Functional characterization of the Mycobacterium abscessus genome coupled with condition specific transcriptomics reveals conserved molecular strategies for host adaptation and persistence

Background: Mycobacterium abscessus subsp. abscessus (MAB) is a highly drug resistant mycobacterium and the most common respiratory pathogen among the rapidly growing non-tuberculous mycobacteria. MAB is also one of the most deadly of the emerging cystic fibrosis (CF) pathogens requiring prolonged treatment with multiple antibiotics. In addition to its “mycobacterial” virulence genes, the genome of MAB harbours a large accessory genome, presumably acquired via lateral gene transfer including homologs shared with the CF pathogens Pseudomonas aeruginosa and Burkholderia cepacia. While multiple genome sequences are available there is little functional genomics data available for this important pathogen. Results: We report here the first multi-omics approach to characterize the primary transcriptome, coding potential and potential regulatory regions of the MAB genome utilizing differential RNA sequencing (dRNA-seq), RNA-seq, Ribosome profiling and LC-MS proteomics. In addition we attempt to address the genomes contribution to the molecular systems that underlie MAB’s adaptation and persistence in the human host through an examination of MABs transcriptional response to a number of clinically relevant conditions. These include hypoxia, exposure to sub-inhibitory concentrations of antibiotics and growth in an artificial sputum designed to mimic the conditions within the cystic fibrosis lung. Conclusions: Our integrated map provides the first comprehensive view of the primary transcriptome of MAB and evidence for the translation of over one hundred new short open reading frames (sORFs). Our map will act as a resource for ongoing functional genomics characterization of MAB and our transcriptome data from clinically relevant stresses informs our understanding of MAB’s adaptation to life in the CF lung. MAB’s adaptation to growth in artificial CF sputum highlights shared metabolic strategies with other CF pathogens including P. aeruginosa and mirrors the transcriptional responses that lead to persistence in mycobacteria. These strategies include an increased reliance on amino acid metabolism, and fatty acid catabolism and highlights the relevance of the glyoxylate shunt to growth in the CF lung. Our data suggests that, similar to what is seen in chronically persisting P. aeruginosa, progression towards a biofilm mode of growth would play a more prominent role in a longer-term MAB infection. Finally, MAB’s transcriptional response to antibiotics highlights the role of antibiotic modifications enzymes, active transport and the evolutionarily conserved WhiB7 driven antibiotic resistance regulon

Evidence for lateral transfer of genes encoding ferredoxins, nitroreductases, NADH oxidase, and alcohol dehydrogenase 3 from anaerobic prokaryotes to Giardia lamblia and Entamoeba histolytica

Author Posting. © American Society for Microbiology, 2002. This article is posted here by permission of American Society for Microbiology for personal use, not for redistribution. The definitive version was published in Eukaryotic Cell 1 (2002): 181-190, doi:10.1128/EC.1.2.181-190.2002.Giardia lamblia and Entamoeba histolytica are amitochondriate, microaerophilic protists which use fermentation enzymes like those of bacteria to survive anaerobic conditions within the intestinal lumen. Genes encoding fermentation enzymes and related electron transport peptides (e.g., ferredoxins) in giardia organisms and amebae are hypothesized to be derived from either an ancient anaerobic eukaryote (amitochondriate fossil hypothesis), a mitochondrial endosymbiont (hydrogen hypothesis), or anaerobic bacteria (lateral transfer hypothesis). The goals here were to complete the molecular characterization of giardial and amebic fermentation enzymes and to determine the origins of the genes encoding them, when possible. A putative giardia [2Fe-2S]ferredoxin which had a hypothetical organelle-targeting sequence at its N terminus showed similarity to mitochondrial ferredoxins and the hydrogenosomal ferredoxin of Trichomonas vaginalis (another luminal protist). However, phylogenetic trees were star shaped, with weak bootstrap support, so we were unable to confirm or rule out the endosymbiotic origin of the giardia [2Fe-2S]ferredoxin gene. Putative giardial and amebic 6-kDa ferredoxins, ferredoxin-nitroreductase fusion proteins, and oxygen-insensitive nitroreductases each tentatively supported the lateral transfer hypothesis. Although there were not enough sequences to perform meaningful phylogenetic analyses, the unique common occurrence of these peptides and enzymes in giardia organisms, amebae, and the few anaerobic prokaryotes suggests the possibility of lateral transfer. In contrast, there was more robust phylogenetic evidence for the lateral transfer of G. lamblia genes encoding an NADH oxidase from a gram-positive coccus and a microbial group 3 alcohol dehydrogenase from thermoanaerobic prokaryotes. In further support of lateral transfer, the G. lamblia NADH oxidase and adh3 genes appeared to have an evolutionary history distinct from those of E. histolytica.This work was supported by NIH grants (AI33492 to J.S., AI43273 to M.L.S., and AI46516 to B.J.L.). Additional support was provided by the G. Unger Vetlesen Foundation and LI-COR Biotechnology

Molecular basis for the specification of floral organs by APETALA3 and PISTILLATA

How different organs are formed from small sets of undifferentiated precursor cells is a key question in developmental biology. To understand the molecular mechanisms underlying organ specification in plants, we studied the function of the homeotic selector genes APETALA3 (AP3) and PISTILLATA (PI), which control the formation of petals and stamens during Arabidopsis flower development. To this end, we characterized the activities of the transcription factors that AP3 and PI encode throughout flower development by using perturbation assays as well as transcript profiling and genomewide localization studies, in combination with a floral induction system that allows a stage-specific analysis of flower development by genomic technologies. We discovered considerable spatial and temporal differences in the requirement for AP3/PI activity during flower formation and show that they control different sets of genes at distinct phases of flower development. The genomewide identification of target genes revealed that AP3/PI act as bifunctional transcription factors: they activate genes involved in the control of numerous developmental processes required for organogenesis and repress key regulators of carpel formation. Our results imply considerable changes in the composition and topology of the gene network controlled by AP3/PI during the course of flower development. We discuss our results in light of a model for the mechanism underlying sex-determination in seed plants, in which AP3/PI orthologues might act as a switch between the activation of male and the repression of female development

Analysis of 14 BAC sequences from the Aedes aegypti genome: a benchmark for genome annotation and assembly

In order to provide a set of manually curated and annotated sequences from the Aedes aegypti genome, mapped BAC clones encompassing 1.57 Mb were sequenced, assembled and manually annotated using computational gene-finding, EST matches as well as comparative protein homology

Sequencing and analysis of an Irish human genome

BACKGROUND: Recent studies generating complete human sequences from Asian, African and European subgroups have revealed population-specific variation and disease susceptibility loci. Here, choosing a DNA sample from a population of interest due to its relative geographical isolation and genetic impact on further populations, we extend the above studies through the generation of 11-fold coverage of the first Irish human genome sequence. RESULTS: Using sequence data from a branch of the European ancestral tree as yet unsequenced, we identify variants that may be specific to this population. Through comparisons with HapMap and previous genetic association studies, we identified novel disease-associated variants, including a novel nonsense variant putatively associated with inflammatory bowel disease. We describe a novel method for improving SNP calling accuracy at low genome coverage using haplotype information. This analysis has implications for future re-sequencing studies and validates the imputation of Irish haplotypes using data from the current Human Genome Diversity Cell Line Panel (HGDP-CEPH). Finally, we identify gene duplication events as constituting significant targets of recent positive selection in the human lineage. CONCLUSIONS: Our findings show that there remains utility in generating whole genome sequences to illustrate both general principles and reveal specific instances of human biology. With increasing access to low cost sequencing we would predict that even armed with the resources of a small research group a number of similar initiatives geared towards answering specific biological questions will emerge

Characterization of the equine skeletal muscle transcriptome identifies novel functional responses to exercise training

<p>Abstract</p> <p>Background</p> <p>Digital gene expression profiling was used to characterize the assembly of genes expressed in equine skeletal muscle and to identify the subset of genes that were differentially expressed following a ten-month period of exercise training. The study cohort comprised seven Thoroughbred racehorses from a single training yard. Skeletal muscle biopsies were collected at rest from the <it>gluteus medius </it>at two time points: T₁- untrained, (9 ± 0.5 months old) and T₂- trained (20 ± 0.7 months old).</p> <p>Results</p> <p>The most abundant mRNA transcripts in the muscle transcriptome were those involved in muscle contraction, aerobic respiration and mitochondrial function. A previously unreported over-representation of genes related to RNA processing, the stress response and proteolysis was observed. Following training 92 tags were differentially expressed of which 74 were annotated. Sixteen genes showed increased expression, including the mitochondrial genes <it>ACADVL</it>, <it>MRPS21 </it>and <it>SLC25A29 </it>encoded by the nuclear genome. Among the 58 genes with decreased expression, <it>MSTN</it>, a negative regulator of muscle growth, had the greatest decrease.</p> <p>Functional analysis of all expressed genes using FatiScan revealed an asymmetric distribution of 482 Gene Ontology (GO) groups and 18 KEGG pathways. Functional groups displaying highly significant (<it>P </it>< 0.0001) increased expression included mitochondrion, oxidative phosphorylation and fatty acid metabolism while functional groups with decreased expression were mainly associated with structural genes and included the sarcoplasm, laminin complex and cytoskeleton.</p> <p>Conclusion</p> <p>Exercise training in Thoroughbred racehorses results in coordinate changes in the gene expression of functional groups of genes related to metabolism, oxidative phosphorylation and muscle structure.</p

Evaluating the successful implementation of evidence into practice using the PARiHS framework : theoretical and practical challenges

Background The PARiHS framework (Promoting Action on Research Implementation in Health Services) has proved to be a useful practical and conceptual heuristic for many researchers and practitioners in framing their research or knowledge translation endeavours. However, as a conceptual framework it still remains untested and therefore its contribution to the overall development and testing of theory in the field of implementation science is largely unquantified. Discussion This being the case, the paper provides an integrated summary of our conceptual and theoretical thinking so far and introduces a typology (derived from social policy analysis) used to distinguish between the terms conceptual framework, theory and model – important definitional and conceptual issues in trying to refine theoretical and methodological approaches to knowledge translation. Secondly, the paper describes the next phase of our work, in particular concentrating on the conceptual thinking and mapping that has led to the generation of the hypothesis that the PARiHS framework is best utilised as a two-stage process: as a preliminary (diagnostic and evaluative) measure of the elements and sub-elements of evidence (E) and context (C), and then using the aggregated data from these measures to determine the most appropriate facilitation method. The exact nature of the intervention is thus determined by the specific actors in the specific context at a specific time and place. In the process of refining this next phase of our work, we have had to consider the wider issues around the use of theories to inform and shape our research activity; the ongoing challenges of developing robust and sensitive measures; facilitation as an intervention for getting research into practice; and finally to note how the current debates around evidence into practice are adopting wider notions that fit innovations more generally. Summary The paper concludes by suggesting that the future direction of the work on the PARiHS framework is to develop a two-stage diagnostic and evaluative approach, where the intervention is shaped and moulded by the information gathered about the specific situation and from participating stakeholders. In order to expedite the generation of new evidence and testing of emerging theories, we suggest the formation of an international research implementation science collaborative that can systematically collect and analyse experiences of using and testing the PARiHS framework and similar conceptual and theoretical approaches. We also recommend further refinement of the definitions around conceptual framework, theory, and model, suggesting a wider discussion that embraces multiple epistemological and ontological perspectives

Genome sequencing of the extinct Eurasian wild aurochs, Bos primigenius, illuminates the phylogeography and evolution of cattle

Background Domestication of the now-extinct wild aurochs, Bos primigenius, gave rise to the two major domestic extant cattle taxa, B. taurus and B. indicus. While previous genetic studies have shed some light on the evolutionary relationships between European aurochs and modern cattle, important questions remain unanswered, including the phylogenetic status of aurochs, whether gene flow from aurochs into early domestic populations occurred, and which genomic regions were subject to selection processes during and after domestication. Here, we address these questions using whole-genome sequencing data generated from an approximately 6,750-year-old British aurochs bone and genome sequence data from 81 additional cattle plus genome-wide single nucleotide polymorphism data from a diverse panel of 1,225 modern animals. Results Phylogenomic analyses place the aurochs as a distinct outgroup to the domestic B. taurus lineage, supporting the predominant Near Eastern origin of European cattle. Conversely, traditional British and Irish breeds share more genetic variants with this aurochs specimen than other European populations, supporting localized gene flow from aurochs into the ancestors of modern British and Irish cattle, perhaps through purposeful restocking by early herders in Britain. Finally, the functions of genes showing evidence for positive selection in B. taurus are enriched for neurobiology, growth, metabolism and immunobiology, suggesting that these biological processes have been important in the domestication of cattle. Conclusions This work provides important new information regarding the origins and functional evolution of modern cattle, revealing that the interface between early European domestic populations and wild aurochs was significantly more complex than previously thought

Methicillin Resistance Alters the Biofilm Phenotype and Attenuates Virulence in Staphylococcus aureus Device-Associated Infections

Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). Biofilm production in methicillin-susceptible S. aureus (MSSA) strains is typically dependent on PIA/PNAG whereas methicillin-resistant isolates express an Atl/FnBP-mediated biofilm phenotype suggesting a relationship between susceptibility to β-lactam antibiotics and biofilm. By introducing the methicillin resistance gene mecA into the PNAG-producing laboratory strain 8325-4 we generated a heterogeneously resistant (HeR) strain, from which a homogeneous, high-level resistant (HoR) derivative was isolated following exposure to oxacillin. The HoR phenotype was associated with a R602H substitution in the DHHA1 domain of GdpP, a recently identified c-di-AMP phosphodiesterase with roles in resistance/tolerance to β-lactam antibiotics and cell envelope stress. Transcription of icaADBC and PNAG production were impaired in the 8325-4 HoR derivative, which instead produced a proteinaceous biofilm that was significantly inhibited by antibodies against the mecA-encoded penicillin binding protein 2a (PBP2a). Conversely excision of the SCCmec element in the MRSA strain BH1CC resulted in oxacillin susceptibility and reduced biofilm production, both of which were complemented by mecA alone. Transcriptional activity of the accessory gene regulator locus was also repressed in the 8325-4 HoR strain, which in turn was accompanied by reduced protease production and significantly reduced virulence in a mouse model of device infection. Thus, homogeneous methicillin resistance has the potential to affect agr- and icaADBC-mediated phenotypes, including altered biofilm expression and virulence, which together are consistent with the adaptation of healthcare-associated MRSA strains to the antibiotic-rich hospital environment in which they are frequently responsible for device-related infections in immuno-compromised patients