• 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.
    • 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.
    • 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.
    • 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.
    • DncV Synthesizes Cyclic GMP-AMP and Regulates Biofilm Formation and Motility in ECOR31.

      Li, Fengyang; Cimdins, Annika; Rohde, Manfred; Jänsch, Lothar; Kaever, Volkhard; Nimtz, Manfred; Römling, Ute; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (ASM, 2019-03-05)
      Cyclic dinucleotides (cDNs) act as intracellular second messengers, modulating bacterial physiology to regulate the fundamental life style transition between motility and sessility commonly known as biofilm formation. Cyclic GMP-AMP (cGAMP), synthesized by the dinucleotide cyclase DncV, is a newly discovered cDN second messenger involved in virulence and chemotaxis in Vibrio cholerae O1 biovar El Tor. Here we report a novel role for horizontally transferred DncV in cGAMP production and regulation of biofilm formation and motility in the animal commensal strain Escherichia coli ECOR31. ECOR31 expresses a semiconstitutive temperature-independent rdar (red, dry, and rough) morphotype on Congo red agar plates characterized by the extracellular matrix components cellulose and curli fimbriae which requires activation by the major biofilm regulator CsgD and cyclic di-GMP signaling. In contrast, C-terminal His-tagged DncV negatively regulates the rdar biofilm morphotype and cell aggregation via downregulation of csgD mRNA steady-state level. Furthermore, DncV sequentially promotes and inhibits adhesion to the abiotic surface after 24 h and 48 h of growth, respectively. DncV also suppresses swimming and swarming motility posttranscriptional of the class 1 flagellum regulon gene flhD Purified DncV produced different cDNs, cyclic di-GMP, cyclic di-AMP, an unknown product(s), and the dominant species 3'3'-cGAMP. In vivo, only the 3'3'-cGAMP concentration was elevated upon short-term overexpression of dncV, making this work a first report on cGAMP production in E. coli Regulation of rdar biofilm formation and motility upon overexpression of untagged DncV in combination with three adjacent cotransferred gene products suggests a novel temperature-dependent cGAMP signaling module in E. coli ECOR31.IMPORTANCE The ability of bacteria to sense and respond to environmental signals is critical for survival. Bacteria use cyclic dinucleotides as second messengers to regulate a number of physiological processes, such as the fundamental life style transition between motility and sessility (biofilm formation). cGAMP, which is synthesized by a dinucleotide cyclase called DncV, is a newly discovered second messenger involved in virulence and chemotaxis in the Vibrio cholerae biovar El Tor causing the current 7th cholera pandemic. However, to what extent cGAMP exists and participates in physiological processes in other bacteria is still unknown. In this study, we found an elevated cGAMP level to possibly regulate biofilm formation and motility in the animal commensal E. coli strain ECOR31. Thus, we detected a novel role for cGAMP signaling in regulation of physiological processes other than those previously reported in proteobacterial species.
    • Microbiome yarns: the Global Phenotype-Genotype Survey: Episode II: laryngeal microbiota and vocal phenotypes (or diction and addiction).

      Timmis, Kenneth; Jebok, Franziska; Rohde, M; Molinari, Gabriella; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (Wiley-Blackwell, 2019-03-01)
    • 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.
    • Corrigendum: 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)
    • Sulfate-Reducing Bacteria That Produce Exopolymers Thrive in the Calcifying Zone of a Hypersaline Cyanobacterial Mat.

      Spring, Stefan; Sorokin, Dimitry Y; Verbarg, Susanne; Rohde, M; Woyke, Tanja; Kyrpides, Nikos C; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (Frontiers, 2019-01-01)
      Calcifying microbial mats in hypersaline environments are important model systems for the study of the earliest ecosystems on Earth that started to appear more than three billion years ago and have been preserved in the fossil record as laminated lithified structures known as stromatolites. It is believed that sulfate-reducing bacteria play a pivotal role in the lithification process by increasing the saturation index of calcium minerals within the mat. Strain L21-Syr-ABT was isolated from anoxic samples of a several centimeters-thick microbialite-forming cyanobacterial mat of a hypersaline lake on the Kiritimati Atoll (Kiribati, Central Pacific). The novel isolate was assigned to the family Desulfovibrionaceae within the Deltaproteobacteria. Available 16S rRNA-based population surveys obtained from discrete layers of the mat indicate that the occurrence of a species-level clade represented by strain L21-Syr-ABT is restricted to a specific layer of the suboxic zone, which is characterized by the presence of aragonitic spherulites. To elucidate a possible function of this sulfate-reducing bacterium in the mineral formation within the mat a comprehensive phenotypic characterization was combined with the results of a comparative genome analysis. Among the determined traits of strain L21-Syr-ABT, several features were identified that could play a role in the precipitation of calcium carbonate: (i) the potential deacetylation of polysaccharides and consumption of substrates such as lactate and sulfate could mobilize free calcium; (ii) under conditions that favor the utilization of formate and hydrogen, the alkalinity engine within the mat is stimulated, thereby increasing the availability of carbonate; (iii) the production of extracellular polysaccharides could provide nucleation sites for calcium mineralization. In addition, our data suggest the proposal of the novel species and genus Desulfohalovibrio reitneri represented by the type strain L21-Syr-ABT (=DSM 26903T = JCM 18662T).
    • Homophilic protein interactions facilitate bacterial aggregation and IgG-dependent complex formation by the Streptococcus canis M protein SCM.

      Nerlich, Andreas; Lapschies, Antje-Maria; Kohler, Thomas P; Cornax, Ingrid; Eichhorn, Inga; Goldmann, Oliver; Krienke, Petra; Bergmann, Simone; Nizet, Victor; Hammerschmidt, Sven; et al. (Taylor & Francis, 2019-01-01)
      Streptococcus canis is a zoonotic agent that causes serious invasive diseases in domestic animals and humans, but knowledge about its pathogenic potential and underlying virulence mechanisms is limited. Here, we report on the ability of certain S. canis isolates to form large bacterial aggregates when grown in liquid broth. Bacterial aggregation was attributed to the presence and the self-binding activity of SCM, the M protein of S. canis, as evaluated by bacterial sedimentation assays, immunofluorescence- and electron microscopic approaches. Using a variety of truncated recombinant SCM fragments, we demonstrated that homophilic SCM interactions occur via the N-terminal, but not the C-terminal part, of the mature M protein. Interestingly, when incubated in human plasma, SCM forms soluble protein complexes comprising its known ligands, immunoglobulin G (IgG) and plasminogen (Plg). Co-incubation studies with purified host proteins revealed that SCM-mediated complex formation is based on the interaction of SCM with itself and with IgG, but not with Plg or fibrinogen (Fbg), well-established constituents of M protein-mediated protein complexes in human-associated streptococci. Notably, these soluble, SCM-mediated plasma complexes harbored complement factor C1q, which can induce complement breakdown in the periphery and therefore represent another immune evasion mechanism of SCM.
    • Microbiome yarns: The Global Phenotype-Genotype Survey: Episode I: all my worldly goods, including my microbiome, I thee endow.

      Timmis, Kenneth; Jebok, Franziska; Rohde, M; Molinari, Gabriella; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley and Sons, 2019-01-01)
    • Molecular typing of Streptococcus suis strains isolated from diseased and healthy pigs between 1996-2016.

      Prüfer, T Louise; Rohde, Judith; Verspohl, Jutta; Rohde, M; de Greeff, Astrid; Willenborg, Jörg; Valentin-Weigand, Peter; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (PLOS, 2019-01-01)
      Streptococcus suis is an economically important pathogen of pigs as well as a zoonotic cause of human disease. Serotyping is used for further characterization of isolates; some serotypes seem to be more virulent and more widely spread than others. This study characterizes a collection of German field isolates of Streptococcus suis from pigs dating from 1996 to 2016 with respect to capsular genes (cps) specific for individual serotypes and pathotype by multiplex PCR and relates results to the clinical background of these isolates. The most prominent finding was the reduction in prevalence of serotype-2/serotype-1/2 among invasive isolates during this sampling period, which might be attributed to widely implemented autogenous vaccination programs in swine against serotype 2 in Germany. In diseased pigs (systemically ill; respiratory disease) isolates of serotype-1/serotype-14, serotype-2/serotype-1/2, serotype 3 to 5 and 7 to 9 were most frequent while in carrier isolates a greater variety of cps types was found. Serotype-1/serotype-14 seemed to be preferentially located in joints, serotype 4 and serotype 3 in the central nervous system, respectively. The virulence associated extracellular protein factor was almost exclusively associated with invasive serotype-1/serotype-14 and serotype-2/serotype-1/2 isolates. In contrast, lung isolates of serotype-2/serotype-1/2 mainly harbored the gene for muramidase-released protein. Serotype 4 and serotype 9 isolates from clinically diseased pigs most frequently carried the muramidase-released protein gene and the suilysin gene. When examined by transmission electron microscopy all but one of the isolates which were non-typable by molecular and serological methods showed various amounts of capsular material indicating potentially new serotypes among these isolates. Given the variety of cps types/serotypes detected in pigs, not only veterinarians but also medical doctors should consider other serotypes than just serotype 2 when investigating potential human cases of Streptococcus suis infection.
    • Intracellular Streptococcal Uptake and Persistence: A Potential Cause of Erysipelas Recurrence.

      Jendoubi, Fatma; Rohde, M; Prinz, Jörg Christoph; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2019-01-01)
      Erysipelas is a severe streptococcal infection of the skin primarily spreading through the lymphatic vessels. Penicillin is the treatment of choice. The most common complication consists in relapses which occur in up to 40% or more of patients despite appropriate antibiotic treatment. They cause lymphatic damage resulting in irreversible lymphedema and ultimately elephantiasis nostras and lead to major health restrictions and high socio-medical costs. Prevention of relapses is an unmet need, because even long-term prophylactic penicillin application does eventually not reduce the risk of recurrence. In this article we assess risk factors and causes of erysipelas recurrence. A systematic literature search for clinical studies addressing potential causes and measures for prevention of erysipelas recurrence was combined with a review of experimental and clinical data assessing the ability and clinical relevance of streptococci for intracellular uptake and persistence. The literature review found that venous insufficiency, lymphedema, and intertrigo from fungal infections are considered to be major risk factors for recurrence of erysipelas but cannot adequately explain the high recurrence rate. As hitherto unrecognized likely cause of erysipelas relapses we identify the ability of streptococci for intracellular uptake into and persistence within epithelial and endothelial cells and macrophages. This creates intracellular streptococcal reservoirs out of reach of penicillins which do not reach sufficient bactericidal intracellular concentrations. Incomplete streptococcal elimination due to intracellular streptococcal persistence has been observed in various deep tissue infections and is considered as cause of relapsing streptococcal pharyngitis despite proper antibiotic treatment. It may also serves as endogenous infectious source of erysipelas relapses. We conclude that the current antibiotic treatment strategies and elimination of conventional risk factors employed in erysipelas management are insufficient to prevent erysipelas recurrence. The reactivation of streptococcal infection from intracellular reservoirs represents a plausible explanation for the frequent occurrence erysipelas relapses. Prevention of erysipelas relapses therefore demands for novel antibiotic strategies capable of eradicating intracellular streptococcal persistence.
    • Microbiome yarns: The Global Phenotype-Genotype Survey. Episode III: importance of microbiota diversification for microbiome function and biome health.

      Timmis, Kenneth; Jebok, Franziska; Rohde, M; Lahti, Leo; Molinari, Gabriella; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (Wiley-Blackwell, 2019-01-01)
    • Day and Night: Metabolic Profiles and Evolutionary Relationships of Six Axenic Non-Marine Cyanobacteria.

      Will, Sabine Eva; Henke, Petra; Boedeker, Christian; Huang, Sixing; Brinkmann, Henner; Rohde, M; Jarek, Michael; Friedl, Thomas; Seufert, Steph; Schumacher, Martin; et al. (Oxford Academic, 2019-01-01)
      Cyanobacteria are dominant primary producers of various ecosystems and they colonize marine as well as freshwater and terrestrial habitats. On the basis of their oxygenic photosynthesis they are known to synthesize a high number of secondary metabolites, which makes them promising for biotechnological applications. State-of-the-art sequencing and analytical techniques and the availability of several axenic strains offer new opportunities for the understanding of the hidden metabolic potential of cyanobacteria beyond those of single model organisms. Here, we report comprehensive genomic and metabolic analyses of five non-marine cyanobacteria, that is, Nostoc sp. DSM 107007, Anabaena variabilis DSM 107003, Calothrix desertica DSM 106972, Chroococcidiopsis cubana DSM 107010, Chlorogloeopsis sp. PCC 6912, and the reference strain Synechocystis sp. PCC 6803. Five strains that are prevalently belonging to the order Nostocales represent the phylogenetic depth of clade B1, a morphologically highly diverse sister lineage of clade B2 that includes strain PCC 6803. Genome sequencing, light and scanning electron microscopy revealed the characteristics and axenicity of the analyzed strains. Phylogenetic comparisons showed the limits of the 16S rRNA gene for the classification of cyanobacteria, but documented the applicability of a multilocus sequence alignment analysis based on 43 conserved protein markers. The analysis of metabolites of the core carbon metabolism showed parts of highly conserved metabolic pathways as well as lineage specific pathways such as the glyoxylate shunt, which was acquired by cyanobacteria at least twice via horizontal gene transfer. Major metabolic changes were observed when we compared alterations between day and night samples. Furthermore, our results showed metabolic potential of cyanobacteria beyond Synechocystis sp. PCC 6803 as model organism and may encourage the cyanobacterial community to broaden their research to related organisms with higher metabolic activity in the desired pathways.
    • Isolation, characterization and analysis of bacteriophages from the haloalkaline lake Elmenteita, Kenya.

      Akhwale, Juliah Khayeli; Rohde, M; Rohde, Christine; Bunk, Boyke; Spröer, Cathrin; Boga, Hamadi Iddi; Klenk, Hans-Peter; Wittmann, Johannes; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (PLOS, 2019-01-01)
      As a step towards better understanding of diversity and biology of phages and their hosts in haloalkaline Lake Elmenteita, phages were isolated from sediment samples and overlying water using indigenous bacteria as hosts. 17 seemingly different phages of diverse morphotypes with different dimensions and partly exhibiting remarkably unusual ultrastructures were revealed by transmission electron microscopy. 12 clonal phage isolates were further characterized. Infection capability of the phages was optimum at 30–35°C and in alkali condition with optimum at pH 10–12. Structural protein profiles and restriction fragment length polymorphism analyses patterns were distinct for each of the phage type. Complete nucleotide sequences of phages vB-VmeM-32, vB_EauS-123 and vB_BhaS-171 genomes varied in size from 30,926–199,912 bp and G + C content of between 36.25–47.73%. A range of 56–260 potential open reading frames were identified and annotated. The results showed that the 12 phages were distinct from each other and confirmed the presence and diversity of phages in extreme environment of haloalkaline Lake Elmenteita. The phages were deposited at the German Collection of Microorganisms and Cell Cultures and three of their genomes uploaded to NCBI GenBank.
    • Comparative genomic analysis of eight novel haloalkaliphilic bacteriophages from Lake Elmenteita, Kenya.

      Akhwale, Juliah Khayeli; Rohde, M; Rohde, Christine; Bunk, Boyke; Spröer, Cathrin; Klenk, Hans-Peter; Boga, Hamadi Iddi; Wittmann, Johannes; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (PLOS, 2019-01-01)
      We report complete genome sequences of eight bacteriophages isolated from Haloalkaline Lake Elmenteita found on the floor of Kenyan Rift Valley. The bacteriophages were sequenced, annotated and a comparative genomic analysis using various Bioinformatics tools carried out to determine relatedness of the bacteriophages to each other, and to those in public databases. Basic genome properties like genome size, percentage coding density, number of open reading frames, percentage GC content and gene organizations revealed the bacteriophages had no relationship to each other. Comparison to other nucleotide sequences in GenBank database showed no significant similarities hence novel. At the amino acid level, phages of our study revealed mosaicism to genes with conserved domains to already described phages. Phylogenetic analyses of large terminase gene responsible for DNA packaging and DNA polymerase gene for replication further showed diversity among the bacteriophages. Our results give insight into diversity of bacteriophages in Lake Elmenteita and provide information on their evolution. By providing primary sequence information, this study not only provides novel sequences for biotechnological exploitation, but also sets stage for future studies aimed at better understanding of virus diversity and genomes from haloalkaline lakes in the Rift Valley.
    • Macrophage entrapped silica coated superparamagnetic iron oxide particles for controlled drug release in a 3D cancer model.

      Ullah, Sami; Seidel, Katja; Türkkan, Sibel; Warwas, Dawid Peter; Dubich, Tatyana; Rohde, Manfred; Hauser, Hansjörg; Behrens, Peter; Kirschning, Andreas; Köster, Mario; et al. (2018-12-23)
      Targeted delivery of drugs is a major challenge in treatment of diverse diseases. Systemically administered drugs demand high doses and are accompanied by poor selectivity and side effects on non-target cells. Here, we introduce a new principle for targeted drug delivery. It is based on macrophages as transporters for nanoparticle-coupled drugs as well as controlled release of drugs by hyperthermia mediated disruption of the cargo cells and simultaneous deliberation of nanoparticle-linked drugs. Hyperthermia is induced by an alternating electromagnetic field (AMF) that induces heat from silica-coated superparamagnetic iron oxide nanoparticles (SPIONs). We show proof-of-principle of controlled release by the simultaneous disruption of the cargo cells and the controlled, AMF induced release of a toxin, which was covalently linked to silica-coated SPIONs via a thermo-sensitive linker. Cells that had not been loaded with SPIONs remain unaffected. Moreover, in a 3D co-culture model we demonstrate specific killing of associated tumour cells when employing a ratio as low as 1:40 (SPION-loaded macrophage: tumour cells). Overall, our results demonstrate that AMF induced drug release from macrophage-entrapped nanoparticles is tightly controlled and may be an attractive novel strategy for targeted drug release.