• Lignipirellula cremea gen. nov., sp. nov., a planctomycete isolated from wood particles in a brackish river estuary.

      Peeters, Stijn H; Wiegand, Sandra; Kallscheuer, Nicolai; Jogler, Mareike; Heuer, Anja; Jetten, Mike S M; Boedeker, Christian; Rohde, Manfred; Jogler, Christian; HZI,Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (Springer, 2020-04-01)
      A novel planctomycetal strain, designated Pla85_3_4T, was isolated from the surface of wood incubated at the discharge of a wastewater treatment plant in the Warnow river near Rostock, Germany. Cells of the novel strain have a cell envelope architecture resembling that of Gram-negative bacteria, are round to pear-shaped (length: 2.2 ± 0.4 µm, width: 1.2 ± 0.3 µm), form aggregates and divide by polar budding. Colonies have a cream colour. Strain Pla85_3_4T grows at ranges of 10-30 °C (optimum 26 °C) and at pH 6.5-10.0 (optimum 7.5), and has a doubling time of 26 h. Phylogenetically, strain Pla85_3_4T (DSM 103796T = LMG 29741T) is concluded to represent a novel species of a novel genus within the family Pirellulaceae, for which we propose the name Lignipirellula cremea gen. nov., sp. nov.
    • Aureliella helgolandensis gen. nov., sp. nov., a novel Planctomycete isolated from a jellyfish at the shore of the island Helgoland.

      Kallscheuer, Nicolai; Wiegand, Sandra; Boedeker, Christian; Peeters, Stijn H; Jogler, Mareike; Rast, Patrick; Heuer, Anja; Jetten, Mike S M; Rohde, Manfred; Jogler, Christian; et al. (Springer, 2020-03-27)
      A novel planctomycetal strain, designated Q31aT, was isolated from a jellyfish at the shore of the island Helgoland in the North Sea. The strain forms lucid white colonies on solid medium and displays typical characteristics of planctomycetal strains, such as division by budding, formation of rosettes, presence of crateriform structures, extracellular matrix or fibre and a holdfast structure. Q31aT is mesophilic (temperature optimum 27 °C), neutrophilic (pH optimum 7.5), aerobic and heterotrophic. A maximal growth rate of 0.017 h- 1 (generation time of 41 h) was observed. Q31aT has a genome size of 8.44 Mb and a G + C content of 55.3%. Phylogenetically, the strain represents a novel genus and species in the recently introduced family Pirellulaceae, order Pirellulales, class Planctomycetia. We propose the name Aureliella helgolandensis gen. nov., sp. nov. for the novel species, represented by Q31aT (= DSM 103537T = LMG 29700T) as the type strain.
    • Capsule and fimbriae modulate the invasion of Haemophilus influenzae in a human blood-cerebrospinal fluid barrier model.

      Häuser, Svenja; Wegele, Christian; Stump-Guthier, Carolin; Borkowski, Julia; Weiss, Christel; Rohde, Manfred; Ishikawa, Hiroshi; Schroten, Horst; Schwerk, Christian; Adam, Rüdiger; et al. (Elsevier, 2018-07-17)
      The Gram-negative bacterium Haemophilus influenzae (H. influenzae) can commensally colonize the upper respiratory tract, but also cause life threatening disease including epiglottitis, sepsis and meningitis. The H. influenzae capsule protects the bacteria against both phagocytosis and opsonization. Encapsulated H. influenzae strains are classified into serotypes ranging from a to f dependent on their distinct polysaccharide capsule. Due to the implementation of vaccination the incidence of invasive H. influenzae type b (Hib) infections has strongly decreased and infections with other capsulated types, including H. influenzae type f (Hif), are emerging. The pathogenesis of H. influenzae meningitis is not clarified. To enter the central nervous system (CNS) the bacteria generally have to cross either the blood-brain barrier (BBB) or the blood-cerebrospinal fluid barrier (BSCFB). Using a cell culture model of the BCSFB based on human choroid plexus papilloma (HIBCPP) cells and different H. influenzae strains we investigated whether Hib and Hif invade the cells, and if invasion differs between encapsulated vs. capsular-deficient and fimbriated vs. non-fimbriated variants. We find that Hib can adhere to and invade into HIBCPP cells. Invasion occurs in a strongly polar fashion, since the bacteria enter the cells preferentially from the basolateral "blood "side. Fimbriae and capsule attenuate invasion into choroid plexus (CP) epithelial cells, and capsulation can influence the bacterial distribution pattern. Finally, analysis of clinical Hib and Hif isolates confirms the detected invasive properties of H. influenzae. Our data point to roles of capsule and fimbriae during invasion of CP epithelial cells.
    • Metabolic Rearrangements Causing Elevated Proline and Polyhydroxybutyrate Accumulation During the Osmotic Adaptation Response of .

      Godard, Thibault; Zühlke, Daniela; Richter, Georg; Wall, Melanie; Rohde, Manfred; Riedel, Katharina; Poblete-Castro, Ignacio; Krull, Rainer; Biedendieck, Rebekka; HZI,Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (Frontiers, 2020-02-21)
      For many years now, Bacillus megaterium serves as a microbial workhorse for the high-level production of recombinant proteins in the g/L-scale. However, efficient and stable production processes require the knowledge of the molecular adaptation strategies of the host organism to establish optimal environmental conditions. Here, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome, and fluxome analyses. An initial transient adaptation consisted of potassium import and glutamate counterion synthesis. The massive synthesis of the compatible solute proline constituted the second longterm adaptation process. Several stress response enzymes involved in iron scavenging and reactive oxygen species (ROS) fighting proteins showed higher levels under prolonged osmotic stress induced by 1.8 M NaCl. At the same time, the downregulation of the expression of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The increased production of lactate accompanied by the reduction of acetate secretion partially compensate for the unbalanced (NADH/NAD+) ratio. Besides, the tricarboxylic acid cycle (TCA) mainly supplies the produced NADH, as indicated by the higher mRNA and protein levels of involved enzymes, and further confirmed by 13C flux analyses. As a consequence of the metabolic flux toward acetyl-CoA and the generation of an excess of NADPH, B. megaterium redirected the produced acetyl-CoA toward the polyhydroxybutyrate (PHB) biosynthetic pathway accumulating around 30% of the cell dry weight (CDW) as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.
    • Proteomic Investigation Uncovers Potential Targets and Target Sites of Pneumococcal Serine-Threonine Kinase StkP and Phosphatase PhpP.

      Hirschfeld, Claudia; Gómez-Mejia, Alejandro; Bartel, Jürgen; Hentschker, Christian; Rohde, Manfred; Maaß, Sandra; Hammerschmidt, Sven; Becher, Dörte; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2020-02-04)
      Like eukaryotes, different bacterial species express one or more Ser/Thr kinases and phosphatases that operate in various signaling networks by catalyzing phosphorylation and dephosphorylation of proteins that can immediately regulate biochemical pathways by altering protein function. The human pathogen Streptococcus pneumoniae encodes a single Ser/Thr kinase-phosphatase couple known as StkP-PhpP, which has shown to be crucial in the regulation of cell wall synthesis and cell division. In this study, we applied proteomics to further understand the physiological role of pneumococcal PhpP and StkP with an emphasis on phosphorylation events on Ser and Thr residues. Therefore, the proteome of the non-encapsulated D39 strain (WT), a kinase (ΔstkP), and phosphatase mutant (ΔphpP) were compared in a mass spectrometry based label-free quantification experiment. Results show that a loss of function of PhpP causes an increased abundance of proteins in the phosphate uptake system Pst. Quantitative proteomic data demonstrated an effect of StkP and PhpP on the two-component systems ComDE, LiaRS, CiaRH, and VicRK. To obtain further information on the function, targets and target sites of PhpP and StkP we combined the advantages of phosphopeptide enrichment using titanium dioxide and spectral library based data evaluation for sensitive detection of changes in the phosphoproteome of the wild type and the mutant strains. According to the role of StkP in cell division we identified several proteins involved in cell wall synthesis and cell division that are apparently phosphorylated by StkP. Unlike StkP, the physiological function of the co-expressed PhpP is poorly understood. For the first time we were able to provide a list of previously unknown putative targets of PhpP. Under these new putative targets of PhpP are, among others, five proteins with direct involvement in cell division (DivIVA, GpsB) and peptidoglycan biosynthesis (MltG, MreC, MacP).
    • Discovery of Paenibacillus larvae ERIC V: Phenotypic and genomic comparison to genotypes ERIC I-IV reveal different inventories of virulence factors which correlate with epidemiological prevalences of American Foulbrood.

      Beims, Hannes; Bunk, Boyke; Erler, Silvio; Mohr, Kathrin I; Spröer, Cathrin; Pradella, Silke; Günther, Gabi; Rohde, Manfred; von der Ohe, Werner; Steinert, Michael; et al. (Elsevier, 2020-02-01)
      Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a highly contagious brood disease of honey bees (Apis mellifera). AFB requires mandatory reporting to the veterinary authority in many countries and until now four genotypes, P. larvae ERIC I-IV, have been identified. We isolated a new genotype, ERIC V, from a Spanish honey sample. After a detailed phenotypic comparison with the reference strains of the ERIC I-IV genotypes, including spore morphology, non-ribosomal peptide (NRP) profiling, and in vivo infections of A. mellifera larvae, we established a genomic DNA Macrorestriction Fragment Pattern Analysis (MRFPA) scheme for future epidemiologic discrimination. Whole genome comparison of the reference strains and the new ERIC V genotype (DSM 106052) revealed that the respective virulence gene inventories of the five genotypes corresponded with the time needed to kill 100 % of the infected bee larvae (LT100) in in vivo infection assays. The rarely isolated P. larvae genotypes ERIC II I-V with a fast-killing phenotype (LT100 3 days) harbor genes with high homology to virulence factors of other insect pathogens. These virulence genes are absent in the epidemiologically prevalent genotypes ERIC I (LT100 12 days) and ERIC II (LT100 7 days), which exhibit slower killing phenotypes. Since killing-retardation is known to reduce the success of hygienic cleaning by nurse bees, the identified absence of virulence factors might explain the epidemiological prevalences of ERIC genotypes. The discovery of the P. larvae ERIC V isolate suggests that more unknown ERIC genotypes exist in bee colonies. Since inactivation or loss of a few genes can transform a fast-killing phenotype into a more dangerous slow-killing phenotype, these rarely isolated genotypes may represent a hidden reservoir for future AFB outbreaks.
    • Hypericibacter terrae gen. nov., sp. nov. and sp. nov., two new members of the family isolated from the rhizosphere of Hypericum perforatum.

      Noviana, Zahra; Vieira, Selma; Pascual, Javier; Fobofou, Serge Alain Tanemossu; Rohde, Manfred; Spröer, Cathrin; Bunk, Boyke; Overmann, Jorg; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.; German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, 38124 Braunschweig, Germany. (Microbiology Society, 2020-01-20)
      Two strains of the family Rhodospirillaceae were isolated from the rhizosphere of the medicinal plant Hypericum perforatum. Cells of both strains were Gram-stain-negative, motile by means of a single polar flagellum, non-spore-forming, non-capsulated, short rods that divided by binary fission. Colonies were small and white. Strains R5913T and R5959T were oxidase-positive, mesophilic, neutrophilic and grew optimally without NaCl. Both grew under aerobic and microaerophilic conditions and on a limited range of substrates with best results on yeast extract. Major fatty acids were C19 : 0 cyclo ω8c and C16 : 0; in addition, C18 : 1ω7c was also found as a predominant fatty acid in strain R5913T. The major respiratory quinone was ubiquinone 10 (Q-10). The DNA G+C contents of strains R5913T and R5959T were 66.0 and 67.4 mol%, respectively. 16S rRNA gene sequence comparison revealed that the closest relatives (<92 % similarity) of the strains are Oceanibaculum pacificum MCCC 1A02656T, Dongia mobilis CGMCC 1.7660T, Dongia soli D78T and Dongia rigui 04SU4-PT. The two novel strains shared 98.6 % sequence similarity and represent different species on the basis of low average nucleotide identity of their genomes (83.8 %). Based on the combined phenotypic, genomic and phylogenetic investigations, the two strains represent two novel species of a new genus in the family Rhodospirillaceae, for which the name Hypericibacter gen. nov. is proposed, comprising the type species Hypericibacter terrae sp. nov. (type strain R5913T=DSM 109816T=CECT 9472T) and Hypericibacter adhaerens sp. nov. (type strain R5959T=DSM 109817T=CECT 9620T).
    • The immunogenic potential of bacterial flagella for Salmonella-mediated tumor therapy.

      Felgner, Sebastian; Spöring, Imke; Pawar, Vinay; Kocijancic, Dino; Preusse, Matthias; Falk, Christine; Rohde, Manfred; Häussler, Susanne; Weiss, Siegfried; Erhardt, Marc; et al. (Wiley-Blackwell, 2019-11-21)
      Genetically engineered Salmonella Typhimurium are potent vectors for prophylactic and therapeutic measures against pathogens as well as cancer. This is based on the potent adjuvanticity that supports strong immune responses. The physiology of Salmonella is well understood. It simplifies engineering of both enhanced immune‐stimulatory properties as well as safety features, thus, resulting in an appropriate balance between attenuation and efficacy for clinical applications. A major virulence factor of Salmonella is the flagellum. It is also a strong pathogen‐associated molecular pattern recognized by extra‐ and intracellular receptors of immune cells of the host. At the same time, it represents a serious metabolic burden. Accordingly, the bacteria evolved tight regulatory mechanisms that control flagella synthesis in vivo. Here, we systematically investigated the immunogenicity and adjuvant properties of various flagella mutants of Salmonella in vitro and in a mouse cancer model in vivo. We found that mutants lacking the flagellum‐specific ATPase FliHIJ or the inner membrane ring FliF displayed the greatest stimulatory capacity and strongest anti‐tumor effects, while remaining safe in vivo. Scanning electron microscopy revealed the presence of outer membrane vesicles in the ΔfliF and ΔfliHIJ mutants. Finally, the combination of the ΔfliF and ΔfliHIJ mutations with our previously described attenuated and immunogenic background strain SF102 displayed strong efficacy against the highly resistant cancer cell line RenCa. We thus conclude that manipulating flagella biosynthesis has great potential for the construction of highly efficacious and versatile Salmonella vector strains.
    • Prothrombotic and Proinflammatory Activities of the β-Hemolytic Group B Streptococcal Pigment.

      Siemens, Nikolai; Oehmcke-Hecht, Sonja; Hoßmann, Jörn; Skorka, Sebastian B; Nijhuis, Roel H T; Ruppen, Corinne; Skrede, Steinar; Rohde, Manfred; Schultz, Daniel; Lalk, Michael; et al. (Karger, 2019-11-19)
      A prominent feature of severe streptococcal infections is the profound inflammatory response that contributes to systemic toxicity. In sepsis the dysregulated host response involves both immunological and nonimmunological pathways. Here, we report a fatal case of an immunocompetent healthy female presenting with toxic shock and purpura fulminans caused by group B streptococcus (GBS; serotype III, CC19). The strain (LUMC16) was pigmented and hyperhemolytic. Stimulation of human primary cells with hyperhemolytic LUMC16 and STSS/NF-HH strains and pigment toxin resulted in a release of proinflammatory mediators, including tumor necrosis factor, interleukin (IL)-1β, and IL-6. In addition, LUMC16 induced blood clotting and showed factor XII activity on its surface, which was linked to the presence of the pigment. The expression of pigment was not linked to a mutation within the CovR/S region. In conclusion, our study shows that the hemolytic lipid toxin contributes to the ability of GBS to cause systemic hyperinflammation and interferes with the coagulation system.
    • Rubinisphaera italica sp. nov. isolated from a hydrothermal area in the Tyrrhenian Sea close to the volcanic island Panarea.

      Kallscheuer, Nicolai; Jogler, Mareike; Wiegand, Sandra; Peeters, Stijn H; Heuer, Anja; Boedeker, Christian; Jetten, Mike S M; Rohde, Manfred; Jogler, Christian (Springer, 2019-11-26)
      Planctomycetes is a fascinating phylum of mostly aquatic bacteria, not only due to the environmental importance in global carbon and nitrogen cycles, but also because of a unique cell biology. Their lifestyle and metabolic capabilities are not well explored, which motivated us to study the role of Planctomycetes in biofilms on marine biotic surfaces. Here, we describe the novel strain Pan54T which was isolated from algae in a hydrothermal area close to the volcanic island Panarea in the Tyrrhenian Sea, north of Sicily in Italy. The strain grew best at pH 9.0 and 26 °C and showed typical characteristics of planctomycetal bacteria, e.g. division by polar budding, formation of aggregates and presence of stalks and crateriform structures. Phylogenetically, the strain belongs to the genus Rubinisphaera. Our analysis suggests that Pan54T represents a novel species of this genus, for which we propose the name Rubinisphaera italica sp. nov. We suggest Pan54T (= DSM 29369 = LMG 29789) as the type strain of the novel species.
    • Non-Invasive Approach for Evaluation of Pulmonary Hypertension Using Extracellular Vesicle-Associated Small Non-Coding RNA.

      Lipps, Christoph; Northe, Philipp; Figueiredo, Ricardo; Rohde, Manfred; Brahmer, Alexandra; Krämer-Albers, Eva-Maria; Liebetrau, Christoph; Wiedenroth, Christoph B; Mayer, Eckhard; Kriechbaum, Steffen D; et al. (MDPI, 2019-10-29)
      Extracellular vesicles are released by numerous cell types of the human body under physiological but also under pathophysiological conditions. They are important for cell-cell communication and carry specific signatures of peptides and RNAs. In this study, we aimed to determine whether extracellular vesicles isolated from patients with pulmonary hypertension show a disease specific signature of small non-coding RNAs and thus have the potential to serve as diagnostic and prognostic biomarkers. Extracellular vesicles were isolated from the serum of 23 patients with chronic thromboembolic pulmonary hypertension (CTEPH) and 23 controls using two individual methods: a column-based method or by precipitation. Extracellular vesicle- associated RNAs were analyzed by next-generation sequencing applying molecular barcoding, and differentially expressed small non-coding RNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). We identified 18 microRNAs and 21 P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) or piRNA clusters that were differentially expressed in CTEPH patients compared with controls. Bioinformatic analysis predicted a contribution of these piRNAs to the progression of cardiac and vascular remodeling. Expression levels of DQ593039 correlated with clinically meaningful parameters such as mean pulmonary arterial pressure, pulmonary vascular resistance, right ventricular systolic pressure, and levels of N-terminal pro-brain natriuretic peptide. Thus, we identified the extracellular vesicle- derived piRNA, DQ593039, as a potential biomarker for pulmonary hypertension and right heart disease.
    • R18C is a new viable P2-like bacteriophage of rabbit origin infecting Citrobacter rodentium and Shigella sonnei strains.

      Sváb, Domonkos; Horváth, Balázs; Rohde, Manfred; Maróti, Gergely; Tóth, István; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer-Nature, 2019-10-23)
      Here, we report a novel virulent P2-like bacteriophage, R18C, isolated from rabbit faeces, which, in addition to Escherichia coli K-12 strains, was able to be propagated on Citrobacter rodentium strain ICC169 and a range of Shigella sonnei strains with high efficiency of plating (EOP). It represents the first lytic bacteriophage originating from rabbit and the first infectious P2-like phage of animal origin. In the three characteristic moron-containing regions of P2-like phages, R18C contains genes with unknown function that have so far only been found in cryptic P2-like prophages.
    • The secRNome of Listeria monocytogenes Harbors Small Noncoding RNAs That Are Potent Inducers of Beta Interferon.

      Frantz, Renate; Teubner, Lisa; Schultze, Tilman; La Pietra, Luigi; Müller, Christin; Gwozdzinski, Konrad; Pillich, Helena; Hain, Torsten; Weber-Gerlach, Michaela; Panagiotidis, Georgios-Dimitrios; et al. (ASM, 2019-10-08)
      Cellular sensing of bacterial RNA is increasingly recognized as a determinant of host-pathogen interactions. The intracellular pathogen Listeria monocytogenes induces high levels of type I interferons (alpha/beta interferons [IFN-α/β]) to create a growth-permissive microenvironment during infection. We previously demonstrated that RNAs secreted by L. monocytogenes (comprising the secRNome) are potent inducers of IFN-β. We determined the composition and diversity of the members of the secRNome and found that they are uniquely enriched for noncoding small RNAs (sRNAs). Testing of individual sRNAs for their ability to induce IFN revealed several sRNAs with this property. We examined ril32, an intracellularly expressed sRNA that is highly conserved for the species L. monocytogenes and that was the most potent inducer of IFN-β expression of all the sRNAs tested in this study, in more detail. The rli32-induced IFN-β response is RIG-I (retinoic acid inducible gene I) dependent, and cells primed with rli32 inhibit influenza virus replication. We determined the rli32 motif required for IFN induction. rli32 overproduction promotes intracellular bacterial growth, and a mutant lacking rli32 is restricted for intracellular growth in macrophages. rli32-overproducing bacteria are resistant to H2O2 and exhibit both increased catalase activity and changes in the cell envelope. Comparative transcriptome sequencing (RNA-Seq) analysis indicated that ril32 regulates expression of the lhrC locus, previously shown to be involved in cell envelope stress. Inhibition of IFN-β signaling by ruxolitinib reduced rli32-dependent intracellular bacterial growth, indicating a link between induction of the interferon system and bacterial physiology. rli32 is, to the best of our knowledge, the first secreted individual bacterial sRNA known to trigger the induction of the type I IFN response.IMPORTANCE Interferons are potent and broadly acting cytokines that stimulate cellular responses to nucleic acids of unusual structures or locations. While protective when induced following viral infections, the induction of interferons is detrimental to the host during L. monocytogenes infection. Here, we identify specific sRNAs, secreted by the bacterium, with the capacity to induce type I IFN. Further analysis of the most potent sRNA, rli32, links the ability to induce RIG-I-dependent induction of the type I IFN response to the intracellular growth properties of the bacterium. Our findings emphasize the significance of released RNA for Listeria infection and shed light on a compartmental strategy used by an intracellular pathogen to modulate host responses to its advantage.
    • The Gram-Positive Bacterial Cell Wall

      Rohde, Manfred; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Society for Microbiology, 2019-05-24)
      The chapter about the Gram-positive bacterial cell wall gives a brief historical background on the discovery of Gram-positive cell walls and their constituents and microscopic methods applied for studying the Gram-positive cell envelope. Followed by the description of the different chemical building blocks of peptidoglycan and the biosynthesis of the peptidoglycan layers and high turnover of peptidoglycan during bacterial growth. Lipoteichoic acids and wall teichoic acids are highlighted as major components of the cell wall. Characterization of capsules and the formation of extracellular vesicles by Gram-positive bacteria close the section on cell envelopes which have a high impact on bacterial pathogenesis. In addition, the specialized complex and unusual cell wall of mycobacteria is introduced thereafter. Next a short back view is given on the development of electron microscopic examinations for studying bacterial cell walls. Different electron microscopic techniques and methods applied to examine bacterial cell envelopes are discussed in the view that most of the illustrated methods should be available in a well-equipped life sciences orientated electron microscopic laboratory. In addition, newly developed and mostly well-established cryo-methods like high-pressure freezing and freeze-substitution (HPF-FS) and cryo-sections of hydrated vitrified bacteria (CEMOVIS, Cryo-electron microscopy of vitreous sections) are described. At last, modern cryo-methods like cryo-electron tomography (CET) and cryo-FIB-SEM milling (focus ion beamscanning electron microscopy) are introduced which are available only in specialized institutions, but at present represent the best available methods and techniques to study Gram-positive cell walls under close-to-nature conditions in great detail and at high resolution.
    • Mitochondria Are a Subset of Extracellular Vesicles Released by Activated Monocytes and Induce Type I IFN and TNF Responses in Endothelial Cells.

      Puhm, Florian; Afonyushkin, Taras; Resch, Ulrike; Obermayer, Georg; Rohde, Manfred; Penz, Thomas; Schuster, Michael; Wagner, Gabriel; Rendeiro, Andre F; Melki, Imene; et al. (Lippincott,Williams & Wilkins, 2019-06-21)
      Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood. OBJECTIVE: To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells. METHODS AND RESULTS: LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO. CONCLUSIONS: Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.
    • Corrigendum: Fuerstia marisgermanicae gen. nov., sp. nov., an Unusual Member of the Phylum Planctomycetes From the German Wadden Sea.

      Kohn, Timo; Heuer, Anja; Jogler, Mareike; Vollmers, John; Boedeker, Christian; Bunk, Boyke; Rast, Patrick; Borchert, Daniela; Glöckner, Ines; Freese, Heike M; et al. (Frontiers, 2019-01-01)
    • Fuerstia marisgermanicae gen. nov., sp. nov., an Unusual Member of the Phylum Planctomycetes from the German Wadden Sea.

      Kohn, Timo; Heuer, Anja; Jogler, Mareike; Vollmers, John; Boedeker, Christian; Bunk, Boyke; Rast, Patrick; Borchert, Daniela; Glöckner, Ines; Freese, Heike M; et al. (Frontiers, 2016-01-01)
      Members of the phylum Planctomycetes are ubiquitous bacteria that dwell in aquatic and terrestrial habitats. While planctomycetal species are important players in the global carbon and nitrogen cycle, this phylum is still undersampled and only few genome sequences are available. Here we describe strain NH11T, a novel planctomycete obtained from a crustacean shell (Wadden Sea, Germany). The phylogenetically closest related cultivated species is Gimesia maris, sharing only 87% 16S rRNA sequence identity. Previous isolation attempts have mostly yielded members of the genus Rhodopirellula from water of the German North Sea. On the other hand, only one axenic culture of the genus Pirellula was obtained from a crustacean thus far. However, the 16S rRNA gene sequence of strain NH11T shares only 80% sequence identity with the closest relative of both genera, Rhodopirellula and Pirellula. Thus, strain NH11T is unique in terms of origin and phylogeny. While the pear to ovoid shaped cells of strain NH11T are typical planctomycetal, light-, and electron microscopic observations point toward an unusual variation of cell division through budding: during the division process daughter- and mother cells are connected by an unseen thin tubular-like structure. Furthermore, the periplasmic space of strain NH11T was unusually enlarged and differed from previously known planctomycetes. The complete genome of strain NH11T, with almost 9 Mb in size, is among the largest planctomycetal genomes sequenced thus far, but harbors only 6645 protein-coding genes. The acquisition of genomic components by horizontal gene transfer is indicated by the presence of numerous putative genomic islands. Strikingly, 45 "giant genes" were found within the genome of NH11T. Subsequent analysis of all available planctomycetal genomes revealed that Planctomycetes as such are especially rich in "giant genes". Furthermore, Multilocus Sequence Analysis (MLSA) tree reconstruction support the phylogenetic distance of strain NH11T from other cultivated Planctomycetes of the same phylogenetic cluster. Thus, based on our findings, we propose to classify strain NH11T as Fuerstia marisgermanicae gen. nov., sp. nov., with the type strain NH11T, within the phylum Planctomycetes.
    • IL-1β Promotes Biofilms on Implants .

      Gutierrez Jauregui, Rodrigo; Fleige, Henrike; Bubke, Anja; Rohde, Manfred; Weiss, Siegfried; Förster, Reinhold; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2019-01-01)
      Implant associated infections represent a serious health burden in clinics since some microorganisms are able to colonize biological surfaces or surfaces of indwelling medical devices and form biofilms. Biofilms represent communities of microorganisms attached to hydrated surfaces and enclosed in self-produced extracellular matrix. This renders them resistant to exogenous assaults like antibiotics or immune effector mechanisms. Little is known regarding the role of the immune system in the formation of biofilms during implant associated infections, largely due to the lack of suitable mouse models. Here we use colonized osmotic pumps in mice to study the interaction of an activated immune system with biofilm-forming Staphylococcus aureus encoding Gaussia luciferase. This approach permits biofilm formation on the osmotic pumps in living animals. It also allows the continuous supply of soluble immune cell activating agents, such as cytokines to study their effect on biofilm formation in vivo. Using non-invasive imaging of the bioluminescent signal emitted by the lux expressing bacteria for quantification of bacterial load in conjunction with light and electron microscopy, we observed that pump-supplied pro-inflammatory cytokine IL-1β strongly increased biofilm formation along with a massive influx of neutrophils adjacent to the biofilm-coated pumps. Thus, our data demonstrate that immune defense mechanisms can augment biofilm formation.
    • Still Something to Discover: Novel Insights into Phage Diversity and Taxonomy.

      Korf, Imke H E; Meier-Kolthoff, Jan P; Adriaenssens, Evelien M; Kropinski, Andrew M; Nimtz, Manfred; Rohde, Manfred; van Raaij, Mark J; Wittmann, Johannes; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2019-05-17)
      The aim of this study was to gain further insight into the diversity of Escherichia coli phagesfollowed by enhanced work on taxonomic issues in that field. Therefore, we present the genomiccharacterization and taxonomic classification of 50 bacteriophages against E. coli isolated fromvarious sources, such as manure or sewage. All phages were examined for their host range on a setof different E. coli strains, originating, e.g., from human diagnostic laboratories or poultry farms.Transmission electron microscopy revealed a diversity of morphotypes (70% Myo-, 22% Sipho-, and8% Podoviruses), and genome sequencing resulted in genomes sizes from ~44 to ~370 kb.Annotation and comparison with databases showed similarities in particular to T4- and T5-likephages, but also to less-known groups. Though various phages against E. coli are already describedin literature and databases, we still isolated phages that showed no or only few similarities to otherphages, namely phages Goslar, PTXU04, and KWBSE43-6. Genome-based phylogeny andclassification of the newly isolated phages using VICTOR resulted in the proposal of new generaand led to an enhanced taxonomic classification of E. coli phages.
    • Three glycosylated serine-rich repeat proteins play a pivotal role in adhesion and colonization of the pioneer commensal bacterium, Streptococcus salivarius.

      Couvigny, Benoit; Lapaque, Nicolas; Rigottier-Gois, Lionel; Guillot, Alain; Chat, Sophie; Meylheuc, Thierry; Kulakauskas, Saulius; Rohde, M; Mistou, Michel-Yves; Renault, Pierre; et al. (Wiley-Blackwell, 2017-01-01)
      Bacterial adhesion is a critical step for colonization of the host. The pioneer colonizer and commensal bacterium of the human gastrointestinal tract, Streptococcus salivarius, has strong adhesive properties but the molecular determinants of this adhesion remain uncharacterized. Serine‐rich repeat (SRR) glycoproteins are a family of adhesins that fulfil an important role in adhesion. In general, Gram‐positive bacterial genomes have a unique SRR glycoprotein‐encoding gene. We demonstrate that S. salivarius expresses three large and glycosylated surface‐exposed proteins – SrpA, SrpB and SrpC – that show characteristics of SRR glycoproteins and are secreted through the accessory SecA2/Y2 system. Two glycosyltransferases – GtfE/F – encoded outside of the secA2/Y2 locus, unusually, perform the first step of the sequential glycosylation process, which is crucial for SRR activity. We show that SrpB and SrpC play complementary adhesive roles involved in several steps of the colonization process: auto‐aggregation, biofilm formation and adhesion to a variety of host epithelial cells and components. We also show that at least one of the S. salivarius SRR glycoproteins is important for colonization in mice. SrpA, SrpB and SrpC are the main factors underlying the multifaceted adhesion of S. salivarius and, therefore, play a major role in host colonization.