Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells*
2015; Elsevier BV; Volume: 14; Issue: 12 Linguagem: Inglês
10.1074/mcp.m115.051938
ISSN1535-9484
AutoresMargarita Villar, Nieves Ayllón, Pilar Alberdi, Andrés Moreno, María Moreno, Raquel Tobes, Lourdes Mateos‐Hernández, Sabine Weisheit, Lesley Bell‐Sakyi, José de la Fuente,
Tópico(s)Viral Infections and Vectors
ResumoAnaplasma phagocytophilum is an emerging zoonotic pathogen that causes human granulocytic anaplasmosis. These intracellular bacteria establish infection by affecting cell function in both the vertebrate host and the tick vector, Ixodes scapularis. Previous studies have characterized the tick transcriptome and proteome in response to A. phagocytophilum infection. However, in the postgenomic era, the integration of omics datasets through a systems biology approach allows network-based analyses to describe the complexity and functionality of biological systems such as host–pathogen interactions and the discovery of new targets for prevention and control of infectious diseases. This study reports the first systems biology integration of metabolomics, transcriptomics, and proteomics data to characterize essential metabolic pathways involved in the tick response to A. phagocytophilum infection. The ISE6 tick cells used in this study constitute a model for hemocytes involved in pathogen infection and immune response. The results showed that infection affected protein processing in endoplasmic reticulum and glucose metabolic pathways in tick cells. These results supported tick–Anaplasma co-evolution by providing new evidence of how tick cells limit pathogen infection, while the pathogen benefits from the tick cell response to establish infection. Additionally, ticks benefit from A. phagocytophilum infection by increasing survival while pathogens guarantee transmission. The results suggested that A. phagocytophilum induces protein misfolding to limit the tick cell response and facilitate infection but requires protein degradation to prevent ER stress and cell apoptosis to survive in infected cells. Additionally, A. phagocytophilum may benefit from the tick cell's ability to limit bacterial infection through PEPCK inhibition leading to decreased glucose metabolism, which also results in the inhibition of cell apoptosis that increases infection of tick cells. These results support the use of this experimental approach to systematically identify cell pathways and molecular mechanisms involved in tick–pathogen interactions. Data are available via ProteomeXchange with identifier PXD002181. Anaplasma phagocytophilum is an emerging zoonotic pathogen that causes human granulocytic anaplasmosis. These intracellular bacteria establish infection by affecting cell function in both the vertebrate host and the tick vector, Ixodes scapularis. Previous studies have characterized the tick transcriptome and proteome in response to A. phagocytophilum infection. However, in the postgenomic era, the integration of omics datasets through a systems biology approach allows network-based analyses to describe the complexity and functionality of biological systems such as host–pathogen interactions and the discovery of new targets for prevention and control of infectious diseases. This study reports the first systems biology integration of metabolomics, transcriptomics, and proteomics data to characterize essential metabolic pathways involved in the tick response to A. phagocytophilum infection. The ISE6 tick cells used in this study constitute a model for hemocytes involved in pathogen infection and immune response. The results showed that infection affected protein processing in endoplasmic reticulum and glucose metabolic pathways in tick cells. These results supported tick–Anaplasma co-evolution by providing new evidence of how tick cells limit pathogen infection, while the pathogen benefits from the tick cell response to establish infection. Additionally, ticks benefit from A. phagocytophilum infection by increasing survival while pathogens guarantee transmission. The results suggested that A. phagocytophilum induces protein misfolding to limit the tick cell response and facilitate infection but requires protein degradation to prevent ER stress and cell apoptosis to survive in infected cells. Additionally, A. phagocytophilum may benefit from the tick cell's ability to limit bacterial infection through PEPCK inhibition leading to decreased glucose metabolism, which also results in the inhibition of cell apoptosis that increases infection of tick cells. These results support the use of this experimental approach to systematically identify cell pathways and molecular mechanisms involved in tick–pathogen interactions. Data are available via ProteomeXchange with identifier PXD002181. A. phagocytophilum (Rickettsiales: Anaplasmataceae) is the causative agent of human granulocytic anaplasmosis, equine and canine granulocytic anaplasmosis and tick-borne fever of ruminants (1.Severo M.S. Pedra J.H. F. Ayllón N. Kocan K.M. de la Fuente J. Molecular Medical Microbiology.Academic Press. 2015; : 2033-2042Google Scholar). A. phagocytophilum has been reported to be one the most common tick-borne pathogens in Europe and the United States where it is vectored by I. ricinus, I. scapularis, and I. pacificus (2.Goodman J.L. Tick-Borne Diseases of Humans.ASM Press. 2005; : 218-238Google Scholar, 3.Stuen S. Anaplasma phagocytophilum–The most widespread tick-borne infection in animals in Europe.Vet. Res. Commun. 2007; 31: 79-84Crossref PubMed Scopus (196) Google Scholar). The wide host range of A. phagocytophilum and the extensive distribution of tick vector populations will likely result in establishment of reservoir hosts, followed by the continued emergence of enzootic human granulocytic anaplasmosis in several regions of the world. In addition, tick vector populations are expanding due to changes in climate and human interventions that impact reservoir host movement and human contact with infected ticks (4.Estrada-Peña A. Ostfeld R.S. Peterson A.T. Poulin R. de la Fuente J. Effects of environmental change on zoonotic disease risk: An ecological primer.Trends Parasitol. 2014; 30: 205-214Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 5.Gortazar C. Reperant L.A. Kuiken T. de la Fuente J. Boadella M. Martínez-Lopez B. Ruiz-Fons F. Estrada-Peña A. Drosten C. Medley G. Ostfeld R. Peterson T. VerCauteren K.C. Menge C. Artois M. Schultsz C. Delahay R. Serra-Cobo J. Poulin R. Keck F. Aguirre A.A. Henttonen H. Dobson A.P. Kutz S. Lubroth J. Mysterud A. Crossing the interspecies barrier: Opening the door to zoonotic pathogens.PLoS Pathog. 2014; 10: e1004129Crossref PubMed Scopus (106) Google Scholar). All these factors increase the risk of acquiring A. phagocytophilum infection, and thus this tick-borne pathogen is likely to be a growing concern for human and animal health. The I. scapularis genome is the only tick genome sequenced and assembled (GenBank accession ABJB010000000) and constitutes a valuable resource for the study of tick biology and tick–pathogen interactions (6.Geraci N.S. Johnston J.S. Robinson J.P. Wikel S.K. Hill C.A. Variation in genome size of argasid and ixodid ticks.Insect. Biochem. Mol. Biol. 2007; 37: 399-408Crossref PubMed Scopus (65) Google Scholar, 7.Genomic Resources Development Consortium Contreras M. de la Fuente J. Estrada-Peña A. Grubhoffer L. Tobes R. Transcriptome sequence divergence between Lyme disease tick vectors, Ixodes scapularisIxodes ricinus.Genomic Resources Notes Mol. Ecol. Resour. 2014; 14: 1095PubMed Google Scholar). Postgenomic experimental approaches such as transcriptomics and proteomics have increased our understanding of tick–pathogen interactions. Recent research by our group has focused on the characterization of the vector competency of ticks for A. phagocytophilum (8.Ayllón N. Villar M. Galindo R.C. Kocan K.M. Šíma R. López J.A. Vázquez J. Alberdi P. Cabezas-Cruz A. Kopáček P. de la Fuente J. Systems biology of tissue-specific response to Anaplasma phagocytophilum reveals differentiated apoptosis in the tick vector Ixodes scapularis.PLoS Genet. 2015; 11: e1005120Crossref PubMed Scopus (100) Google Scholar). Previous results demonstrated that tick vector competency involves molecular interactions that ensure that A. phagocytophilum bacteria infect, develop, and are transmitted by ticks (1.Severo M.S. Pedra J.H. F. Ayllón N. Kocan K.M. de la Fuente J. Molecular Medical Microbiology.Academic Press. 2015; : 2033-2042Google Scholar, 8.Ayllón N. Villar M. Galindo R.C. Kocan K.M. Šíma R. López J.A. Vázquez J. Alberdi P. Cabezas-Cruz A. Kopáček P. de la Fuente J. Systems biology of tissue-specific response to Anaplasma phagocytophilum reveals differentiated apoptosis in the tick vector Ixodes scapularis.PLoS Genet. 2015; 11: e1005120Crossref PubMed Scopus (100) Google Scholar, 9.Sukumaran B. Narasimhan S. Anderson J.F. DePonte K. Marcantonio N. Krishnan M.N. Fish D. Telford S.R. Cantor F.S. Fikrig E. An Ixodes scapularis protein required for survival of Anaplasma phagocytophilum in tick salivary glands.J. Exp. Med. 2006; 203: 1507-1517Crossref PubMed Scopus (93) Google Scholar, 10.Zivkovic Z. Blouin E.F. Manzano-Roman R. Almazán C. Naranjo V. Massung R.F. Jongejan F Kocan K.M. de la Fuente J. Anaplasma phagocytophilumA. marginale elicit different gene expression responses in cultured tick cells.Comp. Funct. Genomics. 2009; 2009: 705034Crossref Scopus (30) Google Scholar, 11.Sultana H. Neelakanta G. Kantor F.S. Malawista S.E. Fish D. Montgomery R.R. Fikrig E. Anaplasma phagocytophilum induces actin phosphorylation to selectively regulate gene transcription in Ixodes scapularis ticks.J. Exp. Med. 2010; 207: 1727-1743Crossref PubMed Scopus (59) Google Scholar, 12.Ayllón N. Villar M. Busby A.T. Kocan K.M. Blouin E.F. Bonzon-Kulichenko E. Galindo R.C. Mangold A.J. Alberdi P. Pérez de la Lastra J.M. Vazquez J. de la Fuente J. Anaplasma phagocytophilum inhibits apoptosis and promotes cytoskeleton rearrangement for infection of tick cells.Infect. Immun. 2013; 81: 2415-2425Crossref PubMed Scopus (56) Google Scholar, 13.Naranjo V. Ayllón N. Pérez de la Lastra J.M. Galindo R.C. Kocan K.M. Blouin E.F. Mitra R. Alberdi P. Villar M. de la Fuente J. Reciprocal regulation of NF-kB (Relish) and Subolesin in the tick vector, Ixodes scapularis.PLoS ONE. 2013; 8: e65915Crossref PubMed Scopus (36) Google Scholar, 14.Hajdušek O. Šíma R. Ayllón N. Jalovecká M. Perner J. de la Fuente J. Kopacek P. Interaction of the tick immune system with transmitted pathogens.Front. Cell. Infect. Microbiol. 2013; 3: 26Crossref PubMed Scopus (150) Google Scholar). Two studies have characterized the tick transcriptome and proteome in response to A. phagocytophilum infection (8.Ayllón N. Villar M. Galindo R.C. Kocan K.M. Šíma R. López J.A. Vázquez J. Alberdi P. Cabezas-Cruz A. Kopáček P. de la Fuente J. Systems biology of tissue-specific response to Anaplasma phagocytophilum reveals differentiated apoptosis in the tick vector Ixodes scapularis.PLoS Genet. 2015; 11: e1005120Crossref PubMed Scopus (100) Google Scholar, 10.Zivkovic Z. Blouin E.F. Manzano-Roman R. Almazán C. Naranjo V. Massung R.F. Jongejan F Kocan K.M. de la Fuente J. Anaplasma phagocytophilumA. marginale elicit different gene expression responses in cultured tick cells.Comp. Funct. Genomics. 2009; 2009: 705034Crossref Scopus (30) Google Scholar) and tick proteins have been identified that mediate A. phagocytophilum infection, multiplication, and transmission (1.Severo M.S. Pedra J.H. F. Ayllón N. Kocan K.M. de la Fuente J. Molecular Medical Microbiology.Academic Press. 2015; : 2033-2042Google Scholar, 8.Ayllón N. Villar M. Galindo R.C. Kocan K.M. Šíma R. López J.A. Vázquez J. Alberdi P. Cabezas-Cruz A. Kopáček P. de la Fuente J. Systems biology of tissue-specific response to Anaplasma phagocytophilum reveals differentiated apoptosis in the tick vector Ixodes scapularis.PLoS Genet. 2015; 11: e1005120Crossref PubMed Scopus (100) Google Scholar, 9.Sukumaran B. Narasimhan S. Anderson J.F. DePonte K. Marcantonio N. Krishnan M.N. Fish D. Telford S.R. Cantor F.S. Fikrig E. An Ixodes scapularis protein required for survival of Anaplasma phagocytophilum in tick salivary glands.J. Exp. Med. 2006; 203: 1507-1517Crossref PubMed Scopus (93) Google Scholar, 11.Sultana H. Neelakanta G. Kantor F.S. Malawista S.E. Fish D. Montgomery R.R. Fikrig E. Anaplasma phagocytophilum induces actin phosphorylation to selectively regulate gene transcription in Ixodes scapularis ticks.J. Exp. Med. 2010; 207: 1727-1743Crossref PubMed Scopus (59) Google Scholar, 12.Ayllón N. Villar M. Busby A.T. Kocan K.M. Blouin E.F. Bonzon-Kulichenko E. Galindo R.C. Mangold A.J. Alberdi P. Pérez de la Lastra J.M. Vazquez J. de la Fuente J. Anaplasma phagocytophilum inhibits apoptosis and promotes cytoskeleton rearrangement for infection of tick cells.Infect. Immun. 2013; 81: 2415-2425Crossref PubMed Scopus (56) Google Scholar, 13.Naranjo V. Ayllón N. Pérez de la Lastra J.M. Galindo R.C. Kocan K.M. Blouin E.F. Mitra R. Alberdi P. Villar M. de la Fuente J. Reciprocal regulation of NF-kB (Relish) and Subolesin in the tick vector, Ixodes scapularis.PLoS ONE. 2013; 8: e65915Crossref PubMed Scopus (36) Google Scholar, 14.Hajdušek O. Šíma R. Ayllón N. Jalovecká M. Perner J. de la Fuente J. Kopacek P. Interaction of the tick immune system with transmitted pathogens.Front. Cell. Infect. Microbiol. 2013; 3: 26Crossref PubMed Scopus (150) Google Scholar). Metabolomics is a postgenomic research field concerned with developing methods for analysis of low molecular weight compounds in biological systems such as cells, organs, and organisms. Metabolomics has been used for the study of infectious diseases (15.Fuchs T.M. Eisenreich W. Heesemann J. Goebel W. Metabolic adaptation of human pathogenic and related nonpathogenic bacteria to extra- and intracellular habitats.FEMS Microbiol. Rev. 2012; 36: 435-462Crossref PubMed Scopus (82) Google Scholar, 16.Eoh H. Metabolomics: a window into the adaptive physiology of Mycobacterium tuberculosis.Tuberculosis. 2014; 94: 538-543Crossref PubMed Scopus (16) Google Scholar), but data are not available for ticks. Only a few studies have been published on selected metabolic pathways in tick-borne pathogens grown in culture (17.Marcelino I. de Almeida A.M. Ventosa M. Pruneau L. Meyer D.F. Martinez D. Lefrançois T. Vachiéry N. Coelho A.V. Tick-borne diseases in cattle: Applications of proteomics to develop new generation vaccines.J. Proteomics. 2012; 75: 4232-4350Crossref PubMed Scopus (62) Google Scholar). Rather than focusing on single omics studies, the integration of omics datasets through a systems biology approach allows network-based analyses to describe the complexity and functionality of biological systems such as host–pathogen interactions (17.Marcelino I. de Almeida A.M. Ventosa M. Pruneau L. Meyer D.F. Martinez D. Lefrançois T. Vachiéry N. Coelho A.V. Tick-borne diseases in cattle: Applications of proteomics to develop new generation vaccines.J. Proteomics. 2012; 75: 4232-4350Crossref PubMed Scopus (62) Google Scholar, 18.Van Assche R. Broeckx V. Boonen K. Maes E. De Haes W. Schoofs L. Temmerman L. Integrating -omics: systems biology as explored through C. elegans research.J. Mol. Biol. 2015; Crossref PubMed Scopus (26) Google Scholar) and the discovery of new targets for prevention and control of infectious diseases (19.de la Fuente J. Merino O. Vaccinomics, the new roads to tick vaccines.Vaccine. 2013; 31: 5923-5929Crossref PubMed Scopus (62) Google Scholar). Our objective was that the integration of metabolomics, transcriptomics, and proteomics data to expand the understanding of tick–Anaplasma interactions with the discovery of tick metabolic pathways playing a critical role at the tick–pathogen interface. To address this objective, a systems biology approach was developed to integrate metabolomics, transcriptomics, and proteomics data collected from uninfected and A. phagocytophilum-infected I. scapularis ISE6 cells, which constitute a model for hemocytes involved in pathogen infection and immune response (20.Munderloh U.G. Liu Y. Wang M. Chen C. Kurtti T.J. Establishment, maintenance and description of cell lines from the tick Ixodes scapularis.J. Parasitol. 1994; 80: 533-543Crossref PubMed Scopus (201) Google Scholar). The results showed that infection with A. phagocytophilum affected protein processing in endoplasmic reticulum (ER) and glucose metabolic pathways in tick cells and suggested new coevolved mechanisms involved in pathogen infection and the tick cell response to infection. The I. scapularis embryo-derived tick cell line ISE6, provided by Ulrike Munderloh, University of Minnesota, was cultured in l-15B300 medium as described previously (20.Munderloh U.G. Liu Y. Wang M. Chen C. Kurtti T.J. Establishment, maintenance and description of cell lines from the tick Ixodes scapularis.J. Parasitol. 1994; 80: 533-543Crossref PubMed Scopus (201) Google Scholar), except that the osmotic pressure was lowered by the addition of one-fourth sterile water by volume. The ISE6 cells were first inoculated with A. phagocytophilum (human NY18 isolate)-infected HL-60 cells (21.de la Fuente J. Ayoubi P. Blouin E.F. Almazán C. Naranjo V. Kocan K.M. Gene expression profiling of human promyelocytic cells in response to infection with Anaplasma phagocytophilum.Cell. Microbiol. 2005; 7: 549-559Crossref PubMed Scopus (62) Google Scholar) and maintained according to Munderloh et al. (22.Munderloh U.G. Jauron S.D. Fingerle V. Leitritz L. Hayes S.F. Hautman J.M. Nelson C.M. Huberty B.W. Kurtti T.J. Ahlstrand G.G. Greig B. Mellencamp M.A. Goodman J.L. Invasion and intracellular development of the human granulocytic ehrlichiosis agent in tick cell culture.J. Clin. Microbiol. 1999; 37: 2518-2524Crossref PubMed Google Scholar) until infection was established and routinely passaged. Infected ISE6 cells were frozen in liquid nitrogen and served as inoculum for uninfected cells. Uninfected and infected cultures (n = 3 independent cultures with ∼107 cells each) were sampled at 7 days postinfection (percentage infected cells 71–77% (Ave 1The abbreviations used are:AveaverageBPbiological processCtPCR cycle thresholdDNAdeoxyribonucleic acidERendoplasmic reticulumFDRfalse discovery rateFITCfluorescein isothiocyanateGOgene ontologyHRPhorseradish peroxidaseHspheat shock proteinIgGimmunoglobulin GMKKmitogen-activated protein kinaseMMmethyl methanesulfonatemRNAmessenger RNANMRnuclear magnetic resonancePEPCKphosphoenolpyruvate carboxykinaseppmparts per millionPSMpeptide-spectrum matchesrDNAribosomal DNARNAribonucleic acidRNAiRNA interferencerRNAribosomal RNASDstandard deviation. ± SD, 74 ± 3). The percentage of cells infected with A. phagocytophilum was calculated by examining at least 200 cells using a 100x oil immersion objective. The cells were centrifuged at 10,000 g for 3 min, and cell pellets were lyophilized for 1H nuclear magnetic resonance (NMR) or frozen in liquid nitrogen until used for protein and RNA extraction. Cells from the three replicates of each condition were pooled in 500 μl lysis buffer (phosphate buffered saline (PBS), 1% Triton X-100, supplemented with Complete protease inhibitor mixture (Roche, Basel, Switzerland), and homogenized by passing through a needle (27G). Samples were sonicated for 1 min in an ultrasonic cooled bath, followed by vortexing for 10 s. After three cycles of sonication–vortex, total cell extracts were centrifuged at 200 × g for 5 min to remove cell debris. The supernatants were collected and protein concentration was determined using the BCA Protein Assay (Life Technologies, Carlsbad, CA) with BSA as standard. Total RNA was extracted from aliquots of the same cell cultures using TriReagent (Sigma, St. Louis, MO) following the manufacturer's recommendations. Proteins were used for proteomics and Western blot analyses. Total RNA was used for transcriptomics and real-time RT-PCR. Supplemental Table I contains all genes characterized by real-time RT-PCR. average biological process PCR cycle threshold deoxyribonucleic acid endoplasmic reticulum false discovery rate fluorescein isothiocyanate gene ontology horseradish peroxidase heat shock protein immunoglobulin G mitogen-activated protein kinase methyl methanesulfonate messenger RNA nuclear magnetic resonance phosphoenolpyruvate carboxykinase parts per million peptide-spectrum matches ribosomal DNA ribonucleic acid RNA interference ribosomal RNA standard deviation. NMR is recognized as one of the main analytical methodologies to obtain both qualitative and quantitative information for low molecular weight metabolites (23.Spraul M. Freund A.S. Nast R.E. Withers R.S. Maas W.E. Corcoran O. Advancing NMR sensitivity for LC-NMR-MS using a cryoflow probe: Application to the analysis of acetaminophen metabolites in urine.Anal. Chem. 2003; 75: 1536-1541Crossref PubMed Scopus (132) Google Scholar, 24.Khoo S.H. Al-Rubeai M. Metabolomics as a complementary tool in cell culture.Biotechnol. Appl. Biochem. 2007; 47: 71-84Crossref PubMed Scopus (68) Google Scholar, 25.Cuperlović-Culf M. Barnett D.A. Culf A.S. Chute I. Cell culture metabolomics: applications and future directions.Drug. Discov. Today. 2010; 15: 610-621Crossref PubMed Scopus (166) Google Scholar, 26.Holzgrabe U. Quantitative NMR spectroscopy in pharmaceutical applications.Prog. Nucl. Magn. Reson. Spectrosc. 2010; 57: 229-240Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). The 1H NMR spectroscopy used in the present study allows a rapid quantitative analysis of complex matrices (27.Bala L. Sharma A. Yellapa R.K. Roy R. Choudhuri G. Khetrapal C.L. (1) H NMR spectroscopy of ascitic fluid: Discrimination between malignant and benign ascites and comparison of the results with conventional methods.NMR Biomed. 2008; 21: 606-614Crossref PubMed Scopus (22) Google Scholar). Furthermore, the 13C nucleus sweeps a wider range of chemical shifts, therefore improving resolution and facilitating assignments and quantitative analysis. For all NMR spectra, the lyophilized tick cell samples were dissolved in 500 μl deuterium oxide and transferred to 5 mm NMR tubes. All proton and carbon NMR spectra were recorded on a Varian Inova 500 MHz spectrometer in deuterium oxide at 278 °K and at 499.769 MHz and 125.678 MHz for 1H and 13C NMR, respectively. Chemical shifts were referenced to trimethylsilyl propionate. 1H NMR experiments were recorded with the spectral width of 7,000 Hz, 90° pulse and 256 scans, the relaxation delay was 25 s, and the number of data points was 30,256. The 13C NMR data were acquired in order to identify the main compounds using the following conditions: spectral width 20,000 Hz, 90° pulse, 256 scans, the relaxation delay was 25 s, and the number of data points were 81,726; inverse gated decoupling sequence was applied for proton decoupling during acquisition. Assignment of spectra was carried out using NOESY-1D, g-COSY and Total Correlation Spectroscopy. The spectra were acquired employing the inverse gated decoupling pulse sequence in order to suppress nuclear overhauser effects. The following parameters were used: spectral width, acquisition time 0.64 s, and 160 scans. In all cases, probe temperatures were adjusted at 25 °C. The pulse programs were taken from the standard Varian pulse sequence library. All spectra were Fourier-transformed with MestReNova 8.1 software (Mestrelab Research S.L., Santiago de Compostela, Spain) and ACD/Specmanager 7.00 software (Advanced Chemistry, Inc., Toronto, ON, Canada). Apodization of 0.2 Hz and zero filling (32,000) were used prior to Fourier transformation. The spectra were phased and baseline corrected by the Whittaker smoother. 1H NMR spectra were integrated manually, choosing the area of each peak of interest. The integrals were normalized in order to carry out relative quantification of the interesting compounds. 1H spectral resonances peak assignment was based on published data (28.Nicholson J.K. Foxall P.J. Spraul M. Farrant R.D. Lindon J.C. 750 MHz 1H and 1H-13C NMR spectroscopy of human blood plasma.Anal. Chem. 1995; 67: 793-811Crossref PubMed Scopus (943) Google Scholar, 29.Wishart D.S. Tzur D. Knox C. Eisner R. Guo A.C. Young N. Cheng D. Jewell K. Arndt D. Sawhney S. Fung C. Nikolai L. Lewis M. Coutouly M.A. Forsythe I. Tang P. Shrivastava S. Jeroncic K. Stothard P. Amegbey G. Block D. Hau D.D. Wagner J. Miniaci J. Clements M. Gebremedhin M. Guo N. Zhang Y. Duggan G.E. Macinnis G.D. Weljie A.M. Dowlatabadi R. Bamforth F. Clive D. Greiner R. Li L. Marrie T. Sykes B.D. Vogel H.J. Querengesser L. HMDB: The Human Metabolome Database.Nucleic Acids Res. 2007; 35: D521-D526Crossref PubMed Scopus (2176) Google Scholar, 30.Wishart D.S. Knox C. Guo A.C. Eisner R. Young N. Gautam B. Hau D.D. Psychogios N. Dong E. Bouatra S. Mandal R. Sinelnikov I. Xia J. Jia L. Cruz J.A. Lim E. Sobsey C.A. Shrivastava S. Huang P. Liu P. Fang L. Peng J. Fradette R. Cheng D. Tzur D. Clements M. Lewis A. De Souza A. Zuniga A. Dawe M. Xiong Y. Clive D. Greiner R. Nazyrova A. Shaykhutdinov R. Li L. Vogel H.J. Forsythe I. HMDB: A knowledgebase for the human metabolome.Nucleic Acids Res. 2009; 37: D603-D610Crossref PubMed Scopus (1497) Google Scholar, 31.Wishart D.S. Jewison T. Guo A.C. Wilson M. Knox C. Liu Y. Djoumbou Y. Mandal R. Aziat F. Dong E. Bouatra S. Sinelnikov I. Arndt D. Xia J. Liu P. Yallou F. Bjorndahl T. Perez-Pineiro R. Eisner R. Allen F. Neveu V. Greiner R. Scalbert A. HMDB 3.0. The Human Metabolome Database in 2013.Nucleic Acids Res. 2013; 41: D801-D807Crossref PubMed Scopus (2280) Google Scholar; Human Metabolome Database version 3.0, http://www.hmdb.ca) and Chenomx Profiler software (Chenomx NMR Suite, Edmonton, Canada). Assignments were confirmed through the addition of pure standard compounds (Sigma). Three biological replicates were used for each of uninfected and infected tick cells, and results were compared between uninfected and infected tick cells by Student's t test for unequal variance (p = .05). Supplemental Table II contains all 1H NMR spectra (parts per million; ppm) for all identified metabolites in analyzed samples and data quantitation. Purified RNAs were used for library preparation using the TruSeq-based NEB-Next Ultra Directional RNA Library Prep kit (New England Biolabs, Ipswick, MA). Briefly, 1 μg total RNA was used as starting material for library preparation. Messenger RNA was captured using oligo-dT magnetic beads (Poly(A) RNA Magnetic Isolation Module; New England Biolabs) and purified poly(A)+ RNA was chemically fragmented and reverse-transcribed. A controlled fragmentation period was applied to obtain an insert size of ∼320 nucleotides, best suited to generate nonoverlapping reads after pair-end sequencing. The second strand was generated and double-stranded DNA was purified using AMPure SPRI-based magnetic beads (Beckman Coulter, IZASA, Spain). Next, samples were end-repaired followed by adaptor ligation and removal of excess oligonucleotides by double AMPure selection, made according to NEB-Next recommendations as a function of library size. Adaptor oligonucleotides contained the signals for subsequent amplification and sequencing as well as sample-specific identifiers, which allowed multiplexing in the sequencing run. An enrichment procedure based on PCR was then performed to ensure that all molecules in the library conserved the adapters at both ends. The number of PCR cycles was adjusted to 13 for all four samples. The final amplified library was checked again on a BioAnalyzer 2100 (Agilent, Santa Clara, CA), pooled, quantified by fluorimetric methods (PicoGreen®), and titrated by real-time PCR using a well-controlled library as standard. For RNAseq, libraries were denatured and seeded on the surface of a Pair-End Flowcell, where clusters were generated and sequenced using a HiSeq2000 equipment (Illumina, San Diego, CA), under a 2 × 100 recipe. Libraries were diluted to obtain 50.7 (Uninfected 1) 52.8 (Uninfected 2) 57.2 (Infected 1) and 57.6 (Infected 2) million pair-end reads per sample. The 1 × 100 bp pair-end reads were mapped to the I. scapularis reference genome sequence (assembly JCVI_ISG_i3_1.0; http://www.ncbi.nlm.nih.gov/nuccore/NZ_ABJB000000000). Reads were mapped to the reference genome using the scaffolds and GFF (General Feature Format) files (https://www.vectorbase.org/) with the tool Bowtie (32.Langmead B. Trapnell C. Pop M. Salzberg S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.Genome Biol. 2009; 10: R25Crossref PubMed Scopus (14996) Google Scholar) integrated in the TopHat suite (33.Trapnell C. Pachter L. Salzberg S.L. TopHat: Discovering splice junctions with RNA-Seq.Bioinformatics. 2009; 25: 1105-1111Crossref PubMed Scopus (8995) Google Scholar). The pipeline used for bioinformatics analysis of RNAseq data was similar to that described by Trapnell et al. (34.Trapnell C. Hendrickson D.G. Sauvageau M. Goff L. Rinn J.L. Pachter L. Differential analysis of gene regulation at transcript resolution with RNA-seq.Nat. Biotechnol. 2013; 31: 46-53Crossref PubMed Scopus (2419) Google Scholar) using a TopHat-Cufflinks-Cuffmerge-Cuffquant-Cuffdiff pipeline (35.Twine N.A. Janitz K. Wilkins M.R. Janitz M. Whole transcriptome sequencing reveals gene expression and splicing differences in brain regions affected by Alzheimer's disease.PLoS ONE. 2011; 6: e16266Crossref PubMed Scopus (227) Google Scholar, 36.Li B. Bi C.L. Lang N. Li Y.Z. Xu C. Zhang Y.Q. Zhai A.X. Cheng Z.F. RNA-seq methods for identifying differentially expressed gene in human pancreatic islet cells treated with pro-inflammatory cytokines.Mol. Biol. Rep. 2014; 4: 1917-1925Crossref Scopus (13) Google Scholar) (Supplemental Fig. 1). Two biological replicates were used for each of uninfected and infected tick cells, and genes differentially expressed in response to A. phagocytophilum infection were selected with p ≤ .05. Gene ontology (GO) analysis for biological process (BP) was done with Blast2GO software (version 3.0; www.blast2go.com) (37.Villar M. Popara M. Ayllón N. Fernández de Mera I.G. Mateos-Hernández L. Galindo R.C. Manrique M. Tobes R. de la Fuente J. A systems biology approach to
Referência(s)