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PubMed | Complutense University of Madrid, Oh no sequences! Research Group, Boston University, Institute Investigacion en Recursos Cinegeticos IREC CSIC UCLM JCCM and 2 more.
Type: Comparative Study | Journal: PLoS neglected tropical diseases | Year: 2015

Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly affect humans and animals worldwide. The life cycle of mycobacteria is complex and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Recently, comparative genomics analyses have provided new insights into the evolution and adaptation of the MTBC to survive inside the host. However, most of this information has been obtained using M. tuberculosis but not other members of the MTBC such as M. bovis and M. caprae. In this study, the genome of three M. bovis (MB1, MB3, MB4) and one M. caprae (MB2) field isolates with different lesion score, prevalence and host distribution phenotypes were sequenced. Genome sequence information was used for whole-genome and protein-targeted comparative genomics analysis with the aim of finding correlates with phenotypic variation with potential implications for tuberculosis (TB) disease risk assessment and control. At the whole-genome level the results of the first comparative genomics study of field isolates of M. bovis including M. caprae showed that as previously reported for M. tuberculosis, sequential chromosomal nucleotide substitutions were the main driver of the M. bovis genome evolution. The phylogenetic analysis provided a strong support for the M. bovis/M. caprae clade, but supported M. caprae as a separate species. The comparison of the MB1 and MB4 isolates revealed differences in genome sequence, including gene families that are important for bacterial infection and transmission, thus highlighting differences with functional implications between isolates otherwise classified with the same spoligotype. Strategic protein-targeted analysis using the ESX or type VII secretion system, proteins linking stress response with lipid metabolism, host T cell epitopes of mycobacteria, antigens and peptidoglycan assembly protein identified new genetic markers and candidate vaccine antigens that warrant further study to develop tools to evaluate risks for TB disease caused by M. bovis/M.caprae and for TB control in humans and animals.

PubMed | Hospital Universitario La Paz, Oh no sequences! Research group, CSIC - National Center for Biotechnology, Charles III University of Madrid and 5 more.
Type: Case Reports | Journal: Mucosal immunology | Year: 2015

Methicillin-resistant Staphylococcus aureus (MRSA) causes chronic pulmonary infections in patients with cystic fibrosis (CF). This study tracks the 13-year evolution (1996-2009) of a single MRSA clone in a male patient with CF, evaluating both the host immunogenic response and the microbial variations. Whole-genome sequencing was performed for the initial (CF-96) and evolved (CF-09) isolates. The immunogenicity of CF-96 and CF-09 was evaluated by incubation with innate immune cells from healthy volunteers. We also studied the patients innate immune response profile, cytokine production, expression of triggering receptor expressed on myeloid cells-1 (TREM-1), and phagocytosis. A total of 30 MRSA ST247-SCCmecI-pvl(-) isolates were collected, which evidenced a genome size reduction from the CF-96 ancestor to the evolved CF-09 strain. Up to six changes in the spa-type were observed over the course of the 13-year evolution. Cytokine production, TREM-1 expression, and phagocytosis were significantly lower for the healthy volunteer monocytes exposed to CF-09, compared with those exposed to CF-96. Patient monocytes exhibited a reduced inflammatory response when challenged with CF-09. Genetic changes in MRSA, leading to reduced immunogenicity and entry into the refractory state, may contribute to the attenuation of virulence and efficient persistence of the bacteria in the CF lung.

Villar M.,Institute Investigacion en Recursos Cinegeticos IREC CSIC UCLM JCCM | Popara M.,Institute Investigacion en Recursos Cinegeticos IREC CSIC UCLM JCCM | Ayllon N.,Institute Investigacion en Recursos Cinegeticos IREC CSIC UCLM JCCM | De Fernandez Mera I.G.,Institute Investigacion en Recursos Cinegeticos IREC CSIC UCLM JCCM | And 6 more authors.
PLoS ONE | Year: 2014

Background: Dermacentor reticulatus (Fabricius, 1794) is distributed in Europe and Asia where it infests and transmits disease-causing pathogens to humans, pets and other domestic and wild animals. However, despite its role as a vector of emerging or re-emerging diseases, very little information is available on the genome, transcriptome and proteome of D. reticulatus. Tick larvae are the first developmental stage to infest hosts, acquire infection and transmit pathogens that are transovarially transmitted and are exposed to extremely stressing conditions. In this study, we used a systems biology approach to get an insight into the mechanisms active in D. reticulatus unfed larvae, with special emphasis on stress response. Principal Findings: The results support the use of paired end RNA sequencing and proteomics informed by transcriptomics (PIT) for the analysis of transcriptomics and proteomics data, particularly for organisms such as D. reticulatus with little sequence information available. The results showed that metabolic and cellular processes involved in protein synthesis were the most active in D. reticulatus unfed larvae, suggesting that ticks are very active during this life stage. The stress response was activated in D. reticulatus unfed larvae and a Rickettsia sp. similar to R. raoultii was identified in these ticks. Significance: The activation of stress responses in D. reticulatus unfed larvae likely counteracts the negative effect of temperature and other stress conditions such as Rickettsia infection and favors tick adaptation to environmental conditions to increase tick survival. These results show mechanisms that have evolved in D. reticulatus ticks to survive under stress conditions and suggest that these mechanisms are conserved across hard tick species. Targeting some of these proteins by vaccination may increase tick susceptibility to natural stress conditions, which in turn reduce tick survival and reproduction, thus reducing tick populations and vector capacity for tick-borne pathogens. © 2014 Villar et al.

Contreras M.,Institute Investigacion en Recursos Cinegeticos IREC CSIC UCLM JCCM | de la Fuente J.,Institute Investigacion en Recursos Cinegeticos IREC CSIC UCLM JCCM | de la Fuente J.,Oklahoma State University | Estrada-Pena A.,University of Zaragoza | And 3 more authors.
Molecular Ecology Resources | Year: 2014

This article documents the public availability of a global transcriptome comparison between Lyme disease tick vectors, Ixodes scapularis and Ixodes ricinus. © 2014 John Wiley & Sons Ltd.

Villar M.,Institute Investigacion en Recursos Cinege ticos IREC CSIC UCLM JCCM | Ayllon N.,Institute Investigacion en Recursos Cinege ticos IREC CSIC UCLM JCCM | Alberdi P.,Institute Investigacion en Recursos Cinege ticos IREC CSIC UCLM JCCM | Moreno A.,University of Castilla - La Mancha | And 9 more authors.
Molecular and Cellular Proteomics | Year: 2015

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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

PubMed | Instituto Vasco Of Investigacion Y Desarrollo Agrario Neiker, Institute Investigacion en Recursos Cinegeticos IREC CSIC UCLM JCCM, French Institute of Health and Medical Research and Oh no sequences! Research Group
Type: Journal Article | Journal: Genome announcements | Year: 2015

Here we report the complete genome sequences of field isolates of Mycobacterium bovis and the related mycobacterial species, Mycobacterium caprae. The genomes of three M.bovis (MB1, MB3, MB4) and one M.caprae (MB2) field isolates with different virulence, prevalence, and host distribution phenotypes were sequenced.

PubMed | Fundacion Parque Cientifico de Madrid, Institute Investigacion en Recursos Cinegeticos IREC CSIC UCLM JCCM, Hospital Nacional Of Paraplejicos, Oh no sequences! Research Group and Oklahoma State University
Type: Case Reports | Journal: Journal of immunology (Baltimore, Md. : 1950) | Year: 2016

Guillain-Barr syndrome (GBS) is an immune-mediated peripheral neuropathy. The goal of this research was the identification of biomarkers associated with recovery from GBS. In this study, we compared the transcriptome of PBMCs from a GBS patient and her healthy twin to discover possible correlates of disease progression and recovery. The study was then extended using GBS and spinal cord injury unrelated patients with similar medications and healthy individuals. The early growth response gene-2 (EGR2) was upregulated in GBS patients during disease recovery. The results provided evidence for the implication of EGR2 in GBS and suggested a role for EGR2 in the regulation of IL-17, IL-22, IL-28A, and TNF- cytokines in GBS patients. These results identified biomarkers associated with GBS recovery and suggested that EGR2 overexpression has a pivotal role in the downregulation of cytokines implicated in the pathophysiology of this acute neuropathy.

Lopez-Camacho E.,Hospital Universitario La Paz | Gomez-Gil R.,Hospital Universitario La Paz | Tobes R.,Oh no sequences Research group | Manrique M.,Oh no sequences Research group | And 11 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2014

Objectives: To characterize at the genomic level the evolution of multiresistance during an outbreak of Klebsiella pneumoniae in a burns intensive care unit. The outbreak involved a DHA-1 β-lactamase-producing strain that later acquired carbapenem and fosfomycin resistance, and in one case colistin resistance. Methods: The genomes of two isolateswere sequenced and compared with a previously sequenced genome. The role of hypermutability was investigated by measuring the mutation frequencies of the isolates and comparison with a collection of control strains. Results: Sequence comparison identified four single-nucleotide variants and two transposon insertions. Analysis of the variants in the whole collection related carbapenem and fosfomycin resistance to a nonsense mutation in the ompK36 porin gene and colistin resistance to an IS1 insertion in the mgrB gene. The plasmid carrying the blaDHA-1 gene was unstable in the absence of antibiotics, and analysis of isolates that had lost the plasmid showed that the porin mutation alone was not sufficient to generate carbapenem resistance. The mutation frequencies were similar among all the strains analysed. Conclusions: Carbapenem resistance required production of the DHA-1 β-lactamase and decreased permeability, but fosfomycin resistance depended only on permeability. Resistance to colistin might be related to an alteration in the regulation of the phoPQ system. Hypermutation is not related to the selection of porin mutants. Plasmid instabilitymight be due to the high numberofmobile elements and suggests amajor role forantibiotic selection pressure in the emergence and evolution of this outbreak. © The Author 2013.

PubMed | Yildiz Technical University, Oh no sequences! Research group and Ohio State University
Type: | Journal: Scientific reports | Year: 2015

Establishing the architecture of gene regulatory networks (GRNs) relies on chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) methods that provide genome-wide transcription factor binding sites (TFBSs). ChIP-Seq furnishes millions of short reads that, after alignment, describe the genome-wide binding sites of a particular TF. However, in all organisms investigated an average of 40% of reads fail to align to the corresponding genome, with some datasets having as much as 80% of reads failing to align. We describe here the provenance of previously unaligned reads in ChIP-Seq experiments from animals and plants. We show that a substantial portion corresponds to sequences of bacterial and metazoan origin, irrespective of the ChIP-Seq chromatin source. Unforeseen was the finding that 30%-40% of unaligned reads were actually alignable. To validate these observations, we investigated the characteristics of the previously unaligned reads corresponding to TAL1, a human TF involved in lineage specification of hemopoietic cells. We show that, while unmapped ChIP-Seq read datasets contain foreign DNA sequences, additional TFBSs can be identified from the previously unaligned ChIP-Seq reads. Our results indicate that the re-evaluation of previously unaligned reads from ChIP-Seq experiments will significantly contribute to TF target identification and determination of emerging properties of GRNs.

Pareja-Tobes P.,Oh no sequences Research group | Manrique M.,Oh no sequences Research group | Pareja-Tobes E.,Oh no sequences Research group | Pareja E.,Oh no sequences Research group | Tobes R.,Oh no sequences Research group
PLoS ONE | Year: 2012

BG7 is a new system for de novo bacterial, archaeal and viral genome annotation based on a new approach specifically designed for annotating genomes sequenced with next generation sequencing technologies. The system is versatile and able to annotate genes even in the step of preliminary assembly of the genome. It is especially efficient detecting unexpected genes horizontally acquired from bacterial or archaeal distant genomes, phages, plasmids, and mobile elements. From the initial phases of the gene annotation process, BG7 exploits the massive availability of annotated protein sequences in databases. BG7 predicts ORFs and infers their function based on protein similarity with a wide set of reference proteins, integrating ORF prediction and functional annotation phases in just one step. BG7 is especially tolerant to sequencing errors in start and stop codons, to frameshifts, and to assembly or scaffolding errors. The system is also tolerant to the high level of gene fragmentation which is frequently found in not fully assembled genomes. BG7 current version - which is developed in Java, takes advantage of Amazon Web Services (AWS) cloud computing features, but it can also be run locally in any operating system. BG7 is a fast, automated and scalable system that can cope with the challenge of analyzing the huge amount of genomes that are being sequenced with NGS technologies. Its capabilities and efficiency were demonstrated in the 2011 EHEC Germany outbreak in which BG7 was used to get the first annotations right the next day after the first entero-hemorrhagic E. coli genome sequences were made publicly available. The suitability of BG7 for genome annotation has been proved for Illumina, 454, Ion Torrent, and PacBio sequencing technologies. Besides, thanks to its plasticity, our system could be very easily adapted to work with new technologies in the future. © 2012 Pareja-Tobes et al.

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