent strains from Argentina and Australia revealed a high degree of sequence conservation in BOV57 among these geographically and pathogenically divergent isolates (97% amino acid sequence identity). Additional genomic comparisons show that the bov57 gene locus is also conserved in Babesia bigemina and Babesia equi. While not identifiable through amino acid or nucleotide sequence similarity, the conserved gene order within this locus in multiple piroplasms may suggest a critical function adapted for each species' unique host and life-cycle.
Veterinary Parasitology 10/2010; 173(3-4):211-8. · 2.58 Impact Factor
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ABSTRACT: 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.
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.
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.
BMC Genomics 01/2010; 11:374. · 4.07 Impact Factor
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ABSTRACT: A population of Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae), designated Coatzacoalcos, sampled from a ranch near Veracruz, Mexico, was found to possess a high level of resistance to pyrethroid-based acaricides. Bioassay and biochemical and molecular analysis had previously shown that resistance in this population could primarily be attributed to expression of a highly active metabolic esterase designated CzEST9. We cloned and sequenced the entire CzEST9 coding region, including introns and > 1.0 kb upstream from the transcription start site, and we compared the upstream region sequence between individual resistant and susceptible ticks from several populations with different pyrethroid resistance characteristics. In the 1.0-kb upstream region sequence, four variant nucleotides were found, and a TGA trinucleotide occurred as either four, five, or nine tandem repeats. However, none of these promoter region sequence differences could be clearly associated with a pyrethroid-resistant phenotype; thus, we concluded that differences in gene promoter sequence were not responsible for the pyrethroid resistance mechanism in the Cz strain. CzEST9 was expressed in recombinant Escherichia coli and Pichia pastoris systems and esterase activity was obtained in recombinant CzEST9 from the P. pastoris system.
Journal of Medical Entomology 08/2008; 45(4):677-85. · 1.76 Impact Factor
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Kelly A Brayton,
Audrey O T Lau,
David R Herndon,
Linda Hannick,
Lowell S Kappmeyer,
Shawn J Berens,
Shelby L Bidwell,
Wendy C Brown,
Jonathan Crabtree,
Doug Fadrosh, [......],
Ian T Paulsen,
Diana Radune,
Qinghu Ren,
Roger K Smith,
Carlos E Suarez,
Owen White,
Jennifer R Wortman,
Donald P Knowles,
Terry F McElwain, Vishvanath M Nene
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ABSTRACT: Babesia bovis is an apicomplexan tick-transmitted pathogen of cattle imposing a global risk and severe constraints to livestock health and economic development. The complete genome sequence was undertaken to facilitate vaccine antigen discovery, and to allow for comparative analysis with the related apicomplexan hemoprotozoa Theileria parva and Plasmodium falciparum. At 8.2 Mbp, the B. bovis genome is similar in size to that of Theileria spp. Structural features of the B. bovis and T. parva genomes are remarkably similar, and extensive synteny is present despite several chromosomal rearrangements. In contrast, B. bovis and P. falciparum, which have similar clinical and pathological features, have major differences in genome size, chromosome number, and gene complement. Chromosomal synteny with P. falciparum is limited to microregions. The B. bovis genome sequence has allowed wide scale analyses of the polymorphic variant erythrocyte surface antigen protein (ves1 gene) family that, similar to the P. falciparum var genes, is postulated to play a role in cytoadhesion, sequestration, and immune evasion. The approximately 150 ves1 genes are found in clusters that are distributed throughout each chromosome, with an increased concentration adjacent to a physical gap on chromosome 1 that contains multiple ves1-like sequences. ves1 clusters are frequently linked to a novel family of variant genes termed smorfs that may themselves contribute to immune evasion, may play a role in variant erythrocyte surface antigen protein biology, or both. Initial expression analysis of ves1 and smorf genes indicates coincident transcription of multiple variants. B. bovis displays a limited metabolic potential, with numerous missing pathways, including two pathways previously described for the P. falciparum apicoplast. This reduced metabolic potential is reflected in the B. bovis apicoplast, which appears to have fewer nuclear genes targeted to it than other apicoplast containing organisms. Finally, comparative analyses have identified several novel vaccine candidates including a positional homolog of p67 and SPAG-1, Theileria sporozoite antigens targeted for vaccine development. The genome sequence provides a greater understanding of B. bovis metabolism and potential avenues for drug therapies and vaccine development.
PLoS Pathogens 11/2007; 3(10):1401-13. · 9.13 Impact Factor
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ABSTRACT: The southern cattle tick, Rhipicephalus (Boophilus) microplus, is an economically important parasite of cattle and can transmit several pathogenic microorganisms to its cattle host during the feeding process. Understanding the biology and genomics of R. microplus is critical to developing novel methods for controlling these ticks.
We present a global comparative genomic analysis of a gene index of R. microplus comprised of 13,643 unique transcripts assembled from 42,512 expressed sequence tags (ESTs), a significant fraction of the complement of R. microplus genes. The source material for these ESTs consisted of polyA RNA from various tissues, lifestages, and strains of R. microplus, including larvae exposed to heat, cold, host odor, and acaricide. Functional annotation using RPS-Blast analysis identified conserved protein domains in the conceptually translated gene index and assigned GO terms to those database transcripts which had informative BlastX hits. Blast Score Ratio and SimiTri analysis compared the conceptual transcriptome of the R. microplus database to other eukaryotic proteomes and EST databases, including those from 3 ticks. The most abundant protein domains in BmiGI were also analyzed by SimiTri methodology.
These results indicate that a large fraction of BmiGI entries have no homologs in other sequenced genomes. Analysis with the PartiGene annotation pipeline showed 64% of the members of BmiGI could not be assigned GO annotation, thus minimal information is available about a significant fraction of the tick genome. This highlights the important insights in tick biology which are likely to result from a tick genome sequencing project. Global comparative analysis identified some tick genes with unexpected phylogenetic relationships which detailed analysis attributed to gene losses in some members of the animal kingdom. Some tick genes were identified which had close orthologues to mammalian genes. Members of this group would likely be poor choices as targets for development of novel tick control technology.
BMC Genomics 02/2007; 8:368. · 4.07 Impact Factor
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ABSTRACT: The southern cattle tick, Boophilus microplus (Canestrini), causes annual economic losses in the hundreds of millions of dollars to cattle producers throughout the world, and ranks as the most economically important tick from a global perspective. Control failures attributable to the development of pesticide resistance have become commonplace, and novel control technologies are needed. The availability of the genome sequence will facilitate the development of these new technologies, and we are proposing sequencing to a 4-6X draft coverage. Many existing biological resources are available to facilitate a genome sequencing project, including several inbred laboratory tick strains, a database of approximately 45,000 expressed sequence tags compiled into a B. microplus Gene Index, a bacterial artificial chromosome (BAC) library, an established B. microplus cell line, and genomic DNA suitable for library synthesis. Collaborative projects are underway to map BACs and cDNAs to specific chromosomes and to sequence selected BAC clones. When completed, the genome sequences from the cow, B. microplus, and the B. microplus-borne pathogens Babesia bovis and Anaplasma marginale will enhance studies of host-vector-pathogen systems. Genes involved in the regeneration of amputated tick limbs and transitions through developmental stages are largely unknown. Studies of these and other interesting biological questions will be advanced by tick genome sequence data. Comparative genomics offers the prospect of new insight into many, perhaps all, aspects of the biology of ticks and the pathogens they transmit to farm animals and people. The B. microplus genome sequence will fill a major gap in comparative genomics: a sequence from the Metastriata lineage of ticks. The purpose of the article is to synergize interest in and provide rationales for sequencing the genome of B. microplus and for publicizing currently available genomic resources for this tick.
Journal of Medical Entomology 02/2006; 43(1):9-16. · 1.76 Impact Factor
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ABSTRACT: I. Justification for an Ixodes scapularis Genome Project This proposal represents the cooperative efforts of the international tick research community to develop the first large scale genomic analysis of a medically significant tick, namely Ixodes scapularis. Ticks transmit the greatest variety of human and animal pathogens of any arthropod vector and are second only to mosquitoes as vectors of human disease (Fivaz et al., 1992; Sonenshine and Mather, 1994). Diseases transmitted by blood feeding ixodid ticks (subphylum Chelicerata; class Arachnida; subclass Acari; family Ixodidae) are global medical and veterinary health problems (Sonenshine, 1993) and include a wide variety of bacterial, rickettsial, viral and protozoan diseases. Other forms of pathogenesis attributed to ticks include anemia, dermatosis, toxemia and paralysis (Gothe, 1999; Roberts and Janovy, 1996; Sonenshine, 1991; Sonenshine, 1993). Important tick-borne diseases include Lyme disease (LD), tick-borne relapsing fever, babesiosis, anaplasmosis, Rocky Mountain spotted fever, Boutonneuse fever, Queensland tick typhus, Q fever, and numerous arboviruses (Sonenshine, 1993). The resurgence of LD and the emergence of other tick-borne diseases such as human granulocytic anaplasmosis (HGA) (Childs and Paddock, 2003; Gratz, 1999; Paddock and Childs, 2003) pose increasing public health concerns. Since the discovery of the causative agent of LD, Borrelia burgdorferi, fifteen previously unrecognized tick-borne bacterial pathogens have been described (Parola and Raoult, 2001). Furthermore, due to their efficiency as vectors of a wide variety of pathogens, broad vertebrate host range and worldwide distribution (Sonenshine, 1991), ixodid ticks are recognized as potential vectors of a number of pathogens considered to be possible bioterrorism agents for use against humans and livestock including Crimean-Congo hemorrhagic fever virus, Rickettsia rickettsii (Rocky Mountain Spotted Fever), the tick-borne encephalitis complex of flaviviruses (Central European tick-borne encephalitis, Far Eastern tick-borne encephalitis, Siberian tick-borne encephalitis, Kyasanur forest disease and Omsk hemorrhagic fever), Coxiella burnetii (Q Fever) and Francisella tularensis (tularemia) (Centers for Disease Control and Prevention Select Biological Agents and Toxins, 2004; http://www.cdc.gov/od/sap/docs/salist.pdf). In the United States, I. scapularis is the most important tick species from a human health perspective. Ixodes scapularis transmits LD in the northeastern and north-central US, HGA and babesiosis and possibly the flaviviral agent of Powassan encephalitis (POW) which is related to West Nile virus. Recent studies by Anderson et al., (2003) also suggest that West Nile virus can be transmitted trans-stadially by I. scapularis although vector competence has yet to be established. LD is arguably one of the most important vector borne diseases in the US, Europe and Asia. Over 17, 000 positive LD cases were reported to the US in 2000 (Centers for Disease Control and Prevention, 2002). LD and other tick-borne diseases have important long term health consequences. Of further concern is the fact that the incidence and geographic spread of LD and other tick-borne disease are increasing and many cases are suspected to be vastly under-reported or misdiagnosed (Walker, 1988). Ixodes scapularis is a member of the Prostriata, an evolutionarily primitive phyletic line of the Acari that includes a number of medically significant tick species. Ixodes scapularis genome data will be widely applicable to studies of other prostriates including I. pacificus, the vector of LD on the US Pacific Coast, I. ricinus and I. persulcatus, the Eurasian Ixodes spp. vectors of LD and tick borne encephalitis and I. holocyclus, an Australian ixodid responsible for transmission of Rickettsia and Borrelia and human cases of tick paralysis. These factors, particularly the wide range of human diseases that it transmits, make I. scapularis the best overall candidate for a genome project that seeks to have an ultimate impact on human welfare through development of novel vector suppression measures, therapeutics and vaccines. Control of arthropod-borne pathogens is complicated by lack of vaccines (Walker, 1998) and the development of drug resistant pathogens (Molyneux, 1998) and acaricide resistant ticks (Mitchell, 1996). Current methods for tick control rely primarily on avoidance of tick bites and the use of approved repellants. Despite the medical significance of I. scapularis and other ixodid ticks, there are currently no 2 large-scale genome efforts dedicated to a tick species of public health importance. This is a major impediment to vector biology and vector-borne disease research and ultimately to the development of new tick and tick-borne disease control strategies. Among the most important outcomes of this genome project will be opportunities to identify new acaricide, drug and anti-tick vaccine targets. Ixodes genome sequence offers an opportunity to investigate aspects of tick biology that can be exploited for tick control. Ticks are thought to have developed unique strategies for obligate hematophagy and parasitism. Tick saliva contains a potent cocktail of pharmacologically active peptides and other molecules that modulate or suppress the haemostatic, inflammatory and immune responses of the host (Valenzuela et al., 2002). These and other molecules "mined" from genome sequence offer an exciting possibility to identify new vaccine targets, potential bio-pharmaceuticals, anti-microbial peptides and other novel human therapeutics. Furthermore, tick saliva and modulation of host defenses is increasingly being linked to pathogen transmission (Nuttall et al., 2000; Schoeler and Wikel, 2001; Wikel et al., 1994; Wikel 1996). Sequence data may lead to the identification of molecules associated with the acquisition, development and transmission of infectious agents in the tick. In combination with the completed Homo sapiens (Lander et al., 2001; Venter et al., 2001) and Borrelia burgdorferi (Fraser et al., 1997) genome projects and the anticipated tick-borne pathogen genomes (Babesia bovis, Anaplasma marginale, Theileria parva and T. annulata), it will be possible to apply the Ixodes genome sequence to unravel the complicated molecular and genetic basis of tick-pathogen-host relationships and tick borne disease transmission. Ticks likely appeared during the Paleozoic or early Mesozoic era, approximately 225 million years ago (Klompen et al., 1996) and are expected to have diverged significantly from the subphylum Mandibulata and the class Insecta. The Ixodes project will provide the first genomic overview of the taxonomically diverse subphylum Chelicerata and will significantly expand the scope of comparative and evolutionary eukaryotic analyses. The I. scapularis genome is predicted to be highly unique in comparison to the other sequenced invertebrate genomes such as Drosophila melanogaster, D. pseudoobscura, the malaria mosquito Anopheles gambiae and the honeybee Apis mellifera, all of which are phylogenetically restricted to the subphylum Mandibulata and the class Insecta. The subdivision of Arthropoda and Mandibulata extends back to approximately 750 million years ago and is therefore one of the most ancient among metazoan animals. This suggests that the predictive power of Insecta sequences for interpreting Chelicerata ESTs may be limited. In fact many ixodid tick ESTs have no matches to the current entries in sequence databases (Hill and Gutierrez, 2000; Nene et al. 2002; Valenzuela et al., 2002). In addition to the Drosophila and A. gambiae genomes, the Ixodes genome project will complement and add value to other vector genome projects including the ongoing Aedes aegypti (yellow fever mosquito) project and the proposed Culex pipiens quinquefasciatus (Southern house mosquito) and Tsetse fly genomes. Genetic information will also facilitate studies of tick phylogenetics, population biology, ecology and behavior; areas of tick research that have been hindered by lack of sequence data. Genome data will improve gene prediction capabilities and enable the identification of conserved arthropod specific genes, divergent orthologs and differentially expanded paralogous gene families and metabolic pathways amongst the arthropoda. As the first large scale genome analysis of an acarine species, it is expected that the Ixodes genome will also act as a powerful catalyst for molecular, genomic and comparative studies between other acarine species and a range of prostriate and metastriate ticks. II. Status of the Ixodes scapularis Genome Effort Efforts to develop the critical preliminary molecular tools to support an I. scapularis genome project are presently funded in a number of labs and are well advanced (refer to sections III, V and VI). There are no efforts underway or currently proposed by other agencies or organizations to determine the genome sequence of I. scapularis.
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