• Community richness of amphibian skin bacteria correlates with bioclimate at the global scale.

      Kueneman, Jordan G; Bletz, Molly C; McKenzie, Valerie J; Becker, C Guilherme; Joseph, Maxwell B; Abarca, Juan G; Archer, Holly; Arellano, Ana Lisette; Bataille, Arnaud; Becker, Matthew; Belden, Lisa K; Crottini, Angelica; Geffers, Robert; Haddad, Célio F B; Harris, Reid N; Holden, Whitney M; Hughey, Myra; Jarek, Michael; Kearns, Patrick J; Kerby, Jacob L; Kielgast, Jos; Kurabayashi, Atsushi; Longo, Ana V; Loudon, Andrew; Medina, Daniel; Nuñez, José J; Perl, R G Bina; Pinto-Tomás, Adrián; Rabemananjara, Falitiana C E; Rebollar, Eria A; Rodríguez, Ariel; Rollins-Smith, Louise; Stevenson, Robert; Tebbe, Christoph C; Vargas Asensio, Gabriel; Waldman, Bruce; Walke, Jenifer B; Whitfield, Steven M; Zamudio, Kelly R; Zúñiga Chaves, Ibrahim; Woodhams, Douglas C; Vences, Miguel; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (Dpringer-Nature, 2019-03-01)
      Animal-associated microbiomes are integral to host health, yet key biotic and abiotic factors that shape host-associated microbial communities at the global scale remain poorly understood. We investigated global patterns in amphibian skin bacterial communities, incorporating samples from 2,349 individuals representing 205 amphibian species across a broad biogeographic range. We analysed how biotic and abiotic factors correlate with skin microbial communities using multiple statistical approaches. Global amphibian skin bacterial richness was consistently correlated with temperature-associated factors. We found more diverse skin microbiomes in environments with colder winters and less stable thermal conditions compared with environments with warm winters and less annual temperature variation. We used bioinformatically predicted bacterial growth rates, dormancy genes and antibiotic synthesis genes, as well as inferred bacterial thermal growth optima to propose mechanistic hypotheses that may explain the observed patterns. We conclude that temporal and spatial characteristics of the host's macro-environment mediate microbial diversity.
    • Identification and quantification of (t)RNA modifications in Pseudomonas aeruginosa by liquid chromatography-tandem mass spectrometry.

      Grobe, Svenja; Doberenz, Sebastian; Ferreira, Kevin; Krueger, Jonas; Brönstrup, Mark; Kaever, Volkhard; Häußler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley-Blackwell, 2019-01-15)
      Transfer RNA (tRNA) modifications impact the structure and function of tRNAs thus affecting the efficiency and fidelity of translation. In the opportunistic pathogen Pseudomonas aeruginosa translational regulation plays an important but less defined role in the adaptation to changing environments. In this study, we explored tRNA modifications in P. aeruginosa using LC-MS/MS based approaches. Neutral Loss Scan (NLS) demonstrated the potential to identify previously unknown modifications, while Multiple Reaction Monitoring (MRM) can detect modifications with high specificity and sensitivity. In this study, the MRM-based external calibration method allowed for quantification of the 4 canonical and 32 modified ribonucleosides, of which 21 tRNA modifications were quantified in the total tRNA pool of P. aeruginosa PA14. We also purified the single tRNA isoacceptors tRNA-ArgUCU, tRNA-LeuCAA and tRNA-TrpCCA and determined, both qualitatively and quantitatively, their specific modification pattern. Deeper insights into the nature and dynamics of tRNA modifications in P. aeruginosa will pave the way for further studies on posttranscriptional gene regulation as a relatively unexplored molecular mechanism of controlling bacterial pathogenicity and life style.
    • Thermoplasmatales and sulfur-oxidizing bacteria dominate the microbial community at the surface water of a CO-rich hydrothermal spring located in Tenorio Volcano National Park, Costa Rica.

      Arce-Rodríguez, Alejandro; Puente-Sánchez, Fernando; Avendaño, Roberto; Martínez-Cruz, María; de Moor, J Maarten; Pieper, Dietmar H; Chavarría, Max; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2019-01-01)
      Here we report the chemical and microbial characterization of the surface water of a CO2-rich hydrothermal vent known in Costa Rica as Borbollones, located at Tenorio Volcano National Park. The Borbollones showed a temperature surrounding 60 °C, a pH of 2.4 and the gas released has a composition of ~ 97% CO2, ~ 0.07% H2S, ~ 2.3% N2 and ~ 0.12% CH4. Other chemical species such as sulfate and iron were found at high levels with respect to typical fresh water bodies. Analysis by 16S rRNA gene metabarcoding revealed that in Borbollones predominates an archaeon from the order Thermoplasmatales and one bacterium from the genus Sulfurimonas. Other sulfur- (genera Thiomonas, Acidithiobacillus, Sulfuriferula, and Sulfuricurvum) and iron-oxidizing bacteria (genera Sideroxydans, Gallionella, and Ferrovum) were identified. Our results show that CO2-influenced surface water of Borbollones contains microorganisms that are usually found in acid rock drainage environments or sulfur-rich hydrothermal vents. To our knowledge, this is the first microbiological characterization of a CO2-dominated hydrothermal spring from Central America and expands our understanding of those extreme ecosystems.
    • Importance of flagella in acute and chronic Pseudomonas aeruginosa infections.

      Lorenz, Anne; Preuße, Matthias; Bruchmann, Sebastian; Pawar, Vinay; Grahl, Nora; Pils, Marina C; Nolan, Laura M; Filloux, Alain; Weiss, Siegfried; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley-Blackwell, 2018-11-08)
      Pseudomonas aeruginosa is an environmental microorganism and a causative agent of diverse acute and chronic, biofilm-associated infections. Advancing research-based knowledge on its adaptation to conditions within the human host is bound to reveal novel strategies and targets for therapeutic intervention. Here, we investigated the traits that P. aeruginosa PA14 as well as a virulence attenuated ΔlasR mutant need to survive in selected murine infection models. Experimentally, the genetic programs that the bacteria use to adapt to biofilm-associated versus acute infections were dissected by passaging transposon mutant libraries through mouse lungs (acute) or mouse tumours (biofilm-infection). Adaptive metabolic changes of P. aeruginosa were generally required during both infection processes. Counter-selection against flagella expression was observed during acute lung infections. Obviously, avoidance of flagella-mediated activation of host immunity is advantageous for the wildtype bacteria. For the ΔlasR mutant, loss of flagella did not confer a selective advantage. Apparently, other pathogenesis mechanisms are active in this virulence attenuated strain. In contrast, the infective process of P. aeruginosa in the chronic biofilm model apparently required expression of flagellin. Together, our findings imply that the host immune reactions against the infectious agent are very decisive for acuteness and duration of the infectious disease. They direct disease outcome.
    • Determining lineage-specific bacterial growth curves with a novel approach based on amplicon reads normalization using internal standard (ARNIS).

      Piwosz, Kasia; Shabarova, Tanja; Tomasch, Jürgen; Šimek, Karel; Kopejtka, Karel; Kahl, Silke; Pieper, Dietmar H; Koblížek, Michal; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-11-01)
      The growth rate is a fundamental characteristic of bacterial species, determining its contributions to the microbial community and carbon flow. High-throughput sequencing can reveal bacterial diversity, but its quantitative inaccuracy precludes estimation of abundances and growth rates from the read numbers. Here, we overcame this limitation by normalizing Illumina-derived amplicon reads using an internal standard: a constant amount of Escherichia coli cells added to samples just before biomass collection. This approach made it possible to reconstruct growth curves for 319 individual OTUs during the grazer-removal experiment conducted in a freshwater reservoir Římov. The high resolution data signalize significant functional heterogeneity inside the commonly investigated bacterial groups. For instance, many Actinobacterial phylotypes, a group considered to harbor slow-growing defense specialists, grew rapidly upon grazers' removal, demonstrating their considerable importance in carbon flow through food webs, while most Verrucomicrobial phylotypes were particle associated. Such differences indicate distinct life strategies and roles in food webs of specific bacterial phylotypes and groups. The impact of grazers on the specific growth rate distributions supports the hypothesis that bacterivory reduces competition and allows existence of diverse bacterial communities. It suggests that the community changes were driven mainly by abundant, fast, or moderately growing, and not by rare fast growing, phylotypes. We believe amplicon read normalization using internal standard (ARNIS) can shed new light on in situ growth dynamics of both abundant and rare bacteria.
    • Breaking the vicious cycle of antibiotic killing and regrowth of biofilm-residing .

      Müsken, Mathias; Pawar, Vinay; Schwebs, Timo; Bähre, Heike; Felgner, Sebastian; Weiss, Siegfried; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-10-08)
      Biofilm-residing bacteria embedded in an extracellular matrix are protected from diverse physico-chemical insults. In addition to the general recalcitrance of biofilm-bacteria, high bacterial loads in biofilm-associated infections significantly diminishes the efficacy of antimicrobials due to a low per-cell antibiotic concentration. Accordingly, present antimicrobial treatment protocols, that have been established to serve the eradication of acute infections, fail to clear biofilm-associated chronic infections. In the present study, we applied automated confocal microscopy on Pseudomonas aeruginosa to monitor dynamic killing of biofilm-grown bacteria by tobramycin and colistin in real-time. We revealed that the time required for surviving bacteria to repopulate the biofilm could be taken as measure for effectiveness of the antimicrobial treatment. It depends on the: i) nature and concentration of the antibiotic, ii) duration of antibiotic treatment; iii) application as mono or combination therapy and iv) time intervals of drug administration. The vicious cycle of killing and repopulation of biofilm bacteria could also be broken in an in vivo model system by applying successive antibiotic dosages with time intervals that do not allow full reconstitution of the biofilm communities. Treatment regimens that consider the important aspects of antimicrobial killing kinetics bear the potential to improve control of biofilm regrowth. This is an important and underestimated factor that is bound to ensure sustainable treatment success of chronic infections.
    • BACTOME-a reference database to explore the sequence- and gene expression-variation landscape of Pseudomonas aeruginosa clinical isolates.

      Hornischer, Klaus; Khaledi, Ariane; Pohl, Sarah; Schniederjans, Monika; Pezoldt, Lorena; Casilag, Fiordiligie; Muthukumarasamy, Uthayakumar; Bruchmann, Sebastian; Thöming, Janne; Kordes, Adrian; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-10-01)
      Extensive use of next-generation sequencing (NGS) for pathogen profiling has the potential to transform our understanding of how genomic plasticity contributes to phenotypic versatility. However, the storage of large amounts of NGS data and visualization tools need to evolve to offer the scientific community fast and convenient access to these data. We introduce BACTOME as a database system that links aligned DNA- and RNA-sequencing reads of clinical Pseudomonas aeruginosa isolates with clinically relevant pathogen phenotypes. The database allows data extraction for any single isolate, gene or phenotype as well as data filtering and phenotypic grouping for specific research questions. With the integration of statistical tools we illustrate the usefulness of a relational database structure for the identification of phenotype-genotype correlations as an essential part of the discovery pipeline in genomic research. Furthermore, the database provides a compilation of DNA sequences and gene expression values of a plethora of clinical isolates to give a consensus DNA sequence and consensus gene expression signature. Deviations from the consensus thereby describe the genomic landscape and the transcriptional plasticity of the species P. aeruginosa. The database is available at https://bactome.helmholtz-hzi.de.
    • Interferon-beta expression and type I interferon receptor signaling of hepatocytes prevent hepatic necrosis and virus dissemination in Coxsackievirus B3-infected mice.

      Koestner, Wolfgang; Spanier, Julia; Klause, Tanja; Tegtmeyer, Pia-K; Becker, Jennifer; Herder, Vanessa; Borst, Katharina; Todt, Daniel; Lienenklaus, Stefan; Gerhauser, Ingo; Detje, Claudia N; Geffers, Robert; Langereis, Martijn A; Vondran, Florian W R; Yuan, Qinggong; van Kuppeveld, Frank J M; Ott, Michael; Staeheli, Peter; Steinmann, Eike; Baumgärtner, Wolfgang; Wacker, Frank; Kalinke, Ulrich; TWINCORE, Zentrum für experimentelle und klinischeInfektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany (2018-08-01)
      During Coxsackievirus B3 (CVB3) infection hepatitis is a potentially life threatening complication, particularly in newborns. Studies with type I interferon (IFN-I) receptor (IFNAR)-deficient mice revealed a key role of the IFN-I axis in the protection against CVB3 infection, whereas the source of IFN-I and cell types that have to be IFNAR triggered in order to promote survival are still unknown. We found that CVB3 infected IFN-β reporter mice showed effective reporter induction, especially in hepatocytes and only to a minor extent in liver-resident macrophages. Accordingly, upon in vitro CVB3 infection of primary hepatocytes from murine or human origin abundant IFN-β responses were induced. To identify sites of IFNAR-triggering we performed experiments with Mx reporter mice, which upon CVB3 infection showed massive luciferase induction in the liver. Immunohistological studies revealed that during CVB3 infection MX1 expression of hepatocytes was induced primarily by IFNAR-, and not by IFN-III receptor (IFNLR)-triggering. CVB3 infection studies with primary human hepatocytes, in which either the IFN-I or the IFN-III axis was inhibited, also indicated that primarily IFNAR-, and to a lesser extent IFNLR-triggering was needed for ISG induction. Interestingly, CVB3 infected mice with a hepatocyte-specific IFNAR ablation showed severe liver cell necrosis and ubiquitous viral dissemination that resulted in lethal disease, as similarly detected in classical IFNAR-/- mice. In conclusion, we found that during CVB3 infection hepatocytes are major IFN-I producers and that the liver is also the organ that shows strong IFNAR-triggering. Importantly, hepatocytes need to be IFNAR-triggered in order to prevent virus dissemination and to assure survival. These data are compatible with the hypothesis that during CVB3 infection hepatocytes serve as important IFN-I producers and sensors not only in the murine, but also in the human system.
    • Metatranscriptome Analysis of the Vaginal Microbiota Reveals Potential Mechanisms for Protection against Metronidazole in Bacterial Vaginosis.

      Deng, Zhi-Luo; Gottschick, Cornelia; Bhuju, Sabin; Masur, Clarissa; Abels, Christoph; Wagner-Döbler, Irene; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-06-27)
      Bacterial vaginosis (BV) is a prevalent multifactorial disease of women in their reproductive years characterized by a shift from the
    • Exome sequencing and case-control analyses identify RCC1 as a candidate breast cancer susceptibility gene.

      Riahi, Aouatef; Radmanesh, Hoda; Schürmann, Peter; Bogdanova, Natalia; Geffers, Robert; Meddeb, Rym; Kharrat, Maher; Dörk, Thilo; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-06-15)
      Breast cancer is a genetic disease but the known genes explain a minority of cases. To elucidate the molecular basis of breast cancer in the Tunisian population, we performed exome sequencing on six BRCA1/BRCA2 mutation-negative patients with familial breast cancer and identified a novel frameshift mutation in RCC1, encoding the Regulator of Chromosome Condensation 1. Subsequent genotyping detected the 19-bp deletion in additional 5 out of 153 (3%) breast cancer patients but in none of 400 female controls (p = 0.0015). The deletion was enriched in patients with a positive family history (5%, p = 0.0009) and co-segregated with breast cancer in the initial pedigree. The mutant allele was lost in 4/6 breast tumors from mutation carriers which may be consistent with the hypothesis that RCC1 dysfunction provides a selective disadvantage at the stage of tumor progression. In summary, we propose RCC1 as a likely breast cancer susceptibility gene in the Tunisian population.
    • Regulation of Flagellum Biosynthesis in Response to Cell Envelope Stress in Serovar Typhimurium.

      Spöring, Imke; Felgner, Sebastian; Preuße, Matthias; Eckweiler, Denitsa; Rohde, Manfred; Häussler, Susanne; Weiss, Siegfried; Erhardt, Marc (2018-05-01)
      Flagellum-driven motility of serovar Typhimurium facilitates host colonization. However, the large extracellular flagellum is also a prime target for the immune system. As consequence, expression of flagella is bistable within a population of , resulting in flagellated and nonflagellated subpopulations. This allows the bacteria to maximize fitness in hostile environments. The degenerate EAL domain protein RflP (formerly YdiV) is responsible for the bistable expression of flagella by directing the flagellar master regulatory complex FlhDC with respect to proteolytic degradation. Information concerning the environmental cues controlling expression of and thus about the bistable flagellar biosynthesis remains ambiguous. Here, we demonstrated that RflP responds to cell envelope stress and alterations of outer membrane integrity. Lipopolysaccharide (LPS) truncation mutants of Typhimurium exhibited increasing motility defects due to downregulation of flagellar gene expression. Transposon mutagenesis and genetic profiling revealed that σ (RpoE) and Rcs phosphorelay-dependent cell envelope stress response systems sense modifications of the lipopolysaccaride, low pH, and activity of the complement system. This subsequently results in activation of RflP expression and degradation of FlhDC via ClpXP. We speculate that the presence of diverse hostile environments inside the host might result in cell envelope damage and would thus trigger the repression of resource-costly and immunogenic flagellum biosynthesis via activation of the cell envelope stress response. Pathogenic bacteria such as Typhimurium sense and adapt to a multitude of changing and stressful environments during host infection. At the initial stage of gastrointestinal colonization, uses flagellum-mediated motility to reach preferred sites of infection. However, the flagellum also constitutes a prime target for the host's immune response. Accordingly, the pathogen needs to determine the spatiotemporal stage of infection and control flagellar biosynthesis in a robust manner. We found that uses signals from cell envelope stress-sensing systems to turn off production of flagella. We speculate that downregulation of flagellum synthesis after cell envelope damage in hostile environments aids survival of during late stages of infection and provides a means to escape recognition by the immune system.
    • Birth, evolution, and transmission of satellite-free mammalian centromeric domains.

      Nergadze, Solomon G; Piras, Francesca M; Gamba, Riccardo; Corbo, Marco; Cerutti, Federico; McCarter, Joseph G W; Cappelletti, Eleonora; Gozzo, Francesco; Harman, Rebecca M; Antczak, Douglas F; Miller, Donald; Scharfe, Maren; Pavesi, Giulio; Raimondi, Elena; Sullivan, Kevin F; Giulotto, Elena; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-01-01)
      Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on
    • Distribution and Evolution of Peroxisomes in Alveolates (Apicomplexa, Dinoflagellates, Ciliates).

      Ludewig-Klingner, Ann-Kathrin; Michael, Victoria; Jarek, Michael; Brinkmann, Henner; Petersen, Jörn; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-01-01)
      The peroxisome was the last organelle to be discovered and five decades later it is still the Cinderella of eukaryotic compartments. Peroxisomes have a crucial role in the detoxification of reactive oxygen species, the beta-oxidation of fatty acids, and the biosynthesis of etherphospholipids, and they are assumed to be present in virtually all aerobic eukaryotes. Apicomplexan parasites including the malaria and toxoplasmosis agents were described as the first group of mitochondriate protists devoid of peroxisomes. This study was initiated to reassess the distribution and evolution of peroxisomes in the superensemble Alveolata (apicomplexans, dinoflagellates, ciliates). We established transcriptome data from two chromerid algae (Chromera velia, Vitrella brassicaformis), and two dinoflagellates (Prorocentrum minimum, Perkinsus olseni) and identified the complete set of essential peroxins in all four reference species. Our comparative genome analysis provides unequivocal evidence for the presence of peroxisomes in Toxoplasma gondii and related genera. Our working hypothesis of a common peroxisomal origin of all alveolates is supported by phylogenetic analyses of essential markers such as the import receptor Pex5. Vitrella harbors the most comprehensive set of peroxisomal proteins including the catalase and the glyoxylate cycle and it is thus a promising model organism to investigate the functional role of this organelle in Apicomplexa.
    • Packaging of Dinoroseobacter shibae DNA into Gene Transfer Agent Particles Is Not Random.

      Tomasch, Jürgen; Wang, Hui; Hall, April T K; Patzelt, Diana; Preusse, Matthias; Petersen, Jörn; Brinkmann, Henner; Bunk, Boyke; Bhuju, Sabin; Jarek, Michael; Geffers, Robert; Lang, Andrew S; Wagner-Döbler, Irene; Helmholtz-Zentrum for Infektion Research GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-01-01)
      Gene transfer agents (GTAs) are phage-like particles which contain a fragment of genomic DNA of the bacterial or archaeal producer and deliver this to a recipient cell. GTA gene clusters are present in the genomes of almost all marine Rhodobacteraceae (Roseobacters) and might be important contributors to horizontal gene transfer in the world's oceans. For all organisms studied so far, no obvious evidence of sequence specificity or other nonrandom process responsible for packaging genomic DNA into GTAs has been found. Here, we show that knock-out of an autoinducer synthase gene of Dinoroseobacter shibae resulted in overproduction and release of functional GTA particles (DsGTA). Next-generation sequencing of the 4.2-kb DNA fragments isolated from DsGTAs revealed that packaging was not random. DNA from low-GC conjugative plasmids but not from high-GC chromids was excluded from packaging. Seven chromosomal regions were strongly overrepresented in DNA isolated from DsGTA. These packaging peaks lacked identifiable conserved sequence motifs that might represent recognition sites for the GTA terminase complex. Low-GC regions of the chromosome, including the origin and terminus of replication, were underrepresented in DNA isolated from DsGTAs. DNA methylation reduced packaging frequency while the level of gene expression had no influence. Chromosomal regions found to be over- and underrepresented in DsGTA-DNA were regularly spaced. We propose that a "headful" type of packaging is initiated at the sites of coverage peaks and, after linearization of the chromosomal DNA, proceeds in both directions from the initiation site. GC-content, DNA-modifications, and chromatin structure might influence at which sides GTA packaging can be initiated.
    • Presence of Infected Gr-1CD11bCD11c Monocytic Myeloid Derived Suppressor Cells Subverts T Cell Response and Is Associated With Impaired Dendritic Cell Function in Mycobacterium avium-Infected Mice.

      Abdissa, Ketema; Nerlich, Andreas; Beineke, Andreas; Ruangkiattikul, Nanthapon; Pawar, Vinay; Heise, Ulrike; Janze, Nina; Falk, Christine; Bruder, Dunja; Schleicher, Ulrike; Bogdan, Christian; Weiss, Siegfried; Goethe, Ralph; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-01-01)
      Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunomodulatory function. To study the mechanism by which MDSC affect antimicrobial immunity, we infected mice with two M. avium strains of differential virulence, highly virulent Mycobacterium avium subsp. avium strain 25291 (MAA) and low virulent Mycobacterium avium subsp. hominissuis strain 104 (MAH). Intraperitoneal infection with MAA, but not MAH, caused severe disease and massive splenic infiltration of monocytic MDSC (M-MDSC; Gr-1intCD11bhiCD11cint) expressing inducible NO synthase (Nos2) and bearing high numbers of mycobacteria. Depletion experiments demonstrated that M-MDSC were essential for disease progression. NO production by M-MDSC influenced antigen-uptake and processing by dendritic cells and proliferation of CD4+ T cells. M-MDSC were also induced in MAA-infected mice lacking Nos2. In these mice CD4+ T cell expansion and control of infection were restored. However, T cell inhibition was only partially relieved and arginase (Arg) 1-expressing M-MDSC were accumulated. Likewise, inhibition of Arg1 also partially rescued T cell proliferation. Thus, mycobacterial virulence results in the induction of M-MDSC that block the T cell response in a Nos2- and Arg1-dependent manner.
    • AgNPs Change Microbial Community Structures of Wastewater.

      Guo, Yuting; Cichocki, Nicolas; Schattenberg, Florian; Geffers, Robert; Harms, Hauke; Müller, Susann; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2018-01-01)
      Due to their strong antimicrobial activity, silver nanoparticles (AgNPs) are massively produced, applied, consumed and, as a negative consequence, released into wastewater treatment plants. Most AgNPs are assumed to be bound by sludge, and thus bear potential risk for microbial performance and stability. In this lab-scale study, flow cytometry as a high-throughput method and 16S rRNA gene amplicon Illumina MiSeq sequencing were used to track microbial community structure changes when being exposed to AgNPs. Both methods allowed deeper investigation of the toxic impact of chemicals on microbial communities than classical EC50 determination. In addition, ecological metrics were used to quantify microbial community variations depending on AgNP types (10 and 30 nm) and concentrations. Only low changes in α- and intra-community β-diversity values were found both in successive negative and positive control batches and batches that were run with AgNPs below the EC50 value. Instead, AgNPs at EC50 concentrations caused upcoming of certain and disappearance of formerly dominant subcommunities. Flavobacteriia were among those that almost disappeared, while phylotypes affiliated with Gammaproteobacteria (3.6-fold) and Bacilli (8.4-fold) increased in cell abundance in comparison to the negative control. Thus, silver amounts at the EC50 value affected community structure suggesting a potential negative impact on functions in wastewater treatment systems.
    • Type I Interferon Signaling Is Required for CpG-Oligodesoxynucleotide-Induced Control of Leishmania major, but Not for Spontaneous Cure of Subcutaneous Primary or Secondary L. major Infection.

      Schleicher, Ulrike; Liese, Jan; Justies, Nicole; Mischke, Thomas; Haeberlein, Simone; Sebald, Heidi; Kalinke, Ulrich; Weiss, Siegfried; Bogdan, Christian; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018)
      We previously showed that in mice infected with Leishmania major type I interferons (IFNs) initiate the innate immune response to the parasite at day 1 and 2 of infection. Here, we investigated which type I IFN subtypes are expressed during the first 8 weeks of L. major infection and whether type I IFNs are essential for a protective immune response and clinical cure of the disease. In self-healing C57BL/6 mice infected with a high dose of L. major, IFN-α4, IFN-α5, IFN-α11, IFN-α13, and IFN-β mRNA were most prominently regulated during the course of infection. In C57BL/6 mice deficient for IFN-β or the IFN-α/β-receptor chain 1 (IFNAR1), development of skin lesions and parasite loads in skin, draining lymph node, and spleen was indistinguishable from wild-type (WT) mice. In line with the clinical findings, C57BL/6 IFN-β-/-, IFNAR1-/-, and WT mice exhibited similar mRNA expression levels of IFN-γ, interleukin (IL)-4, IL-12, IL-13, inducible nitric oxide synthase, and arginase 1 during the acute and late phase of the infection. Also, myeloid dendritic cells from WT and IFNAR1-/- mice produced comparable amounts of IL-12p40/p70 protein upon exposure to L. major in vitro. In non-healing BALB/c WT mice, the mRNAs of IFN-α subtypes (α2, α4, α5, α6, and α9) were rapidly induced after high-dose L. major infection. However, genetic deletion of IFNAR1 or IFN-β did not alter the progressive course of infection seen in WT BALB/c mice. Finally, we tested whether type I IFNs and/or IL-12 are required for the prophylactic effect of CpG-oligodesoxynucleotides (ODN) in BALB/c mice. Local and systemic administration of CpG-ODN 1668 protected WT and IFN-β-/- mice equally well from progressive leishmaniasis. By contrast, the protective effect of CpG-ODN 1668 was lost in BALB/c IFNAR1-/- (despite a sustained suppression of IL-4) and in BALB/c IL-12p35-/- mice. From these data, we conclude that IFN-β and IFNAR1 signaling are dispensable for a curative immune response to L. major in C57BL/6 mice and irrelevant for disease development in BALB/c mice, whereas IL-12 and IFN-α subtypes are essential for the disease prevention by CpG-ODNs in this mouse strain.
    • Decreased production of class-switched antibodies in neonatal B cells is associated with increased expression of miR-181b.

      Glaesener, Stephanie; Jaenke, Christine; Habener, Anika; Geffers, Robert; Hagendorff, Petra; Witzlau, Katrin; Imelmann, Esther; Krueger, Andreas; Meyer-Bahlburg, Almut; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018)
      The increased susceptibility to infections of neonates is caused by an immaturity of the immune system as a result of both qualitative and quantitative differences between neonatal and adult immune cells. With respect to B cells, neonatal antibody responses are known to be decreased. Accountable for this is an altered composition of the neonatal B cell compartment towards more immature B cells. However, it remains unclear whether the functionality of individual neonatal B cell subsets is altered as well. In the current study we therefore compared phenotypical and functional characteristics of corresponding neonatal and adult B cell subpopulations. No phenotypic differences could be identified with the exception of higher IgM expression in neonatal B cells. Functional analysis revealed differences in proliferation, survival, and B cell receptor signaling. Most importantly, neonatal B cells showed severely impaired class-switch recombination (CSR) to IgG and IgA. This was associated with increased expression of miR-181b in neonatal B cells. Deficiency of miR-181b resulted in increased CSR. With this, our results highlight intrinsic differences that contribute to weaker B cell antibody responses in newborns.
    • Global micro RNA expression profiling in the liver biopsies of Hepatitis B Virus infected patients suggests specific miRNA signatures for viral persistence and hepatocellular injury.

      Singh, Avishek Kumar; Rooge, Sheetalnath Babasaheb; Varshney, Aditi; Vasudevan, Madavan; Bhardwaj, Ankit; Venugopal, Senthil Kumar; Trehanpati, Nirupama; Kumar, Manoj; Geffers, Robert; Kumar, Vijay; Sarin, Shiv Kumar; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-11-30)
      Hepatitis B virus (HBV) can manipulate the miRNA regulatory networks in infected cells to create a permissive environment for viral replication, cellular injury, disease onset and its progression. The aim of the present study was to understand the miRNA networks and their target genes in the liver of hepatitis B patients involved in HBV replication, liver injury and liver fibrosis. We investigated differentially expressed miRNAs by microarray in the liver biopsy samples from different stages of HBV infection and liver disease [immune tolerant (IT; n= 8); acute viral hepatitis (AVH; n=8); no fibrosis (n=16); early (F1+F2) (n=19) or late fibrosis (F3+F4) (n=14) and healthy controls (n=7)]. The miRNA expression levels were analyzed by the unsupervised principal component analysis (PCA) and hierarchical clustering. Analysis of miRNA-mRNA regulatory networks identified 17 miRNAs and 18 target gene interactions with four distinct nodes each representing a stage-specific gene regulation during disease progression. The IT group showed elevated miR-199a-5p, miR-221-3p and Let-7a-3p levels which could target genes involved in innate immune response and viral replication. In AVH group, miR-125b-5p and miR-3613-3p were up whereas miR-940 was down which might affect cell proliferation via STAT3 pathway. In early fibrosis, miR-34b-3p, miR-1224-3p and miR-1227-3p were up while miR-499a-5p was down which together, possibly mediate chronic inflammation. In advanced fibrosis, miR-1, miR-10b-5p, miR-96-5p, miR-133b and miR-671-5p were up while miR-20b-5p and miR-455-3p were down, possibly allowing chronic disease progression. Interestingly, only 8 of 17 liver-specific miRNAs exhibited a similar expression pattern in patient sera.
    • The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of Escherichia coli.

      Donner, Jannik; Reck, Michael; Bunk, Boyke; Jarek, Michael; App, Constantin Benjamin; Meier-Kolthoff, Jan P; Overmann, Jörg; Müller, Rolf; Kirschning, Andreas; Wagner-Döbler, Irene; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr, 7,38124 Braunschweig, Germany. (2017-11-01)
      The myxobacterial secondary metabolite carolacton inhibits growth of Streptococcus pneumoniae and kills biofilm cells of the caries- and endocarditis-associated pathogen Streptococcus mutans at nanomolar concentrations. Here, we studied the response to carolacton of an Escherichia coli strain that lacked the outer membrane protein TolC. Whole-genome sequencing of the laboratory E. coli strain TolC revealed the integration of an insertion element, IS5, at the tolC locus and a close phylogenetic relationship to the ancient E. coli K-12. We demonstrated via transcriptome sequencing (RNA-seq) and determination of MIC values that carolacton penetrates the phospholipid bilayer of the Gram-negative cell envelope and inhibits growth of E. coli TolC at similar concentrations as for streptococci. This inhibition is completely lost for a C-9 (R) epimer of carolacton, a derivative with an inverted stereocenter at carbon atom 9 [(S) → (R)] as the sole difference from the native molecule, which is also inactive in S. pneumoniae and S. mutans, suggesting a specific interaction of native carolacton with a conserved cellular target present in bacterial phyla as distantly related as Firmicutes and Proteobacteria. The efflux pump inhibitor (EPI) phenylalanine arginine β-naphthylamide (PAβN), which specifically inhibits AcrAB-TolC, renders E. coli susceptible to carolacton. Our data indicate that carolacton has potential for use in antimicrobial chemotherapy against Gram-negative bacteria, as a single drug or in combination with EPIs. Strain E. coli TolC has been deposited at the DSMZ; together with the associated RNA-seq data and MIC values, it can be used as a reference during future screenings for novel bioactive compounds. IMPORTANCE The emergence of pathogens resistant against most or all of the antibiotics currently used in human therapy is a global threat, and therefore the search for antimicrobials with novel targets and modes of action is of utmost importance. The myxobacterial secondary metabolite carolacton had previously been shown to inhibit biofilm formation and growth of streptococci. Here, we investigated if carolacton could act against Gram-negative bacteria, which are difficult targets because of their double-layered cytoplasmic envelope. We found that the model organism Escherichia coli is susceptible to carolacton, similar to the Gram-positive Streptococcus pneumoniae, if its multidrug efflux system AcrAB-TolC is either inactivated genetically, by disruption of the tolC gene, or physiologically by coadministering an efflux pump inhibitor. A carolacton epimer that has a different steric configuration at carbon atom 9 is completely inactive, suggesting that carolacton may interact with the same molecular target in both Gram-positive and Gram-negative bacteria.