TY  - JOUR
A1  - Calderon, Dayana
A1  - Peña, Luis
A1  - Suarez, Angélica
A1  - Villamil, Carolina
A1  - Ramirez-Rojas, Adan
A1  - Anzola, Juan M.
A1  - García-Betancur, Juan C.
A1  - Cepeda, Martha L.
A1  - Uribe, Daniel
A1  - Del Portillo, Patricia
A1  - Mongui, Alvaro
T1  - Recovery and functional validation of hidden soil enzymes in metagenomic libraries
JF  - MicrobiologyOpen
N2  - The vast microbial diversity on the planet represents an invaluable source for identifying novel activities with potential industrial and therapeutic application. In this regard, metagenomics has emerged as a group of strategies that have significantly facilitated the analysis of DNA from multiple environments and has expanded the limits of known microbial diversity. However, the functional characterization of enzymes, metabolites, and products encoded by diverse microbial genomes is limited by the inefficient heterologous expression of foreign genes. We have implemented a pipeline that combines NGS and Sanger sequencing as a way to identify fosmids within metagenomic libraries. This strategy facilitated the identification of putative proteins, subcloning of targeted genes and preliminary characterization of selected proteins. Overall, the in silico approach followed by the experimental validation allowed us to efficiently recover the activity of previously hidden enzymes derived from agricultural soil samples. Therefore, the methodology workflow described herein can be applied to recover activities encoded by environmental DNA from multiple sources.
KW  - environmental microbiology
KW  - functional genomics
KW  - metagenomics
KW  - microbial genomics
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222016
VL  - 8
ER  - 
TY  - JOUR
A1  - Czimmerer, Zsolt
A1  - Daniel, Bence
A1  - Horvath, Attila
A1  - Rückerl, Dominik
A1  - Nagy, Gergely
A1  - Kiss, Mate
A1  - Peloquin, Matthew
A1  - Budai, Marietta M.
A1  - Cuaranta-Monroy, Ixchelt
A1  - Simandi, Zoltan
A1  - Steiner, Laszlo
A1  - Nagy Jr., Bela
A1  - Poliska, Szilard
A1  - Banko, Csaba
A1  - Bacso, Zsolt
A1  - Schulman, Ira G.
A1  - Sauer, Sascha
A1  - Deleuze, Jean-Francois
A1  - Allen, Judith E.
A1  - Benko, Szilvia
A1  - Nagy, Laszlo
T1  - The Transcription Factor STAT6 Mediates Direct Repression of Inflammatory Enhancers and Limits Activation of Alternatively Polarized Macrophages
JF  - Immunity
N2  - The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1β production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli.
KW  - IL-4
KW  - STAT6
KW  - alternative macrophage polarization
KW  - transcription
KW  - repression
KW  - inflammation
KW  - inflammasome activation
KW  - pyroptosis
KW  - IL-1β
KW  - macrophage epigenomics
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223380
VL  - 48
ER  - 
TY  - JOUR
A1  - Trübe, Patricia
A1  - Hertlein, Tobias
A1  - Mrochen, Daniel M.
A1  - Schulz, Daniel
A1  - Jorde, Ilka
A1  - Krause, Bettina
A1  - Zeun, Julia
A1  - Fischer, Stefan
A1  - Wolf, Silver A.
A1  - Walther, Birgit
A1  - Semmler, Torsten
A1  - Bröker, Barbara M.
A1  - Ulrich, Rainer G.
A1  - Ohlsen, Knut
A1  - Holtfreter, Silva
T1  - Bringing together what belongs together: Optimizing murine infection models by using mouse-adapted Staphylococcus aureus strains
JF  - International Journal of Medical Microbiology
N2  - Staphylococcus (S.) aureus is a leading cause of bacterial infection world-wide, and currently no vaccine is available for humans. Vaccine development relies heavily on clinically relevant infection models. However, the suitability of mice for S. aureus infection models has often been questioned, because experimental infection of mice with human-adapted S. aureus requires very high infection doses. Moreover, mice were not considered to be natural hosts of S. aureus. The latter has been disproven by our recent findings, showing that both laboratory mice, as well as wild small mammals including mice, voles, and shrews, are naturally colonized with S. aureus. Here, we investigated whether mouse-and vole-derived S. aureus strains show an enhanced virulence in mice as compared to the human-adapted strain Newman. Using a step-wise approach based on the bacterial genotype and in vitro assays for host adaptation, we selected the most promising candidates for murine infection models out of a total of 254 S. aureus isolates from laboratory mice as well as wild rodents and shrews. Four strains representing the clonal complexes (CC) 8, 49, and 88 (n = 2) were selected and compared to the human-adapted S. aureus strain Newman (CC8) in murine pneumonia and bacteremia models. Notably, a bank vole-derived CC49 strain, named DIP, was highly virulent in BALB/c mice in pneumonia and bacteremia models, whereas the other murine and vole strains showed virulence similar to or lower than that of Newman. At one tenth of the standard infection dose DIP induced disease severity, bacterial load and host cytokine and chemokine responses in the murine bacteremia model similar to that of Newman. In the pneumonia model, DIP was also more virulent than Newman but the effect was less pronounced. Whole genome sequencing data analysis identified a pore-forming toxin gene, lukF-PV(P83)/lukM, in DIP but not in the other tested S. aureus isolates. To conclude, the mouse-adapted S. aureus strain DIP allows a significant reduction of the inoculation dose in mice and is hence a promising tool to develop clinically more relevant infection models.
KW  - Staphylococcus aureus
KW  - host-adapted
KW  - infection model
KW  - mouse
KW  - vole
KW  - CC49
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229081
VL  - 309
ER  - 
TY  - JOUR
A1  - Nguyen, Minh-Thu
A1  - Saising, Jongkon
A1  - Tribelli, Paula Maria
A1  - Nega, Mulugeta
A1  - Diene, Seydina M.
A1  - François, Patrice
A1  - Schrenzel, Jacques
A1  - Spröer, Cathrin
A1  - Bunk, Boyke
A1  - Ebner, Patrick
A1  - Hertlein, Tobias
A1  - Kumari, Nimerta
A1  - Härtner, Thomas
A1  - Wistuba, Dorothee
A1  - Voravuthikunchai, Supayang P.
A1  - Mäder, Ulrike
A1  - Ohlsen, Knut
A1  - Götz, Friedrich
T1  - Inactivation of farR Causes High Rhodomyrtone Resistance and Increased Pathogenicity in Staphylococcus aureus
JF  - Frontiers in Microbiology
N2  - Rhodomyrtone (Rom) is an acylphloroglucinol antibiotic originally isolated from leaves of Rhodomyrtus tomentosa. Rom targets the bacterial membrane and is active against a wide range of Gram-positive bacteria but the exact mode of action remains obscure. Here we isolated and characterized a spontaneous Rom-resistant mutant from the model strain Staphylococcus aureus HG001 (RomR) to learn more about the resistance mechanism. We showed that Rom-resistance is based on a single point mutation in the coding region of farR [regulator of fatty acid (FA) resistance] that causes an amino acid change from Cys to Arg at position 116 in FarR, that affects FarR activity. Comparative transcriptome analysis revealed that mutated farR affects transcription of many genes in distinct pathways. FarR represses for example the expression of its own gene (farR), its flanking gene farE (effector of FA resistance), and other global regulators such as agr and sarA. All these genes were consequently upregulated in the RomR clone. Particularly the upregulation of agr and sarA leads to increased expression of virulence genes rendering the RomR clone more cytotoxic and more pathogenic in a mouse infection model. The Rom-resistance is largely due to the de-repression of farE. FarE is described as an efflux pump for linoleic and arachidonic acids. We observed an increased release of lipids in the RomR clone compared to its parental strain HG001. If farE is deleted in the RomR clone, or, if native farR is expressed in the RomR strain, the corresponding strains become hypersensitive to Rom. Overall, we show here that the high Rom-resistance is mediated by overexpression of farE in the RomR clone, that FarR is an important regulator, and that the point mutation in farR (RomR clone) makes the clone hyper-virulent.
KW  - antibiotic
KW  - Gram-positive bacteria
KW  - rhodomyrtone
KW  - Staphylococcus
KW  - membrane active
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224117
VL  - 10
ER  - 
TY  - JOUR
A1  - Müller, Laura S. M.
A1  - Cosentino, Raúl O.
A1  - Förstner, Konrad U.
A1  - Guizetti, Julien
A1  - Wedel, Carolin
A1  - Kaplan, Noam
A1  - Janzen, Christian J.
A1  - Arampatzi, Panagiota
A1  - Vogel, Jörg
A1  - Steinbiss, Sascha
A1  - Otto, Thomas D.
A1  - Saliba, Antoine-Emmanuel
A1  - Sebra, Robert P.
A1  - Siegel, T. Nicolai
T1  - Genome organization and DNA accessibility control antigenic variation in trypanosomes
JF  - Nature
N2  - Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses—Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing—that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.
KW  - histone variants
KW  - genome architecture
KW  - single molecule real time (SMRT)
KW  - brucei genome
KW  - distance-dependent decay
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224265
VL  - 563
ER  - 
TY  - JOUR
A1  - Liao, Chunyu
A1  - Ttofali, Fani
A1  - Slotkowski, Rebecca A.
A1  - Denny, Steven R.
A1  - Cecil, Taylor D.
A1  - Leenay, Ryan T.
A1  - Keung, Albert J.
A1  - Beisel, Chase L.
T1  - Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis
JF  - Nature Communications
N2  - CRISPR-Cas systems inherently multiplex through CRISPR arrays—whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.
KW  - biotechnology
KW  - CRISPR-Cas systems
KW  - microbiology
KW  - small RNAs
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236843
VL  - 10
ER  - 
TY  - JOUR
A1  - Bury, Susanne
A1  - Soundararajan, Manonmani
A1  - Bharti, Richa
A1  - von Bünau, Rudolf
A1  - Förstner, Konrad U.
A1  - Oelschlaeger, Tobias A.
T1  - The probiotic escherichia coli strain Nissle 1917 combats lambdoid bacteriophages stx and lambda
JF  - Frontiers in Microbiology
N2  - Shiga toxin (Stx) producing E. coli (STEC) such as Enterohemorrhagic E. coli (EHEC) are the major cause of foodborne illness in humans. In vitro studies showed the probiotic Escherichia coil strain Nissle 1917 (EcN) to efficiently inhibit the production of Stx. Life threatening EHEC strains as for example the serotype 0104:H4, responsible for the great outbreak in 2011 in Germany, evolutionary developed from certain E. coll strains which got infected by stx2-encoding lambdoid phages turning the E. coil into lysogenic and subsequently Stx producing strains. Since antibiotics induce stx genes and Stx production, EHEC infected persons are not recommended to be treated with antibiotics. Therefore, EcN might be an alternative medication. However, because even commensal E. coli strains might be converted into Stx-producers after becoming host to a stx encoding prophage, we tested EcN for stx-phage genome integration. Our experiments revealed the resistance of EcN toward not only stx-phages but also against lambda-phages. This resistance was not based on the lack of or by mutated phage receptors. Rather it involved the expression of a phage repressor (pr) gene of a defective prophage in EcN which was able to partially protect E. coli K-12 strain MG1655 against stx and lambda phage infection. Furthermore, we observed EcN to inactivate phages and thereby to protect E. coli K-12 strains against infection by stx- as well as lambda-phages. Inactivation of lambda-phages was due to binding of lambda-phages to LamB of EcN whereas inactivation of stx-phages was caused by a thermostable protein of EcN. These properties together with its ability to inhibit Stx production make EcN a good candidate for the prevention of illness caused by EHEC and probably for the treatment of already infected people.
KW  - probiotic
KW  - E. coli Nissle 1917
KW  - EHEC
KW  - Shiga toxin producing E. coli
KW  - stx-phages
KW  - lambda-phages
KW  - lambdoid prophage
KW  - LamB
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221960
VL  - 9
ER  - 
TY  - JOUR
A1  - Balasubramanian, Srikkanth
A1  - Skaf, Joseph
A1  - Holzgrabe, Ulrike
A1  - Bharti, Richa
A1  - Förstner, Konrad U.
A1  - Ziebuhr, Wilma
A1  - Humeida, Ute H.
A1  - Abdelmohsen, Usama R.
A1  - Oelschlaeger, Tobias A.
T1  - A new bioactive compound from the marine sponge-derived Streptomyces sp. SBT348 inhibits staphylococcal growth and biofilm formation
JF  - Frontiers in Microbiology
N2  - Staphylococcus epidermidis, the common inhabitant of human skin and mucosal surfaces has emerged as an important pathogen in patients carrying surgical implants and medical devices. Entering the body via surgical sites and colonizing the medical devices through formation of multi-layered biofilms leads to refractory and persistent device-related infections (DRIs). Staphylococci organized in biofilms are more tolerant to antibiotics and immune responses, and thus are difficult-to-treat. The consequent morbidity and mortality, and economic losses in health care systems has strongly necessitated the need for development of new anti-bacterial and anti-biofilm-based therapeutics. In this study, we describe the biological activity of a marine sponge-derived Streptomyces sp. SBT348 extract in restraining staphylococcal growth and biofilm formation on polystyrene, glass, medically relevant titan metal, and silicone surfaces. A bioassay-guided fractionation was performed to isolate the active compound (SKC3) from the crude SBT348 extract. Our results demonstrated that SKC3 effectively inhibits the growth (MIC: 31.25 \(\mu\)g/ml) and biofilm formation (sub-MIC range: 1.95-<31.25 \(\mu\)g/ml) of S. epidermidis RP62A in vitro. Chemical characterization of SKC3 by heat and enzyme treatments, and mass spectrometry (HRMS) revealed its heat-stable and non-proteinaceous nature, and high molecular weight (1258.3 Da). Cytotoxicity profiling of SKC3 in vitro on mouse fibroblast (NIH/3T3) and macrophage (J774.1) cell lines, and in vivo on the greater wax moth larvae Galleria mellonella revealed its non-toxic nature at the effective dose. Transcriptome analysis of SKC3 treated S. epidermidis RP62A has further unmasked its negative effect on central metabolism such as carbon flux as well as, amino acid, lipid, and energy metabolism. Taken together, these findings suggest a potential of SKC3 as a putative drug to prevent staphylococcal DRIs.
KW  - marine sponges
KW  - Streptomyces
KW  - Staphylococci
KW  - device-related infections
KW  - bioassay-guided fractionation
KW  - transcriptome
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221408
VL  - 9
ER  - 
TY  - JOUR
A1  - Kraus, Amelie J.
A1  - Brink, Benedikt G.
A1  - Siegel, T. Nicolai
T1  - Efficient and specific oligo-based depletion of rRNA
JF  - Scientific Reports
N2  - In most organisms, ribosomal RNA (rRNA) contributes to >85% of total RNA. Thus, to obtain useful information from RNA-sequencing (RNA-seq) analyses at reasonable sequencing depth, typically, mature polyadenylated transcripts are enriched or rRNA molecules are depleted. Targeted depletion of rRNA is particularly useful when studying transcripts lacking a poly(A) tail, such as some non-coding RNAs (ncRNAs), most bacterial RNAs and partially degraded or immature transcripts. While several commercially available kits allow effective rRNA depletion, their efficiency relies on a high degree of sequence homology between oligonucleotide probes and the target RNA. This restricts the use of such kits to a limited number of organisms with conserved rRNA sequences. In this study we describe the use of biotinylated oligos and streptavidin-coated paramagnetic beads for the efficient and specific depletion of trypanosomal rRNA. Our approach reduces the levels of the most abundant rRNA transcripts to less than 5% with minimal off-target effects. By adjusting the sequence of the oligonucleotide probes, our approach can be used to deplete rRNAs or other abundant transcripts independent of species. Thus, our protocol provides a useful alternative for rRNA removal where enrichment of polyadenylated transcripts is not an option and commercial kits for rRNA are not available.
KW  - parasite biology
KW  - RNA sequencing
KW  - transcriptomics
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224829
VL  - 9
ER  - 
TY  - JOUR
A1  - Allert, Stefanie
A1  - Förster, Toni M.
A1  - Svensson, Carl-Magnus
A1  - Richardson, Jonathan P.
A1  - Pawlik, Tony
A1  - Hebecker, Betty
A1  - Rudolphi, Sven
A1  - Juraschitz, Marc
A1  - Schaller, Martin
A1  - Blagojevic, Mariana
A1  - Morschhäuser, Joachim
A1  - Figge, Marc Thilo
A1  - Jacobsen, Ilse D.
A1  - Naglik, Julian R.
A1  - Kasper, Lydia
A1  - Mogavero, Selene
A1  - Hube, Bernhard
T1  - \(Candida\) \(albicans\)-Induced Epithelial Damage Mediates Translocation through Intestinal Barriers
JF  - mBio
N2  - Life-threatening systemic infections often occur due to the translocation of pathogens across the gut barrier and into the bloodstream. While the microbial and host mechanisms permitting bacterial gut translocation are well characterized, these mechanisms are still unclear for fungal pathogens such as Candida albicans, a leading cause of nosocomial fungal bloodstream infections. In this study, we dissected the cellular mechanisms of translocation of C. albicans across intestinal epithelia in vitro and identified fungal genes associated with this process. We show that fungal translocation is a dynamic process initiated by invasion and followed by cellular damage and loss of epithelial integrity. A screen of >2,000 C. albicans deletion mutants identified genes required for cellular damage of and translocation across enterocytes. Correlation analysis suggests that hypha formation, barrier damage above a minimum threshold level, and a decreased epithelial integrity are required for efficient fungal translocation. Translocation occurs predominantly via a transcellular route, which is associated with fungus-induced necrotic epithelial damage, but not apoptotic cell death. The cytolytic peptide toxin of C. albicans, candidalysin, was found to be essential for damage of enterocytes and was a key factor in subsequent fungal translocation, suggesting that transcellular translocation of C. albicans through intestinal layers is mediated by candidalysin. However, fungal invasion and low-level translocation can also occur via non-transcellular routes in a candidalysin-independent manner. This is the first study showing translocation of a human-pathogenic fungus across the intestinal barrier being mediated by a peptide toxin. IMPORTANCE Candida albicans, usually a harmless fungus colonizing human mucosae, can cause lethal bloodstream infections when it manages to translocate across the intestinal epithelium. This can result from antibiotic treatment, immune dysfunction, or intestinal damage (e.g., during surgery). However, fungal processes may also contribute. In this study, we investigated the translocation process of C. albicans using in vitro cell culture models. Translocation occurs as a stepwise process starting with invasion, followed by epithelial damage and loss of epithelial integrity. The ability to secrete candidalysin, a peptide toxin deriving from the hyphal protein Ece1, is key: C. albicans hyphae, secreting candidalysin, take advantage of a necrotic weakened epithelium to translocate through the intestinal layer.
KW  - Candida albicans
KW  - candidalysin
KW  - host cell damage
KW  - host cell invasion
KW  - intestinal barrier
KW  - necrosis
KW  - translocation
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221084
VL  - 9
IS  - 3
ER  -