• No impact of a short-term climatic "El Niño" fluctuation on gut microbial diversity in populations of the Galápagos marine iguana (Amblyrhynchus cristatus).

      Ibáñez, Alejandro; Bletz, Molly C; Quezada, Galo; Geffers, Robert; Jarek, Michael; Vences, Miguel; Steinfartz, Sebastian; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Springer, 2021-02-02)
      Gut microorganisms are crucial for many biological functions playing a pivotal role in the host's well-being. We studied gut bacterial community structure of marine iguana populations across the Galápagos archipelago. Marine iguanas depend heavily on their specialized gut microbiome for the digestion of dietary algae, a resource whose growth was strongly reduced by severe "El Niño"-related climatic fluctuations in 2015/2016. As a consequence, marine iguana populations showed signs of starvation as expressed by a poor body condition. Body condition indices (BCI) varied between island populations indicating that food resources (i.e., algae) are affected differently across the archipelago during 'El Niño' events. Though this event impacted food availability for marine iguanas, we found that reductions in body condition due to "El Niño"-related starvation did not result in differences in bacterial gut community structure. Species richness of gut microorganisms was instead correlated with levels of neutral genetic diversity in the distinct host populations. Our data suggest that marine iguana populations with a higher level of gene diversity and allelic richness may harbor a more diverse gut microbiome than those populations with lower genetic diversity. Since low values of these diversity parameters usually correlate with small census and effective population sizes, we use our results to propose a novel hypothesis according to which small and genetically less diverse host populations might be characterized by less diverse microbiomes. Whether such genetically depauperate populations may experience additional threats from reduced dietary flexibility due to a limited intestinal microbiome is currently unclear and calls for further investigation.
    • 3D culture conditions support Kaposi's sarcoma herpesvirus (KSHV) maintenance and viral spread in endothelial cells.

      Dubich, Tatyana; Dittrich, Anne; Bousset, Kristine; Geffers, Robert; Büsche, Guntram; Köster, Mario; Hauser, Hansjörg; Schulz, Thomas F; Wirth, Dagmar; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer International, 2021-01-23)
      Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumorigenic virus and the etiological agent of an endothelial tumor (Kaposi's sarcoma) and two B cell proliferative diseases (primary effusion lymphoma and multicentric Castleman's disease). While in patients with late stage of Kaposi's sarcoma the majority of spindle cells are KSHV-infected, viral copies are rapidly lost in vitro, both upon culture of tumor-derived cells or from newly infected endothelial cells. We addressed this discrepancy by investigating a KSHV-infected endothelial cell line in various culture conditions and in tumors of xenografted mice. We show that, in contrast to two-dimensional endothelial cell cultures, KSHV genomes are maintained under 3D cell culture conditions and in vivo. Additionally, an increased rate of newly infected cells was detected in 3D cell culture. Furthermore, we show that the PI3K/Akt/mTOR and ATM/γH2AX pathways are modulated and support an improved KSHV persistence in 3D cell culture. These mechanisms may contribute to the persistence of KSHV in tumor tissue in vivo and provide a novel target for KS specific therapeutic interventions. KEY MESSAGES: In vivo maintenance of episomal KSHV can be mimicked in 3D spheroid cultures 3D maintenance of KSHV is associated with an increased de novo infection frequency PI3K/Akt/mTOR and ATM/ γH2AX pathways contribute to viral maintenance.
    • p53-Independent Induction of p21 Fails to Control Regeneration and Hepatocarcinogenesis in a Murine Liver Injury Model.

      Buitrago-Molina, Laura Elisa; Marhenke, Silke; Becker, Diana; Geffers, Robert; Itzel, Timo; Teufel, Andreas; Jaeschke, Hartmut; Lechel, André; Unger, Kristian; Markovic, Jovana; et al. (Elsevier, 2021-01-21)
      Background & aims: A coordinated stress and regenerative response is important after hepatocyte damage. Here, we investigate the phenotypes that result from genetic abrogation of individual components of the checkpoint kinase 2/transformation-related protein 53 (p53)/cyclin-dependent kinase inhibitor 1A (p21) pathway in a murine model of metabolic liver injury. Methods: Nitisinone was reduced or withdrawn in Fah-/- mice lacking Chk2, p53, or p21, and survival, tumor development, liver injury, and regeneration were analyzed. Partial hepatectomies were performed and mice were challenged with the Fas antibody Jo2. Results: In a model of metabolic liver injury, loss of p53, but not Chk2, impairs the oxidative stress response and aggravates liver damage, indicative of a direct p53-dependent protective effect on hepatocytes. Cell-cycle control during chronic liver injury critically depends on the presence of both p53 and its downstream effector p21. In p53-deficient hepatocytes, unchecked proliferation occurs despite a strong induction of p21, showing a complex interdependency between p21 and p53. The increased regenerative potential in the absence of p53 cannot fully compensate the surplus injury and is not sufficient to promote survival. Despite the distinct phenotypes associated with the loss of individual components of the DNA damage response, gene expression patterns are dominated by the severity of liver injury, but reflect distinct effects of p53 on proliferation and the anti-oxidative stress response. Conclusions: Characteristic phenotypes result from the genetic abrogation of individual components of the DNA damage-response cascade in a liver injury model. The extent to which loss of gene function can be compensated, or affects injury and proliferation, is related to the level at which the cascade is interrupted. Accession numbers of repository for expression microarray data: GSE156983, GSE156263, GSE156852, and GSE156252.
    • Complete Genome Sequences of Streptococcus suis Pig-Pathogenic Strains 10, 13-00283-02, and 16085/3b.

      Bunk, Boyke; Jakóbczak, Beata; Florian, Volker; Dittmar, Denise; Mäder, Ulrike; Jarek, Michael; Häußler, Susanne; Baums, Christoph Georg; Völker, Uwe; Michalik, Stephan; et al. (American Society for Microbiology, 2021-01-14)
      Streptococcus suis is an important pathogen of pigs that, as a zoonotic agent, can also cause severe disease in humans, including meningitis, endocarditis, and septicemia. We report complete and annotated genomes of S. suis strains 10, 13-00283-02, and 16085/3b, which represent the highly prevalent serotypes cps2, cps7, and cps9, respectively.
    • Serum Response Factor (SRF) Drives the Transcriptional Upregulation of the MDM4 Oncogene in HCC.

      Pellegrino, Rossella; Thavamani, Abhishek; Calvisi, Diego F; Budczies, Jan; Neumann, Ariane; Geffers, Robert; Kroemer, Jasmin; Greule, Damaris; Schirmacher, Peter; Nordheim, Alfred; et al. (MDPI, 2021-01-08)
      Different molecular mechanisms support the overexpression of the mouse double minute homolog 4 (MDM4), a functional p53 inhibitor, in human hepatocellular carcinoma (HCC). However, the transcription factors (TFs) leading to its transcriptional upregulation remain unknown. Following promoter and gene expression analyses, putative TFs were investigated using gene-specific siRNAs, cDNAs, luciferase reporter assays, chromatin immunoprecipitation, and XI-011 drug treatment in vitro. Additionally, MDM4 expression was investigated in SRF-VP16iHep transgenic mice. We observed a copy-number-independent upregulation of MDM4 in human HCCs. Serum response factor (SRF), ELK1 and ELK4 were identified as TFs activating MDM4 transcription. While SRF was constitutively detected in TF complexes at the MDM4 promoter, presence of ELK1 and ELK4 was cell-type dependent. Furthermore, MDM4 was upregulated in SRF-VP16-driven murine liver tumors. The pharmacological inhibitor XI-011 exhibited anti-MDM4 activity by downregulating the TFs driving MDM4 transcription, which decreased HCC cell viability and increased apoptosis. In conclusion, SRF drives transcriptional MDM4 upregulation in HCC, acting in concert with either ELK1 or ELK4. The transcriptional regulation of MDM4 may be a promising target for precision oncology of human HCC, as XI-011 treatment exerts anti-MDM4 activity independent from the MDM4 copy number and the p53 status.
    • Quantitative image analysis of microbial communities with BiofilmQ.

      Hartmann, Raimo; Jeckel, Hannah; Jelli, Eric; Singh, Praveen K; Vaidya, Sanika; Bayer, Miriam; Rode, Daniel K H; Vidakovic, Lucia; Díaz-Pascual, Francisco; Fong, Jiunn C N; et al. (Nature research, 2021-01-04)
      Biofilms are microbial communities that represent a highly abundant form of microbial life on Earth. Inside biofilms, phenotypic and genotypic variations occur in three-dimensional space and time; microscopy and quantitative image analysis are therefore crucial for elucidating their functions. Here, we present BiofilmQ-a comprehensive image cytometry software tool for the automated and high-throughput quantification, analysis and visualization of numerous biofilm-internal and whole-biofilm properties in three-dimensional space and time.
    • Combined high-throughput library screening and next generation RNA sequencing uncover microRNAs controlling human cardiac fibroblast biology

      Schimmel, Katharina; Stojanović, Stevan D.; Huang, Cheng Kai; Jung, Mira; Meyer, Martin H.; Xiao, Ke; Grote-Levi, Lea; Bär, Christian; Pfanne, Angelika; Mitzka, Saskia; et al. (Elsevier, 2021-01-01)
      Background: Myocardial fibrosis is a hallmark of the failing heart, contributing to the most common causes of deaths worldwide. Several microRNAs (miRNAs, miRs) controlling cardiac fibrosis were identified in recent years; however, a more global approach to identify miRNAs involved in fibrosis is missing. Methods and results: Functional miRNA mimic library screens were applied in human cardiac fibroblasts (HCFs) to identify annotated miRNAs inducing proliferation. In parallel, miRNA deep sequencing was performed after subjecting HCFs to proliferating and resting stimuli, additionally enabling discovery of novel miRNAs. In-depth in vitro analysis confirmed the pro-fibrotic nature of selected, highly conserved miRNAs miR-20a-5p and miR-132-3p. To determine downstream cellular pathways and their role in the fibrotic response, targets of the annotated miRNA candidates were modulated by synthetic siRNA. We here provide evidence that repression of autophagy and detoxification of reactive oxygen species by miR-20a-5p and miR-132-3p explain some of their pro-fibrotic nature on a mechanistic level. Conclusion: We here identified both miR-20a-5p and miR-132-3p as crucial regulators of fibrotic pathways in an in vitro model of human cardiac fibroblast biology.
    • Simultaneous Presence of Bacteriochlorophyll and Xanthorhodopsin Genes in a Freshwater Bacterium.

      Kopejtka, Karel; Tomasch, Jürgen; Zeng, Yonghui; Selyanin, Vadim; Dachev, Marko; Piwosz, Kasia; Tichý, Martin; Bína, David; Gardian, Zdenko; Bunk, Boyke; et al. (ASM, 2020-12-22)
      Photoheterotrophic bacteria represent an important part of aquatic microbial communities. There exist two fundamentally different light-harvesting systems: bacteriochlorophyll-containing reaction centers or rhodopsins. Here, we report a photoheterotrophic Sphingomonas strain isolated from an oligotrophic lake, which contains complete sets of genes for both rhodopsin-based and bacteriochlorophyll-based phototrophy. Interestingly, the identified genes were not expressed when cultured in liquid organic media. Using reverse transcription quantitative PCR (RT-qPCR), RNA sequencing, and bacteriochlorophyll a quantification, we document that bacteriochlorophyll synthesis was repressed by high concentrations of glucose or galactose in the medium. Coactivation of photosynthesis genes together with genes for TonB-dependent transporters suggests the utilization of light energy for nutrient import. The photosynthetic units were formed by ring-shaped light-harvesting complex 1 and reaction centers with bacteriochlorophyll a and spirilloxanthin as the main light-harvesting pigments. The identified rhodopsin gene belonged to the xanthorhodopsin family, but it lacks salinixanthin antenna. In contrast to bacteriochlorophyll, the expression of xanthorhodopsin remained minimal under all experimental conditions tested. Since the gene was found in the same operon as a histidine kinase, we propose that it might serve as a light sensor. Our results document that photoheterotrophic Sphingomonas bacteria use the energy of light under carbon-limited conditions, while under carbon-replete conditions, they cover all their metabolic needs through oxidative phosphorylation.IMPORTANCE Phototrophic organisms are key components of many natural environments. There exist two main phototrophic groups: species that collect light energy using various kinds of (bacterio)chlorophylls and species that utilize rhodopsins. Here, we present a freshwater bacterium Sphingomonas sp. strain AAP5 which contains genes for both light-harvesting systems. We show that bacteriochlorophyll-based reaction centers are repressed by light and/or glucose. On the other hand, the rhodopsin gene was not expressed significantly under any of the experimental conditions. This may indicate that rhodopsin in Sphingomonas may have other functions not linked to bioenergetics.
    • Germline variation of Ribonuclease H2 genes in ovarian cancer patients.

      Polaczek, Rahel; Schürmann, Peter; Speith, Lisa-Marie; Geffers, Robert; Dürst, Matthias; Hillemanns, Peter; Park-Simon, Tjoung-Won; Liebrich, Clemens; Dörk, Thilo; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (BMC, 2020-12-22)
      Epithelial ovarian carcinoma (EOC) is a genetically heterogeneous disease that is partly driven by molecular defects in mismatch repair (MMR) or homology-directed DNA repair (HDR). Ribonuclease H2 serves to remove mis-incorporated ribonucleotides from DNA which alleviates HDR mechanisms and guides the MMR machinery. Although Ribonuclease H2 has been implicated in cancer, the role of germline variants for ovarian cancer is unknown. In the present case-control study, we sequenced the coding and flanking untranslated regions of the RNASEH2A, RNASEH2B and RNASEH2C genes, encoding all three subunits of Ribonuclease H2, in a total of 602 German patients with EOC and of 940 healthy females from the same population. We identified one patient with a truncating variant in RNASEH2B, p.C44X, resulting in a premature stop codon. This patient had high-grade serous EOC with an 8 years survival after platinum/taxane-based therapy. Subsequent analysis of TCGA data similarly showed a significantly longer progression-free survival in ovarian cancer patients with low RNASEH2B or RNASEH2C expression levels. In conclusion, loss-of-function variants in Ribonuclease H2 genes are not common predisposing factors in ovarian cancer but the possibility that they modulate therapeutic platinum response deserves further investigation.
    • Determining the effects of trastuzumab, cetuximab and afatinib by phosphoprotein, gene expression and phenotypic analysis in gastric cancer cell lines.

      Ebert, Karolin; Zwingenberger, Gwen; Barbaria, Elena; Keller, Simone; Heck, Corinna; Arnold, Rouven; Hollerieth, Vanessa; Mattes, Julian; Geffers, Robert; Raimúndez, Elba; et al. (BMC, 2020-10-28)
      Background: Gastric cancer is the fifth most frequently diagnosed cancer and the third leading cause of cancer death worldwide. The molecular mechanisms of action for anti-HER-family drugs in gastric cancer cells are incompletely understood. We compared the molecular effects of trastuzumab and the other HER-family targeting drugs cetuximab and afatinib on phosphoprotein and gene expression level to gain insights into the regulated pathways. Moreover, we intended to identify genes involved in phenotypic effects of anti-HER therapies. Methods: A time-resolved analysis of downstream intracellular kinases following EGF, cetuximab, trastuzumab and afatinib treatment was performed by Luminex analysis in the gastric cancer cell lines Hs746T, MKN1, MKN7 and NCI-N87. The changes in gene expression after treatment of the gastric cancer cell lines with EGF, cetuximab, trastuzumab or afatinib for 4 or 24 h were analyzed by RNA sequencing. Significantly enriched pathways and gene ontology terms were identified by functional enrichment analysis. Furthermore, effects of trastuzumab and afatinib on cell motility and apoptosis were analyzed by time-lapse microscopy and western blot for cleaved caspase 3. Results: The Luminex analysis of kinase activity revealed no effects of trastuzumab, while alterations of AKT1, MAPK3, MEK1 and p70S6K1 activations were observed under cetuximab and afatinib treatment. On gene expression level, cetuximab mainly affected the signaling pathways, whereas afatinib had an effect on both signaling and cell cycle pathways. In contrast, trastuzumab had little effects on gene expression. Afatinib reduced average speed in MKN1 and MKN7 cells and induced apoptosis in NCI-N87 cells. Following treatment with afatinib, a list of 14 genes that might be involved in the decrease of cell motility and a list of 44 genes that might have a potential role in induction of apoptosis was suggested. The importance of one of these genes (HBEGF) as regulator of motility was confirmed by knockdown experiments. Conclusions: Taken together, we described the different molecular effects of trastuzumab, cetuximab and afatinib on kinase activity and gene expression. The phenotypic changes following afatinib treatment were reflected by altered biological functions indicated by overrepresentation of gene ontology terms. The importance of identified genes for cell motility was validated in case of HBEGF.
    • Identification of miRNAs associated with dendritic cell dysfunction during Acute and Chronic Hepatitis B virus infection.

      Singh, Avishek Kumar; Rooge, Sheetalnath Babasaheb; Varshney, Aditi; Vasudevan, Madavan; Kumar, Manoj; Geffers, Robert; Kumar, Vijay; Sarin, Shiv Kumar; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-10-27)
      The uptake or expression of hepatitis B virus proteins by Dendritic cells (DCs) is considered important for disease outcome. Differential expression of microRNA may have a role in viral persistence and hepatocellular injury. The miRNA expression was investigated by microarray in DCs from different stages of HBV infection and liver disease viz., immune active (IA; n=20); low replicative (LR; n=20); HBeAg negative (n=20); acute viral hepatitis (AVH, n=20) and healthy controls (n=20). miRNA levels were analyzed by unsupervised hierarchical clustering and principal component analyses and validated by qPCR. The miRNA-mRNA regulatory networks identified 19 miRNAs and 12 target gene interactions in MHC and other immune pathways. miR-2278, miR-615-3p and miR-3681-3p were down-regulated in IA group compared to healthy control, miR-152-3p and miR-3613-3p in LR group compared to IA group and miR-152-3p and miR-503-3p in HBe negative compared to LR group. However, miR-7-1-1-3p, miR-192-5p, miR-195-5p and miR-32-5p in LR, miR-342-3p and miR-940 in HBe negative, and miR-34a-5p, miR-130b-3p, miR-221-3p, miR-320a, miR-324-5p and miR-484 in AVH were up-regulated. Further, qPCR confirmed changes in miRNA levels and their target genes associated with antigen processing and presentation. Thus, a deregulated network of miRNAs-mRNAs in DCs seems responsible for impaired immune response during HBV pathogenesis. This article is protected by copyright. All rights reserved.
    • Analysis of the organization and expression patterns of the convergent pseudomonas aeruginosa lasr/rsal gene pair uncovers mutual influence.

      Schinner, Silvia; Preusse, Matthias; Kesthely, Christopher; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-10-19)
      The two adjacent genes encoding the major Pseudomonas aeruginosa quorum sensing regulator, LasR, and its opponent, RsaL, overlap in their coding 3´ends and produce mRNA transcripts with long untranslated 3´ends that overlap with the sense transcripts of the gene on the opposing DNA strand. In this study, we evaluated whether the overlapping genes are involved in mutual regulatory events and studied interference by natural antisense transcripts. We introduced various gene expression constructs into a P. aeruginosa PA14 lasR/rsaL double deletion mutant, and found that although complementary RNA is produced, this does not interfere with the sense gene expression levels of lasR and rsaL and does not have functional consequences on down-stream gene regulation. Nevertheless, expression of lasR, but not of rsaL, was shown to be enhanced if transcription was terminated at the end of the respective gene so that no overlapping transcription was allowed. Our data indicate that the natural organization with a partial overlap at the 3´ends of the lasR/rsaL genes gives rise to a system of checks and balances to prevent dominant and unilateral control by LasR over the RsaL transcriptional regulator of opposing function.
    • Mining zebrafish microbiota reveals key community-level resistance against fish pathogen infection.

      Stressmann, Franziska A; Bernal-Bayard, Joaquín; Perez-Pascual, David; Audrain, Bianca; Rendueles, Olaya; Briolat, Valérie; Bruchmann, Sebastian; Volant, Stevenn; Ghozlane, Amine; Häussler, Susanne; et al. (Springer Nature, 2020-10-19)
      The long-known resistance to pathogens provided by host-associated microbiota fostered the notion that adding protective bacteria could prevent or attenuate infection. However, the identification of endogenous or exogenous bacteria conferring such protection is often hindered by the complexity of host microbial communities. Here, we used zebrafish and the fish pathogen Flavobacterium columnare as a model system to study the determinants of microbiota-associated colonization resistance. We compared infection susceptibility in germ-free, conventional and reconventionalized larvae and showed that a consortium of 10 culturable bacterial species are sufficient to protect zebrafish. Whereas survival to F. columnare infection does not rely on host innate immunity, we used antibiotic dysbiosis to alter zebrafish microbiota composition, leading to the identification of two different protection strategies. We first identified that the bacterium Chryseobacterium massiliae individually protects both larvae and adult zebrafish. We also showed that an assembly of 9 endogenous zebrafish species that do not otherwise protect individually confer a community-level resistance to infection. Our study therefore provides a rational approach to identify key endogenous protecting bacteria and promising candidates to engineer resilient microbial communities. It also shows how direct experimental analysis of colonization resistance in low-complexity in vivo models can reveal unsuspected ecological strategies at play in microbiota-based protection against pathogens.
    • The Peptide Chain Release Factor Methyltransferase PrmC Influences the Pseudomonas aeruginosa PA14 Endo- and Exometabolome.

      Depke, Tobias; Häussler, Susanne; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-10-18)
      Pseudomonas aeruginosa is one of the most important nosocomial pathogens and understanding its virulence is the key to effective control of P. aeruginosa infections. The regulatory network governing virulence factor production in P. aeruginosa is exceptionally complex. Previous studies have shown that the peptide chain release factor methyltransferase PrmC plays an important role in bacterial pathogenicity. Yet, the underlying molecular mechanism is incompletely understood. In this study, we used untargeted liquid and gas chromatography coupled to mass spectrometry to characterise the metabolome of a prmC defective P. aeruginosa PA14 strain in comparison with the corresponding strain complemented with prmC in trans. The comprehensive metabolomics data provided new insight into the influence of prmC on virulence and metabolism. prmC deficiency had broad effects on the endo- and exometabolome of P. aeruginosa PA14, with a marked decrease of the levels of aromatic compounds accompanied by reduced precursor supply from the shikimate pathway. Furthermore, a pronounced decrease of phenazine production was observed as well as lower abundance of alkylquinolones. Unexpectedly, the metabolomics data showed no prmC-dependent effect on rhamnolipid production and an increase in pyochelin levels. A putative virulence biomarker identified in a previous study was significantly less abundant in the prmC deficient strain.
    • Expression of the MexXY aminoglycoside efflux pump and presence of an aminoglycoside modifying enzyme in clinical isolates are highly correlated.

      Seupt, Alexander; Schniederjans, Monika; Tomasch, Jürgen; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (ASM, 2020-10-12)
      The impact of MexXY efflux pump expression on aminoglycoside resistance in clinical Pseudomonas aeruginosa isolates has been debated. In this study, we found that in general, elevated mexXY gene expression levels in clinical P. aeruginosa isolates confer to slight increases in aminoglycoside MIC levels, however those levels rarely lead to clinically relevant resistance phenotypes. The main driver of resistance in the clinical isolates studied here was the acquisition of aminoglycoside modifying enzymes (AMEs). Nevertheless, acquisition of an AME was strongly associated with mexY overexpression. In line with this observation, we demonstrate that the introduction of a gentamicin acetyl-transferase confers to full gentamicin resistance levels in a P. aeruginosa type strain only if the MexXY efflux pump was active. We discuss that increased mexXY activity in clinical AME harboring P. aeruginosa isolates might affect ion fluxes at the bacterial cell membrane and thus might play a role in the establishment of enhanced fitness that extends beyond aminoglycoside resistance.
    • YB-1 Interferes with TNFα-TNFR Binding and Modulates Progranulin-Mediated Inhibition of TNFα Signaling.

      Hessman, Christopher L; Hildebrandt, Josephine; Shah, Aneri; Brandt, Sabine; Bock, Antonia; Frye, Björn C; Raffetseder, Ute; Geffers, Robert; Brunner-Weinzierl, Monika C; Isermann, Berend; et al. (MDPI, 2020-09-25)
      Inflammation and an influx of macrophages are common elements in many diseases. Among pro-inflammatory cytokines, tumor necrosis factor α (TNFα) plays a central role by amplifying the cytokine network. Progranulin (PGRN) is a growth factor that binds to TNF receptors and interferes with TNFα-mediated signaling. Extracellular PGRN is processed into granulins by proteases released from immune cells. PGRN exerts anti-inflammatory effects, whereas granulins are pro-inflammatory. The factors coordinating these ambivalent functions remain unclear. In our study, we identify Y-box binding protein-1 (YB-1) as a candidate for this immune-modulating activity. Using a yeast-2-hybrid assay with YB-1 protein as bait, clones encoding for progranulin were selected using stringent criteria for strong interaction. We demonstrate that at physiological concentrations, YB-1 interferes with the binding of TNFα to its receptors in a dose-dependent manner using a flow cytometry-based binding assay. We show that YB-1 in combination with progranulin interferes with TNFα-mediated signaling, supporting the functionality with an NF-κB luciferase reporter assay. Together, we show that YB-1 displays immunomodulating functions by affecting the binding of TNFα to its receptors and influencing TNFα-mediated signaling via its interaction with progranulin.
    • Host-induced spermidine production in motile triggers phagocytic uptake.

      Felgner, Sebastian; Preusse, Matthias; Beutling, Ulrike; Stahnke, Stephanie; Pawar, Vinay; Rohde, Manfred; Brönstrup, Mark; Stradal, Theresia; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (elifeSciences, 2020-09-22)
      Exploring the complexity of host-pathogen communication is vital to understand why microbes persist within a host, while others are cleared. Here, we employed a dual-sequencing approach to unravel conversational turn-taking of dynamic host-pathogen communications. We demonstrate that upon hitting a host cell, motile Pseudomonas aeruginosa induce a specific gene expression program. This results in the expression of spermidine on the surface, which specifically activates the PIP3-pathway to induce phagocytic uptake into primary or immortalized murine cells. Non-motile bacteria are more immunogenic due to a lower expression of arnT upon host-cell contact, but do not produce spermidine and are phagocytosed less. We demonstrate that not only the presence of pathogen inherent molecular patterns induces immune responses, but that bacterial motility is linked to a host-cell-induced expression of additional immune modulators. Our results emphasize on the value of integrating microbiological and immunological findings to unravel complex and dynamic host-pathogen interactions.
    • Organism-specific depletion of highly abundant RNA species from bacterial total RNA.

      Engelhardt, Florian; Tomasch, Jürgen; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Microbiology Society, 2020-09-09)
      High-throughput sequencing has become a standard tool for transcriptome analysis. The depletion of overrepresented RNA species from sequencing libraries plays a key role in establishing potent and cost-efficient RNA-seq routines. Commercially available kits are known to obtain good results for the reduction of ribosomal RNA (rRNA). However, we found that the transfer-messenger RNA (tmRNA) was frequently highly abundant in rRNA-depleted samples of Pseudomonas aeruginosa , consuming up to 25 % of the obtained reads. The tmRNA fraction was particularly high in samples taken from stationary cultures. This suggests that overrepresentation of this RNA species reduces the mRNA fraction when cells are grown under challenging conditions. Here, we present an RNase-H-based depletion protocol that targets the tmRNA in addition to ribosomal RNAs. We were able to increase the mRNA fraction to 93-99% and therefore outperform not only the commercially Ribo-off kit (Vazyme) operating by the same principle but also the formerly widely used Ribo-Zero kit (Illumina). Maximizing the read share of scientifically interesting RNA species enhances the discriminatory potential of next-generation RNA-seq experiments and, therefore, can contribute to a better understanding of the transcriptomic landscape of bacterial pathogens and their used mechanisms in host infection.
    • Complete Genome Sequence and Manual Reannotation of Mycobacterium avium subsp. Strain DSM 44135.

      Goethe, Ralph; Basler, Tina; Meissner, Thorsten; Goethe, Elke; Spröer, Cathrin; Swiderski, Jolantha; Gerlach, Gerald-F; Weiss, Siegfried; Jarek, Michael; Bunk, Boyke; et al. (ASM, 2020-08-13)
      Here, we report the complete genome sequence of the Mycobacterium avium subsp. paratuberculosis reference strain DSM 44135, amended with a manual genome reannotation. The strain was originally described as M. paratuberculosis strain 6783. It was isolated from feces from a dairy cow in northern Germany.
    • Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

      Schulte-Schrepping, Jonas; Reusch, Nico; Paclik, Daniela; Baßler, Kevin; Schlickeiser, Stephan; Zhang, Bowen; Krämer, Benjamin; Krammer, Tobias; Brumhard, Sophia; Bonaguro, Lorenzo; et al. (Elsevier /Cell Press), 2020-08-05)
      Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.