All Publications

  • The Cystic Fibrosis Upper and Lower Airway Metagenome MICROBIOLOGY SPECTRUM Pienkowska, K., Pust, M., Gessner, M., Gaedcke, S., Thavarasa, A., Rosenboom, I., Moran Losada, P., Minso, R., Arnold, C., Hedtfeld, S., Dorda, M., Wiehlmann, L., Mainz, J. G., Klockgether, J., Tuemmler, B. 2023: e0363322


    The microbial metagenome in cystic fibrosis (CF) airways was investigated by whole-genome shotgun sequencing of total DNA isolated from nasal lavage samples, oropharyngeal swabs, and induced sputum samples collected from 65 individuals with CF aged 7 to 50 years. Each patient harbored a personalized microbial metagenome unique in microbial load and composition, the exception being monocultures of the most common CF pathogens Staphylococcus aureus and Pseudomonas aeruginosa from patients with advanced lung disease. The sampling of the upper airways by nasal lavage uncovered the fungus Malassezia restricta and the bacterium Staphylococcus epidermidis as prominent species. Healthy and CF donors harbored qualitatively and quantitatively different spectra of commensal bacteria in their sputa, even in the absence of any typical CF pathogen. If P. aeruginosa, S. aureus, or Stenotrophomonas maltophilia belonged to the trio of the most abundant species in the CF sputum metagenome, common inhabitants of the respiratory tract of healthy subjects, i.e., Eubacterium sulci, Fusobacterium periodonticum, and Neisseria subflava, were present only in low numbers or not detectable. Random forest analysis identified the numerical ecological parameters of the bacterial community, such as Shannon and Simpson diversity, as the key parameters that globally distinguish sputum samples from CF and healthy donors. IMPORTANCE Cystic fibrosis (CF) is the most common life-limiting monogenetic disease in European populations and is caused by mutations in the CFTR gene. Chronic airway infections with opportunistic pathogens are the major morbidity that determines prognosis and quality of life in most people with CF. We examined the composition of the microbial communities of the oral cavity and upper and lower airways in CF patients across all age groups. From early on, the spectrum of commensals is different in health and CF. Later on, when the common CF pathogens take up residence in the lungs, we observed differential modes of depletion of the commensal microbiota in the presence of S. aureus, P. aeruginosa, S. maltophilia, or combinations thereof. It remains to be seen whether the implementation of lifelong CFTR (cystic fibrosis transmembrane conductance regulator) modulation will change the temporal evolution of the CF airway metagenome.

    View details for DOI 10.1128/spectrum.03633-22

    View details for Web of Science ID 000946019000001

    View details for PubMedID 36892308

  • Undulating changes in human plasma proteome profiles across the lifespan. Nature medicine Lehallier, B. n., Gate, D. n., Schaum, N. n., Nanasi, T. n., Lee, S. E., Yousef, H. n., Moran Losada, P. n., Berdnik, D. n., Keller, A. n., Verghese, J. n., Sathyan, S. n., Franceschi, C. n., Milman, S. n., Barzilai, N. n., Wyss-Coray, T. n. 2019; 25 (12): 1843–50


    Aging is a predominant risk factor for several chronic diseases that limit healthspan1. Mechanisms of aging are thus increasingly recognized as potential therapeutic targets. Blood from young mice reverses aspects of aging and disease across multiple tissues2-10, which supports a hypothesis that age-related molecular changes in blood could provide new insights into age-related disease biology. We measured 2,925 plasma proteins from 4,263 young adults to nonagenarians (18-95 years old) and developed a new bioinformatics approach that uncovered marked non-linear alterations in the human plasma proteome with age. Waves of changes in the proteome in the fourth, seventh and eighth decades of life reflected distinct biological pathways and revealed differential associations with the genome and proteome of age-related diseases and phenotypic traits. This new approach to the study of aging led to the identification of unexpected signatures and pathways that might offer potential targets for age-related diseases.

    View details for DOI 10.1038/s41591-019-0673-2

    View details for PubMedID 31806903

  • Transcriptome-wide isoform-level dysregulation in ASD, schizophrenia, and bipolar disorder SCIENCE Gandal, M. J., Zhang, P., Hadjimichael, E., Walker, R. L., Chen, C., Liu, S., Won, H., van Bakel, H., Varghese, M., Wang, Y., Shieh, A. W., Haney, J., Parhami, S., Belmont, J., Kim, M., Losada, P., Khan, Z., Mleczko, J., Xia, Y., Dai, R., Wang, D., Yang, Y. T., Xu, M., Fish, K., Hof, P. R., Warrell, J., Fitzgerald, D., White, K., Jaffe, A. E., Peters, M. A., Gerstein, M., Liu, C., Iakoucheva, L. M., Pinto, D., Geschwind, D. H., PsychENCODE Consortium 2018; 362 (6420): 1265-+


    Most genetic risk for psychiatric disease lies in regulatory regions, implicating pathogenic dysregulation of gene expression and splicing. However, comprehensive assessments of transcriptomic organization in diseased brains are limited. In this work, we integrated genotypes and RNA sequencing in brain samples from 1695 individuals with autism spectrum disorder (ASD), schizophrenia, and bipolar disorder, as well as controls. More than 25% of the transcriptome exhibits differential splicing or expression, with isoform-level changes capturing the largest disease effects and genetic enrichments. Coexpression networks isolate disease-specific neuronal alterations, as well as microglial, astrocyte, and interferon-response modules defining previously unidentified neural-immune mechanisms. We integrated genetic and genomic data to perform a transcriptome-wide association study, prioritizing disease loci likely mediated by cis effects on brain expression. This transcriptome-wide characterization of the molecular pathology across three major psychiatric disorders provides a comprehensive resource for mechanistic insight and therapeutic development.

    View details for DOI 10.1126/science.aat8127

    View details for Web of Science ID 000452994400042

    View details for PubMedID 30545856

    View details for PubMedCentralID PMC6443102

  • Filtration and Normalization of Sequencing Read Data in Whole-Metagenome Shotgun Samples PLOS ONE Chouvarine, P., Wiehlmann, L., Losada, P., DeLuca, D. S., Tuemmler, B. 2016; 11 (10): e0165015


    Ever-increasing affordability of next-generation sequencing makes whole-metagenome sequencing an attractive alternative to traditional 16S rDNA, RFLP, or culturing approaches for the analysis of microbiome samples. The advantage of whole-metagenome sequencing is that it allows direct inference of the metabolic capacity and physiological features of the studied metagenome without reliance on the knowledge of genotypes and phenotypes of the members of the bacterial community. It also makes it possible to overcome problems of 16S rDNA sequencing, such as unknown copy number of the 16S gene and lack of sufficient sequence similarity of the "universal" 16S primers to some of the target 16S genes. On the other hand, next-generation sequencing suffers from biases resulting in non-uniform coverage of the sequenced genomes. To overcome this difficulty, we present a model of GC-bias in sequencing metagenomic samples as well as filtration and normalization techniques necessary for accurate quantification of microbial organisms. While there has been substantial research in normalization and filtration of read-count data in such techniques as RNA-seq or Chip-seq, to our knowledge, this has not been the case for the field of whole-metagenome shotgun sequencing. The presented methods assume that complete genome references are available for most microorganisms of interest present in metagenomic samples. This is often a valid assumption in such fields as medical diagnostics of patient microbiota. Testing the model on two validation datasets showed four-fold reduction in root-mean-square error compared to non-normalized data in both cases. The presented methods can be applied to any pipeline for whole metagenome sequencing analysis relying on complete microbial genome references. We demonstrate that such pre-processing reduces the number of false positive hits and increases accuracy of abundance estimates.

    View details for DOI 10.1371/journal.pone.0165015

    View details for Web of Science ID 000386204000075

    View details for PubMedID 27760173

    View details for PubMedCentralID PMC5070866

  • SNP synteny analysis of Staphylococcus aureus and Pseudomonas aeruginosa population genomics FEMS MICROBIOLOGY LETTERS Losada, P., Tuemmler, B. 2016; 363 (19)
  • Intraclonal genome diversity of the major Pseudomonas aeruginosa clones C and PA14 ENVIRONMENTAL MICROBIOLOGY REPORTS Fischer, S., Klockgether, J., Losada, P., Chouvarine, P., Cramer, N., Davenport, C. F., Dethlefsen, S., Dorda, M., Goesmann, A., Hilker, R., Mielke, S., Schoenfelder, T., Suerbaum, S., Tuerk, O., Woltemate, S., Wiehlmann, L., Tuemmler, B. 2016; 8 (2): 227–34


    Bacterial populations differentiate at the subspecies level into clonal complexes. Intraclonal genome diversity was studied in 100 isolates of the two dominant Pseudomonas aeruginosa clones C and PA14 collected from the inanimate environment, acute and chronic infections. The core genome was highly conserved among clone members with a median pairwise within-clone single nucleotide sequence diversity of 8 × 10(-6) for clone C and 2 × 10(-5) for clone PA14. The composition of the accessory genome was, on the other hand, as variable within the clone as between unrelated clones. Each strain carried a large cargo of unique genes. The two dominant worldwide distributed P. aeruginosa clones combine an almost invariant core with the flexible gain and loss of genetic elements that spread by horizontal transfer.

    View details for DOI 10.1111/1758-2229.12372

    View details for Web of Science ID 000372931000008

    View details for PubMedID 26711897

    View details for PubMedCentralID PMC4819714

  • The cystic fibrosis lower airways microbial metagenome. ERJ open research Moran Losada, P., Chouvarine, P., Dorda, M., Hedtfeld, S., Mielke, S., Schulz, A., Wiehlmann, L., Tümmler, B. 2016; 2 (2)


    Chronic airway infections determine most morbidity in people with cystic fibrosis (CF). Herein, we present unbiased quantitative data about the frequency and abundance of DNA viruses, archaea, bacteria, moulds and fungi in CF lower airways. Induced sputa were collected on several occasions from children, adolescents and adults with CF. Deep sputum metagenome sequencing identified, on average, approximately 10 DNA viruses or fungi and several hundred bacterial taxa. The metagenome of a CF patient was typically found to be made up of an individual signature of multiple, lowly abundant species superimposed by few disease-associated pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, as major components. The host-associated signatures ranged from inconspicuous polymicrobial communities in healthy subjects to low-complexity microbiomes dominated by the typical CF pathogens in patients with advanced lung disease. The DNA virus community in CF lungs mainly consisted of phages and occasionally of human pathogens, such as adeno- and herpesviruses. The S. aureus and P. aeruginosa populations were composed of one major and numerous minor clone types. The rare clones constitute a low copy genetic resource that could rapidly expand as a response to habitat alterations, such as antimicrobial chemotherapy or invasion of novel microbes.

    View details for DOI 10.1183/23120541.00096-2015

    View details for PubMedID 27730195

    View details for PubMedCentralID PMC5005179

  • Interclonal gradient of virulence in the Pseudomonas aeruginosa pangenome from disease and environment ENVIRONMENTAL MICROBIOLOGY Hilker, R., Munder, A., Klockgether, J., Losada, P., Chouvarine, P., Cramer, N., Davenport, C. F., Dethlefsen, S., Fischer, S., Peng, H., Schoenfelder, T., Tuerk, O., Wiehlmann, L., Woelbeling, F., Gulbins, E., Goesmann, A., Tuemmler, B. 2015; 17 (1): 29–46


    The population genomics of Pseudomonas aeruginosa was analysed by genome sequencing of representative strains of the 15 most frequent clonal complexes in the P. aeruginosa population and of the five most common clones from the environment of which so far no isolate from a human infection has been detected. Gene annotation identified 5892-7187 open reading frame (ORFs; median 6381 ORFs) in the 20 6.4-7.4 Mbp large genomes. The P. aeruginosa pangenome consists of a conserved core of at least 4000 genes, a combinatorial accessory genome of a further 10 000 genes and 30 000 or more rare genes that are present in only a few strains or clonal complexes. Whole genome comparisons of single nucleotide polymorphism synteny indicated unrestricted gene flow between clonal complexes by recombination. Using standardized acute lettuce, Galleria mellonella and murine airway infection models the full spectrum of possible host responses to P. aeruginosa was observed with the 20 strains ranging from unimpaired health following infection to 100% lethality. Genome comparisons indicate that the differential genetic repertoire of clones maintains a habitat-independent gradient of virulence in the P. aeruginosa population.

    View details for DOI 10.1111/1462-2920.12606

    View details for Web of Science ID 000349152800005

    View details for PubMedID 25156090

  • THOC5, a member of the mRNA export complex, contributes to processing of a subset of wingless/integrated (Wnt) target mRNAs and integrity of the gut epithelial barrier BMC CELL BIOLOGY Saran, S., Tran, D. H., Klebba-Faerber, S., Moran-Losada, P., Wiehlmann, L., Koch, A., Chopra, H., Pabst, O., Hoffmann, A., Klopfleisch, R., Tamura, T. 2013; 14: 51


    THO (Suppressors of the transcriptional defects of hpr1 delta by overexpression) complex 5 (THOC5), an mRNA export protein, is involved in the expression of only 1% of all genes. Using an interferon inducible knockout mouse system, we have previously shown that THOC5 is an essential element in the maintenance of hematopoietic stem cells and cytokine-mediated hematopoiesis in adult mice. Here we interrogate THOC5 function in cell differentiation beyond the hematopoietic system and study pathological changes caused by THOC5 deficiency.To examine whether THOC5 plays a role in general differentiation processes, we generated tamoxifen inducible THOC5 knockout mice. We show here that the depletion of THOC5 impaired not only hematopoietic differentiation, but also differentiation and self renewal of the gut epithelium. Depletion of the THOC5 gene did not cause pathological alterations in liver or kidney. We further show that THOC5 is indispensable for processing of mRNAs induced by Wnt (wingless/integrated) signaling which play key roles in epithelial cell differentiation/proliferation. A subset of Wnt target mRNAs, SRY-box containing gene 9 (Sox9), and achaete-scute complex homolog 2 (Ascl2), but not Fibronectin 1 (Fn1), were down-regulated in THOC5 knockout intestinal cells. The down-regulated Wnt target mRNAs were able to bind to THOC5. Furthermore, pathological alterations in the gastrointestinal tract induced translocation of intestinal bacteria and caused sepsis in mice. The bacteria translocation may cause Toll-like receptor activation. We identified one of the Toll-like receptor inducible genes, prostaglandin-endoperoxidase synthase 2 (Ptgs2 or COX2) transcript as THOC5 target mRNA.THOC5 is indispensable for processing of only a subset of mRNAs, but plays a key role in processing of mRNAs inducible by Wnt signals. Furthermore, THOC5 is dispensable for general mRNA export in terminally differentiated organs, indicating that multiple mRNA export pathways exist. These data imply that THOC5 may be a useful tool for studying intestinal stem cells, for modifying the differentiation processes and for cancer therapy.

    View details for DOI 10.1186/1471-2121-14-51

    View details for Web of Science ID 000328430500001

    View details for PubMedID 24267292

    View details for PubMedCentralID PMC4222586