• Critical Role of Zur and SmtB in Zinc Homeostasis of Mycobacterium smegmatis.

      Goethe, Elke; Laarmann, Kristin; Lührs, Janita; Jarek, Michael; Meens, Jochen; Lewin, Astrid; Goethe, Ralph (2020-04-21)
      Zinc homeostasis is crucial for bacterial cells, since imbalances affect viability. However, in mycobacteria, knowledge of zinc metabolism is incomplete. Mycobacterium smegmatis (MSMEG) is an environmental, nonpathogenic Mycobacterium that is widely used as a model organism to study mycobacterial metabolism and pathogenicity. How MSMEG maintains zinc homeostasis is largely unknown. SmtB and Zur are important regulators of bacterial zinc metabolism. In mycobacteria, these regulators are encoded by an operon, whereas in other bacterial species, SmtB and Zur are encoded on separate loci. Here, we show that the smtB-zur operon is consistently present within the genus Mycobacterium but otherwise found only in Nocardia, Saccharothrix, and Corynebacterium diphtheriae By RNA deep sequencing, we determined the Zur and SmtB regulons of MSMEG and compared them with transcriptional responses after zinc starvation or excess. We found an exceptional genomic clustering of genes whose expression was strongly induced by zur deletion and zinc starvation. These genes encoded zinc importers such as ZnuABC and three additional putative zinc transporters, including the porin MspD, as well as alternative ribosomal proteins. In contrast, only a few genes were affected by deletion of smtB and zinc excess. The zinc exporter ZitA was most prominently regulated by SmtB. Moreover, transcriptional analyses in combination with promoter and chromatin immunoprecipitation assays revealed a special regulation of the smtB-zur operon itself: an apparently zinc-independent, constitutive expression of smtB-zur resulted from sensitive coregulation by both SmtB and Zur. Overall, our data revealed yet unknown peculiarities of mycobacterial zinc homeostasis.IMPORTANCE Zinc is crucial for many biological processes, as it is an essential cofactor of enzymes and a structural component of regulatory and DNA binding proteins. Hence, all living cells require zinc to maintain constant intracellular levels. However, in excess, zinc is toxic. Therefore, cellular zinc homeostasis needs to be tightly controlled. In bacteria, this is achieved by transcriptional regulators whose activity is mediated via zinc-dependent conformational changes promoting or preventing their binding to DNA. SmtB and Zur are important antagonistically acting bacterial regulators in mycobacteria. They sense changes in zinc concentrations in the femtomolar range and regulate transcription of genes for zinc acquisition, storage, and export. Here, we analyzed the role of SmtB and Zur in zinc homeostasis in Mycobacterium smegmatis Our results revealed novel insights into the transcriptional processes of zinc homeostasis in mycobacteria and their regulation.
    • Predicting antimicrobial resistance in Pseudomonas aeruginosa with machine learning‐enabled molecular diagnostics

      Khaledi, Ariane; Weimann, Aaron; Schniederjans, Monika; Asgari, Ehsaneddin; Kuo, Tzu‐Hao; Oliver, Antonio; Cabot, Gabriel; Kola, Axel; Gastmeier, Petra; Hogardt, Michael; et al. (EMBO, 2020-02-12)
      Limited therapy options due to antibiotic resistance underscore the need for optimization of current diagnostics. In some bacterial species, antimicrobial resistance can be unambiguously predicted based on their genome sequence. In this study, we sequenced the genomes and transcriptomes of 414 drug-resistant clinical Pseudomonas aeruginosa isolates. By training machine learning classifiers on information about the presence or absence of genes, their sequence variation, and expression profiles, we generated predictive models and identified biomarkers of resistance to four commonly administered antimicrobial drugs. Using these data types alone or in combination resulted in high (0.8–0.9) or very high (> 0.9) sensitivity and predictive values. For all drugs except for ciprofloxacin, gene expression information improved diagnostic performance. Our results pave the way for the development of a molecular resistance profiling tool that reliably predicts antimicrobial susceptibility based on genomic and transcriptomic markers. The implementation of a molecular susceptibility test system in routine microbiology diagnostics holds promise to provide earlier and more detailed information on antibiotic resistance profiles of bacterial pathogens and thus could change how physicians treat bacterial infections.
    • Fibrosis and Immune Cell Infiltration Are Separate Events Regulated by Cell-Specific Receptor Notch3 Expression.

      Brandt, Sabine; Ballhause, Tobias M; Bernhardt, Anja; Becker, Annika; Salaru, Delia; Le-Deffge, Hien Minh; Fehr, Alexander; Fu, Yan; Philipsen, Lars; Djudjaj, Sonja; et al. (2020-08-28)
      Kidney injuries that result in chronic inflammation initiate crosstalk between stressed resident cells and infiltrating immune cells. In animal models, whole-body receptor Notch3 deficiency protects from leukocyte infiltration and organ fibrosis. However, the relative contribution of Notch3 expression in tissue versus infiltrating immune cells is unknown.
    • Non-Typeable Invade Choroid Plexus Epithelial Cells in a Polar Fashion.

      Wegele, Christian; Stump-Guthier, Carolin; Moroniak, Selina; Weiss, Christel; Rohde, Manfred; Ishikawa, Hiroshi; Schroten, Horst; Schwerk, Christian; Karremann, Michael; Borkowski, Julia (MDPI, 2020-08-10)
      Non-typeable Haemophilus influenzae (NTHI) is a pathogen of the human respiratory tract causing the majority of invasive H. influenzae infections. Severe invasive infections such as septicemia and meningitis occur rarely, but the lack of a protecting vaccine and the increasing antibiotic resistance of NTHI impede treatment and emphasize its relevance as a potential meningitis causing pathogen. Meningitis results from pathogens crossing blood-brain barriers and invading the immune privileged central nervous system (CNS). In this study, we addressed the potential of NTHI to enter the brain by invading cells of the choroid plexus (CP) prior to meningeal inflammation to enlighten NTHI pathophysiological mechanisms. A cell culture model of human CP epithelial cells, which form the blood-cerebrospinal fluid barrier (BCSFB) in vivo, was used to analyze adhesion and invasion by immunofluorescence and electron microscopy. NTHI invade CP cells in vitro in a polar fashion from the blood-facing side. Furthermore, NTHI invasion rates are increased compared to encapsulated HiB and HiF strains. Fimbriae occurrence attenuated adhesion and invasion. Thus, our findings underline the role of the BCSFB as a potential entry port for NTHI into the brain and provide strong evidence for a function of the CP during NTHI invasion into the CNS during the course of meningitis.
    • Crypt residing bacteria and proximal colonic carcinogenesis in a mouse model of Lynch syndrome.

      Lang, Michaela; Baumgartner, Maximilian; Rożalska, Aleksandra; Frick, Adrian; Riva, Alessandra; Jarek, Michael; Berry, David; Gasche, Christoph (Wiley, 2020-05-18)
      Colorectal cancer is a multifactorial disease involving inherited DNA mutations, environmental factors, gut inflammation and intestinal microbiota. Certain germline mutations within the DNA mismatch repair system are associated with Lynch syndrome tumors including right-sided colorectal cancer with mucinous phenotype and presence of an inflammatory infiltrate. Such tumors are more often associated with bacterial biofilms, which may contribute to disease onset and progression. Inflammatory bowel diseases are also associated with colorectal cancer and intestinal dysbiosis. Herein we addressed the question, whether inflammation can aggravate colorectal cancer development under mismatch repair deficiency. MSH2loxP/loxP Vill-cre mice were crossed into the IL-10-/- background to study the importance of inflammation and mucosal bacteria as a driver of tumorigenesis in a Lynch syndrome mouse model. An increase in large bowel tumorigenesis was found in double knockout mice both under conventional housing and under specific pathogen-free conditions. This increase was mostly due to the development of proximal tumors, a hotspot for tumorigenesis in Lynch syndrome, and was associated with a higher degree of inflammation. Additionally, bacterial invasion into the mucus of tumor crypts was observed in the proximal tumors. Inflammation shifted fecal and mucosal microbiota composition and was associated with enrichment in Escherichia-Shigella as well as Akkermansia, Bacteroides and Parabacteroides genera in fecal samples. Tumor-bearing double knockout mice showed a similar enrichment for Escherichia-Shigella and Parabacteroides. Lactobacilli, Lachnospiraceae and Muribaculaceae family members were depleted upon inflammation. In summary, chronic inflammation aggravates colonic tumorigenesis under mismatch repair deficiency and is associated with a shift in microbiota composition.
    • Itaconate and derivatives reduce interferon responses and inflammation in influenza A virus infection.

      Sohail, Aaqib; Iqbal, Azeem A; Sahini, Nishika; Chen, Fangfang; Tantawy, Mohamed; Waqas, Fakhar; Winterhoff, Moritz; Ebensen, Thomas; Schultz, Kristin; Geffers, Robert; et al. (PLOS, 2022-01-13)
      Excessive inflammation is a major cause of morbidity and mortality in many viral infections including influenza. Therefore, there is a need for therapeutic interventions that dampen and redirect inflammatory responses and, ideally, exert antiviral effects. Itaconate is an immunomodulatory metabolite which also reprograms cell metabolism and inflammatory responses when applied exogenously. We evaluated effects of endogenous itaconate and exogenous application of itaconate and its variants dimethyl- and 4-octyl-itaconate (DI, 4OI) on host responses to influenza A virus (IAV). Infection induced expression of ACOD1, the enzyme catalyzing itaconate synthesis, in monocytes and macrophages, which correlated with viral replication and was abrogated by DI and 4OI treatment. In IAV-infected mice, pulmonary inflammation and weight loss were greater in Acod1-/- than in wild-type mice, and DI treatment reduced pulmonary inflammation and mortality. The compounds reversed infection-triggered interferon responses and modulated inflammation in human cells supporting non-productive and productive infection, in peripheral blood mononuclear cells, and in human lung tissue. Itaconates reduced ROS levels and STAT1 phosphorylation, whereas AKT phosphorylation was reduced by 4OI and DI but increased by itaconate. Single-cell RNA sequencing identified monocytes as the main target of infection and the exclusive source of ACOD1 mRNA in peripheral blood. DI treatment silenced IFN-responses predominantly in monocytes, but also in lymphocytes and natural killer cells. Ectopic synthesis of itaconate in A549 cells, which do not physiologically express ACOD1, reduced infection-driven inflammation, and DI reduced IAV- and IFNγ-induced CXCL10 expression in murine macrophages independent of the presence of endogenous ACOD1. The compounds differed greatly in their effects on cellular gene homeostasis and released cytokines/chemokines, but all three markedly reduced release of the pro-inflammatory chemokines CXCL10 (IP-10) and CCL2 (MCP-1). Viral replication did not increase under treatment despite the dramatically repressed IFN responses. In fact, 4OI strongly inhibited viral transcription in peripheral blood mononuclear cells, and the compounds reduced viral titers (4OI>Ita>DI) in A549 cells whereas viral transcription was unaffected. Taken together, these results reveal itaconates as immunomodulatory and antiviral interventions for influenza virus infection.
    • Inhibition of MCL1 induces apoptosis in anaplastic large cell lymphoma and in primary effusion lymphoma.

      Quentmeier, Hilmar; Geffers, Robert; Hauer, Vivien; Nagel, Stefan; Pommerenke, Claudia; Uphoff, Cord C; Zaborski, Margarete; Drexler, Hans G; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Nature, 2022-01-20)
      Overexpression of antiapoptotic BCL2 family proteins occurs in various hematologic malignancies and contributes to tumorigenesis by inhibiting the apoptotic machinery of the cells. Antagonizing BH3 mimetics provide an option for medication, with venetoclax as the first drug applied for chronic lymphocytic leukemia and for acute myeloid leukemia. To find additional hematologic entities with ectopic expression of BCL2 family members, we performed expression screening of cell lines applying the LL-100 panel. Anaplastic large cell lymphoma (ALCL) and primary effusion lymphoma (PEL), 2/22 entities covered by this panel, stood out by high expression of MCL1 and low expression of BCL2. The MCL1 inhibitor AZD-5991 induced apoptosis in cell lines from both malignancies, suggesting that this BH3 mimetic might be efficient as drug for these diseases. The ALCL cell lines also expressed BCLXL and BCL2A1, both contributing to survival of the cells. The combination of specific BH3 mimetics yielded synergistic effects, pointing to a novel strategy for the treatment of ALCL. The PI3K/mTOR inhibitor BEZ-235 could also efficiently be applied in combination with AZD-5991, offering an alternative to avoid thrombocytopenia which is associated with the use of BCLXL inhibitors.
    • SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis.

      Wendisch, Daniel; Dietrich, Oliver; Mari, Tommaso; von Stillfried, Saskia; Ibarra, Ignacio L; Mittermaier, Mirja; Mache, Christin; Chua, Robert Lorenz; Knoll, Rainer; Timm, Sara; et al. (Cell Press, 2021-11-27)
      COVID-19-induced "acute respiratory distress syndrome" (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.
    • Orthodontic Forced Eruption of Permanent Anterior Teeth with Subgingival Fractures: A Systematic Review.

      Reichardt, Elisabeth; Krug, Ralf; Bornstein, Michael M; Tomasch, Jürgen; Verna, Carlalberta; Krastl, Gabriel; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2021-11-29)
      (1) Background: To assess orthodontic forced eruption (OFE) as a pre-restorative procedure for non-restorable permanent teeth with subgingival dental hard tissue defects after dental trauma. (2) Methods: A systematic electronic search of three databases, namely, MEDLINE, Cochrane Library, and EMBASE, revealed a total of 2757 eligible publications. Randomized controlled clinical trials (RCT), retro- and prospective clinical studies, or case series (with a minimum of three patients) were reviewed. (3) Results: Thirteen full-text papers were included: one RCT, one prospective clinical trial, two retrospective cohort studies, and nine case series. Within case series, statistical significance between age and cause of fracture (p < 0.03) was determined. The mean extrusion rate of OFE was 1.5 mm a week within a four to six weeks treatment period followed by retention. Three OFE protocols for maxillary single teeth are available: 1. OFE without migration of gingiva and alveolar bone, 2. OFE with gingival migration and slight alveolar bone migration, and 3. OFE with migration of both gingiva and alveolar bone. (4) Conclusions: The current state of the evidence suggests that OFE is a feasible pre-treatment option for non-restorable permanent teeth. OFE can promote the migration of tooth surrounding hard and soft tissues in the esthetic zone. Root resorption does not seem to be a relevant side effect of OFE.
    • Interaction of myxobacteria-derived outer membrane vesicles with biofilms: antiadhesive and antibacterial effects.

      Goes, Adriely; Vidakovic, Lucia; Drescher, Knut; Fuhrmann, Gregor; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany.; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Royal Society of Chemistry, 2021-08-02)
      Bacterial biofilms are widespread in nature and in medical settings and display a high tolerance to antibiotics and disinfectants. Extracellular vesicles have been increasingly studied to characterise their origins and assess their potential for use as a versatile drug delivery system; however, it remains unclear whether they also have antibiofilm effects. Outer membrane vesicles are lipid vesicles shed by Gram-negative bacteria and, in the case of myxobacteria, carry natural antimicrobial compounds produced by these microorganisms. In this study, we demonstrate that vesicles derived from the myxobacteria Cystobacter velatus Cbv34 and Cystobacter ferrugineus Cbfe23 are highly effective at inhibiting the formation and disrupting biofilms by different bacterial species.
    • Congenital deficiency reveals critical role of ISG15 in skin homeostasis.

      Malik, Muhammad Nasir Hayat; Waqas, Syed F Hassnain; Zeitvogel, Jana; Cheng, Jingyuan; Geffers, Robert; Gouda, Zeinab Abu-Elbaha; Elsaman, Ahmed Mahrous; Radwan, Ahmed R; Schefzyk, Matthias; Braubach, Peter; et al. (Society of clinical investigation, 2021-11-30)
      Ulcerating skin lesions are manifestations of human ISG15 deficiency, a type I interferonopathy. However, chronic inflammation may not be their exclusive cause. We describe two siblings with recurrent skin ulcers that healed with scar formation upon corticosteroid treatment. Both had a homozygous nonsense mutation in the ISG15 gene, leading to unstable ISG15 protein lacking the functional domain. We characterized ISG15-/- dermal fibroblasts, HaCaT keratinocytes, and human induced pluripotent stem cell-derived vascular endothelial cells. ISG15-deficient cells exhibited the expected hyperinflammatory phenotype, but also dysregulated expression of molecules critical for connective tissue and epidermis integrity, including reduced collagens and adhesion molecules, but increased matrix metalloproteases. ISG15-/- fibroblasts exhibited elevated ROS levels and reduced ROS scavenger expression. As opposed to hyperinflammation, defective collagen and integrin synthesis was not rescued by conjugation-deficient ISG15. Cell migration was retarded in ISG15-/- fibroblasts and HaCaT keratinocytes, but normalized under ruxolitinib treatment. Desmosome density was reduced in an ISG15-/- 3D epidermis model. Additionally, there were loose architecture and reduced collagen and desmoglein expression, which could be reversed by treatment with ruxolitinib/doxycycline/TGF-β1. These results reveal critical roles of ISG15 in maintaining cell migration and epidermis and connective tissue homeostasis, whereby the latter likely requires its conjugation to yet unidentified targets.
    • Dysregulated Immunometabolism Is Associated with the Generation of Myeloid-Derived Suppressor Cells in Staphylococcus aureus Chronic Infection.

      Dietrich, Oliver; Heinz, Alexander; Goldmann, Oliver; Geffers, Robert; Beineke, Andreas; Hiller, Karsten; Saliba, Antoine-Emmanuel; Medina, Eva; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Karger, 2021-11-11)
      Myeloid-derived suppressor cells (MDSCs) are a compendium of immature myeloid cells that exhibit potent T-cell suppressive capacity and expand during pathological conditions such as cancer and chronic infections. Although well-characterized in cancer, the physiology of MDSCs in the infection setting remains enigmatic. Here, we integrated single-cell RNA sequencing (scRNA-seq) and functional metabolic profiling to gain deeper insights into the factors governing the generation and maintenance of MDSCs in chronic Staphylococcus aureus infection. We found that MDSCs originate not only in the bone marrow but also at extramedullary sites in S. aureus-infected mice. scRNA-seq showed that infection-driven MDSCs encompass a spectrum of myeloid precursors in different stages of differentiation, ranging from promyelocytes to mature neutrophils. Furthermore, the scRNA-seq analysis has also uncovered valuable phenotypic markers to distinguish mature myeloid cells from immature MDSCs. Metabolic profiling indicates that MDSCs exhibit high glycolytic activity and high glucose consumption rates, which are required for undergoing terminal maturation. However, rapid glucose consumption by MDSCs added to infection-induced perturbations in the glucose supplies in infected mice hinders the terminal maturation of MDSCs and promotes their accumulation in an immature stage. In a proof-of-concept in vivo experiment, we demonstrate the beneficial effect of increasing glucose availability in promoting MDSC terminal differentiation in infected mice. Our results provide valuable information of how metabolic alterations induced by infection influence reprogramming and differentiation of MDSCs.
    • Connection Between Chromosomal Location and Function of CtrA Phosphorelay Genes in Alphaproteobacteria.

      Tomasch, Jürgen; Koppenhöfer, Sonja; Lang, Andrew S; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2021-04-29)
      Most bacterial chromosomes are circular, with replication starting at one origin (ori) and proceeding on both replichores toward the terminus (ter). Several studies have shown that the location of genes relative to ori and ter can have profound effects on regulatory networks and physiological processes. The CtrA phosphorelay is a gene regulatory system conserved in most alphaproteobacteria. It was first discovered in Caulobacter crescentus where it controls replication and division into a stalked and a motile cell in coordination with other factors. The locations of the ctrA gene and targets of this response regulator on the chromosome affect their expression through replication-induced DNA hemi-methylation and specific positioning along a CtrA activity gradient in the dividing cell, respectively. Here we asked to what extent the location of CtrA regulatory network genes might be conserved in the alphaproteobacteria. We determined the locations of the CtrA phosphorelay and associated genes in closed genomes with unambiguously identifiable ori from members of five alphaproteobacterial orders. The location of the phosphorelay genes was the least conserved in the Rhodospirillales followed by the Sphingomonadales. In the Rhizobiales a trend toward certain chromosomal positions could be observed. Compared to the other orders, the CtrA phosphorelay genes were conserved closer to ori in the Caulobacterales. In contrast, the genes were highly conserved closer to ter in the Rhodobacterales. Our data suggest selection pressure results in differential positioning of CtrA phosphorelay and associated genes in alphaproteobacteria, particularly in the orders Rhodobacterales, Caulobacterales and Rhizobiales that is worth deeper investigation.
    • Quo vadis clinical diagnostic microbiology?

      Haag, Sara; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2021-07-26)
      No abstract available
    • Targeting Bacterial Gyrase with Cystobactamid, Fluoroquinolone, and Aminocoumarin Antibiotics Induces Distinct Molecular Signatures in Pseudomonas aeruginosa.

      Franke, Raimo; Overwin, Heike; Häussler, Susanne; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (ASM, 2021-07-13)
      The design of novel antibiotics relies on a profound understanding of their mechanism of action. While it has been shown that cellular effects of antibiotics cluster according to their molecular targets, we investigated whether compounds binding to different sites of the same target can be differentiated by their transcriptome or metabolome signatures. The effects of three fluoroquinolones, two aminocoumarins, and two cystobactamids, all inhibiting bacterial gyrase, on Pseudomonas aeruginosa at subinhibitory concentrations could be distinguished clearly by RNA sequencing as well as metabolomics. We observed a strong (2.8- to 212-fold) induction of autolysis-triggering pyocins in all gyrase inhibitors, which correlated with extracellular DNA (eDNA) release. Gyrase B-binding aminocoumarins induced the most pronounced changes, including a strong downregulation of phenazine and rhamnolipid virulence factors. Cystobactamids led to a downregulation of a glucose catabolism pathway. The study implies that clustering cellular mechanisms of action according to the primary target needs to take class-dependent variances into account. IMPORTANCE Novel antibiotics are urgently needed to tackle the growing worldwide problem of antimicrobial resistance. Bacterial pathogens possess few privileged targets for a successful therapy: the majority of existing antibiotics as well as current candidates in development target the complex bacterial machinery for cell wall synthesis, protein synthesis, or DNA replication. An important mechanistic question addressed by this study is whether inhibiting such a complex target at different sites with different compounds has similar or differentiated cellular consequences. Using transcriptomics and metabolomics, we demonstrate that three different classes of gyrase inhibitors can be distinguished by their molecular signatures in P. aeruginosa. We describe the cellular effects of a promising, recently identified gyrase inhibitor class, the cystobactamids, in comparison to those of the established gyrase A-binding fluoroquinolones and the gyrase B-binding aminocoumarins. The study results have implications for mode-of-action discovery approaches based on target-specific reference compounds, as they highlight the intraclass variability of cellular compound effects.
    • Loss of Hem1 disrupts macrophage function and impacts migration, phagocytosis, and integrin-mediated adhesion.

      Stahnke, Stephanie; Döring, Hermann; Kusch, Charly; de Gorter, David J J; Dütting, Sebastian; Guledani, Aleks; Pleines, Irina; Schnoor, Michael; Sixt, Michael; Geffers, Robert; et al. (Wiley-VCH, 2021-03-11)
      Hematopoietic-specific protein 1 (Hem1) is an essential subunit of the WAVE regulatory complex (WRC) in immune cells. WRC is crucial for Arp2/3 complex activation and the protrusion of branched actin filament networks. Moreover, Hem1 loss of function in immune cells causes autoimmune diseases in humans. Here, we show that genetic removal of Hem1 in macrophages diminishes frequency and efficacy of phagocytosis as well as phagocytic cup formation in addition to defects in lamellipodial protrusion and migration. Moreover, Hem1-null macrophages displayed strong defects in cell adhesion despite unaltered podosome formation and concomitant extracellular matrix degradation. Specifically, dynamics of both adhesion and de-adhesion as well as concomitant phosphorylation of paxillin and focal adhesion kinase (FAK) were significantly compromised. Accordingly, disruption of WRC function in non-hematopoietic cells coincided with both defects in adhesion turnover and altered FAK and paxillin phosphorylation. Consistently, platelets exhibited reduced adhesion and diminished integrin αIIbβ3 activation upon WRC removal. Interestingly, adhesion phenotypes, but not lamellipodia formation, were partially rescued by small molecule activation of FAK. A full rescue of the phenotype, including lamellipodia formation, required not only the presence of WRCs but also their binding to and activation by Rac. Collectively, our results uncover that WRC impacts on integrin-dependent processes in a FAK-dependent manner, controlling formation and dismantling of adhesions, relevant for properly grabbing onto extracellular surfaces and particles during cell edge expansion, like in migration or phagocytosis.
    • Fibroblast GATA-4 and GATA-6 promote myocardial adaptation to pressure overload by enhancing cardiac angiogenesis.

      Dittrich, Gesine M; Froese, Natali; Wang, Xue; Kroeger, Hannah; Wang, Honghui; Szaroszyk, Malgorzata; Malek-Mohammadi, Mona; Cordero, Julio; Keles, Merve; Korf-Klingebiel, Mortimer; et al. (Springer Nature, 2021-04-19)
      Heart failure due to high blood pressure or ischemic injury remains a major problem for millions of patients worldwide. Despite enormous advances in deciphering the molecular mechanisms underlying heart failure progression, the cell-type specific adaptations and especially intercellular signaling remain poorly understood. Cardiac fibroblasts express high levels of cardiogenic transcription factors such as GATA-4 and GATA-6, but their role in fibroblasts during stress is not known. Here, we show that fibroblast GATA-4 and GATA-6 promote adaptive remodeling in pressure overload induced cardiac hypertrophy. Using a mouse model with specific single or double deletion of Gata4 and Gata6 in stress activated fibroblasts, we found a reduced myocardial capillarization in mice with Gata4/6 double deletion following pressure overload, while single deletion of Gata4 or Gata6 had no effect. Importantly, we confirmed the reduced angiogenic response using an in vitro co-culture system with Gata4/6 deleted cardiac fibroblasts and endothelial cells. A comprehensive RNA-sequencing analysis revealed an upregulation of anti-angiogenic genes upon Gata4/6 deletion in fibroblasts, and siRNA mediated downregulation of these genes restored endothelial cell growth. In conclusion, we identified a novel role for the cardiogenic transcription factors GATA-4 and GATA-6 in heart fibroblasts, where both proteins act in concert to promote myocardial capillarization and heart function by directing intercellular crosstalk.
    • Role of endothelial microRNA 155 on capillary leakage in systemic inflammation.

      Etzrodt, Valerie; Idowu, Temitayo O; Schenk, Heiko; Seeliger, Benjamin; Prasse, Antje; Thamm, Kristina; Pape, Thorben; Müller-Deile, Janina; van Meurs, Matijs; Thum, Thomas; et al. (BMC, 2021-02-22)
      Background: Capillary leakage is a key contributor to the pathological host response to infections. The underlying mechanisms remain incompletely understood, and the role of microRNAs (MIR) has not been investigated in detail. We hypothesized that specific MIRs might be regulated directly in the endothelium thereby contributing to vascular leakage. Methods: SmallRNA sequencing of endotoxemic murine pulmonary endothelial cells (ECs) was done to detect regulated vascular MIRs. In vivo models: transgenic zebrafish (flk1:mCherry/l-fabp:eGFP-DPB), knockout/wildtype mouse (B6.Cg-Mir155tm1.1Rsky/J); disease models: LPS 17.5 mg/kgBW and cecal ligation and puncture (CLP); in vitro models: stimulated human umbilical vein EC (HUVECs), transendothelial electrical resistance. Results: Endothelial MIR155 was identified as a promising candidate in endotoxemic murine pulmonary ECs (25 × upregulation). Experimental overexpression in a transgenic zebrafish line and in HUVECs was sufficient to induce spontaneous vascular leakage. To the contrary, genetic MIR155 reduction protects against permeability both in vitro and in endotoxemia in vivo in MIR155 heterozygote knockout mice thereby improving survival by 40%. A tight junction protein, Claudin-1, was down-regulated both in endotoxemia and by experimental MIR155 overexpression. Translationally, MIR155 was detectable at high levels in bronchoalveolar fluid of patients with ARDS compared to healthy human subjects. Conclusions: We found that MIR155 is upregulated in the endothelium in mouse and men as part of a systemic inflammatory response and might contribute to the pathophysiology of vascular leakage in a Claudin-1-dependent manner. Future studies have to clarify whether MIR155 could be a potential therapeutic target.
    • Removable denture is a risk indicator for peri-implantitis and facilitates expansion of specific periodontopathogens: a cross-sectional study.

      Grischke, Jasmin; Szafrański, Szymon P; Muthukumarasamy, Uthayakumar; Häussler, Susanne; Stiesch, Meike; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (BMC, 2021-04-01)
      Background: The prevalence of peri-implantitis ranges between 7 and 38.4% depending on risk indicators such as smoking, diabetes mellitus, lack of periodontal maintenance program, and history or presence of periodontitis. Currently, the possible effect of the type of superstructure on peri-implant health is unclear. This cross-sectional study aims to investigate the influence of the superstructure on the prevalence of peri-implant mucositis, peri-implantitis and peri-implant dysbiosis. Methods: During a 32-month recruitment period dental implants were assessed to diagnose healthy peri-implant tissues, mucositis or peri-implantitis. The study included 1097 implants in 196 patients. Out of all peri-implantitis cases 20 randomly chosen submucosal biofilms from implants with fixed denture (FD) originating from 13 patients and 11 biofilms from implants with removable dentures (RD) originating from 3 patients were studied for microbiome analysis. Composition of transcriptionally active biofilms was revealed by RNAseq. Metatranscriptomic profiles were created for thirty-one peri-implant biofilms suffering from peri-implantitis and microbiome changes associated with superstructure types were identified. Results: 16.41% of the implants were diagnosed with peri-implantitis, 25.00% of implants with RD and 12.68% of implants with FD, respectively. Multivariate analysis showed a significant positive association on patient (p = < 0.001) and implant level (p = 0.03) between the prevalence of peri-implantitis and RD. Eight bacterial species were associated either with FD or RD by linear discriminant analysis effect size method. However, significant intergroup confounders (e.g. smoking) were present. Conclusions: Within the limitations of the present work, RDs appear to be a risk indicator for peri-implantitis and seem to facilitate expansion of specific periodontopathogens. Potential ecological and pathological consequences of shift in microbiome from RDs towards higher activity of Fusobacterium nucleatum subspecies animalis and Prevotella intermedia require further investigation.
    • The Two-Component System 09 Regulates Pneumococcal Carbohydrate Metabolism and Capsule Expression.

      Hirschmann, Stephanie; Gómez-Mejia, Alejandro; Mäder, Ulrike; Karsunke, Julia; Driesch, Dominik; Rohde, Manfred; Häussler, Susanne; Burchhardt, Gerhard; Hammerschmidt, Sven; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2021-02-24)
      Streptococcus pneumoniae two-component regulatory systems (TCSs) are important systems that perceive and respond to various host environmental stimuli. In this study, we have explored the role of TCS09 on gene expression and phenotypic alterations in S. pneumoniae D39. Our comparative transcriptomic analyses identified 67 differently expressed genes in total. Among those, agaR and the aga operon involved in galactose metabolism showed the highest changes. Intriguingly, the encapsulated and nonencapsulated hk09-mutants showed significant growth defects under nutrient-defined conditions, in particular with galactose as a carbon source. Phenotypic analyses revealed alterations in the morphology of the nonencapsulated hk09- and tcs09-mutants, whereas the encapsulated hk09- and tcs09-mutants produced higher amounts of capsule. Interestingly, the encapsulated D39∆hk09 showed only the opaque colony morphology, while the D39∆rr09- and D39∆tcs09-mutants had a higher proportion of transparent variants. The phenotypic variations of D39ΔcpsΔhk09 and D39ΔcpsΔtcs09 are in accordance with their higher numbers of outer membrane vesicles, higher sensitivity against Triton X-100 induced autolysis, and lower resistance against oxidative stress. In conclusion, these results indicate the importance of TCS09 for pneumococcal metabolic fitness and resistance against oxidative stress by regulating the carbohydrate metabolism and thereby, most likely indirectly, the cell wall integrity and amount of capsular polysaccharide.