• Highly stable monodisperse PEGylated iron oxide nanoparticle aqueous suspensions: a nontoxic tracer for homogeneous magnetic bioassays.

      Lak, Aidin; Dieckhoff, Jan; Ludwig, Frank; Scholtyssek, Jan M; Goldmann, Oliver; Lünsdorf, Heinrich; Eberbeck, Dietmar; Kornowski, Andreas; Kraken, Mathias; Litterst, F J; et al. (2013-11-07)
      Uniformly sized and shaped iron oxide nanoparticles with a mean size of 25 nm were synthesized via decomposition of iron-oleate. High resolution transmission electron microscopy and Mössbauer spectroscopy investigations revealed that the particles are spheres primarily composed of Fe3O4 with a small fraction of FeO. From Mössbauer and static magnetization measurements, it was deduced that the particles are superparamagnetic at room temperature. The hydrophobic particles were successfully transferred into water via PEGylation using nitrodopamine as an anchoring group. IR spectroscopy and thermogravimetric analysis showed the success and efficiency of the phase transfer reaction. After PEGylation, the particles retained monodispersity and their magnetic core remained intact as proven by photon cross-correlation spectroscopy, ac susceptibility, and transmission electron microscopy. The particle aqueous suspensions revealed excellent water stability over a month of monitoring and also against temperature up to 40 °C. The particles exhibited a moderate cytotoxic effect on in vitro cultured bone marrow-derived macrophages and no release of inflammatory or anti-inflammatory cytokines. The PEGylated particles were functionalized with Herceptin antibodies via a conjugation chemistry, their response to a rotating magnetic field was studied using a fluxgate-based setup and was compared with the one recorded for hydrophobic and PEGylated particles. The particle phase lag rose after labeling with Herceptin, indicating the successful conjugation of Herceptin antibodies to the particles.
    • Host-derived extracellular RNA promotes adhesion of Streptococcus pneumoniae to endothelial and epithelial cells.

      Zakrzewicz, Dariusz; Bergmann, Simone; Didiasova, Miroslava; Giaimo, Benedetto Daniele; Borggrefe, Tilman; Mieth, Maren; Hocke, Andreas C; Lochnit, Guenter; Schaefer, Liliana; Hammerschmidt, Sven; et al. (2016-11-28)
      Streptococcus pneumoniae is the most frequent cause of community-acquired pneumonia. The infection process involves bacterial cell surface receptors, which interact with host extracellular matrix components to facilitate colonization and dissemination of bacteria. Here, we investigated the role of host-derived extracellular RNA (eRNA) in the process of pneumococcal alveolar epithelial cell infection. Our study demonstrates that eRNA dose-dependently increased S. pneumoniae invasion of alveolar epithelial cells. Extracellular enolase (Eno), a plasminogen (Plg) receptor, was identified as a novel eRNA-binding protein on S. pneumoniae surface, and six Eno eRNA-binding sites including a C-terminal 15 amino acid motif containing lysine residue 434 were characterized. Although the substitution of lysine 434 for glycine (K434G) markedly diminished the binding of eRNA to Eno, the adherence to and internalization into alveolar epithelial cells of S. pneumoniae strain carrying the C-terminal lysine deletion and the mutation of internal Plg-binding motif were only marginally impaired. Accordingly, using a mass spectrometric approach, we identified seven novel eRNA-binding proteins in pneumococcal cell wall. Given the high number of eRNA-interacting proteins on pneumococci, treatment with RNase1 completely inhibited eRNA-mediated pneumococcal alveolar epithelial cell infection. Our data support further efforts to employ RNAse1 as an antimicrobial agent to combat pneumococcal infectious diseases.
    • Host-pathogen interactions in streptococcal immune sequelae.

      Nitsche-Schmitz, D Patric; Chhatwal, Gursharan S; Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124 Braunschweig, Germany. Patric.Nitsche@helmholtz-hzi.de (2013)
      Otherwise uncomplicated infections with Streptococcus pyogenes can cause two insidious immune sequelae known as post-streptococcal glomerulonephritis (PSGN) and acute rheumatic fever (ARF). These diseases follow with a latency of a few weeks or months after primary infection and are responsible for high mortality and morbidity. PSGN has also been linked to infections with group C streptococci of the species S. equi ssp. zooepidemicus (SESZ). Moreover, there are some indications that infection with group C and G streptococci (GCGS) of the subspecies Streptococcus dysgalactiae ssp. equisimilis (SDSE) leads to ARF. Despite decades of research, the picture of the molecular pathogenesis of streptococcal immune sequelae resembles a jigsaw puzzle. Herein we try to put some of the puzzle bits together that have been collected till date.
    • Hydrogen Peroxide-Mediated Killing of Caenorhabditis elegans by Streptococcus pyogenes

      Jansen, W. T. M.; Bolm, M.; Balling, R.; Chhatwal, G. S.; Schnabel, R. (American Society for Microbiology, 2002-09)
    • Hydrogen Peroxide-Mediated Killing of Caenorhabditis elegans: a Common Feature of Different Streptococcal Species

      Bolm, Maike; Jansen, Wouter T. M.; Schnabel, Ralf; Chhatwal, Gursharan S. (American Society for Microbiology, 2004-02)
    • Identification of a streptococcal octapeptide motif involved in acute rheumatic fever.

      Dinkla, Katrin; Nitsche-Schmitz, D Patric; Barroso, Vanessa; Reissmann, Silvana; Johansson, Helena M; Frick, Inga-Maria; Rohde, Manfred; Chhatwal, Gursharan S; Department of Microbial Pathogenesis, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany. (2007-06-29)
      Acute rheumatic fever is a serious autoimmune sequela of pharyngitis caused by certain group A streptococci. One mechanism applied by streptococcal strains capable of causing acute rheumatic fever is formation of an autoantigenic complex with human collagen IV. In some geographic regions with a high incidence of acute rheumatic fever pharyngeal carriage of group C and group G streptococci prevails. Examination of such strains revealed the presence of M-like surface proteins that bind human collagen. Using a peptide array and recombinant proteins with targeted amino acid substitutions, we could demonstrate that formation of collagen complexes during streptococcal infections depends on an octapeptide motif, which is present in collagen binding M and M-like proteins of different beta-hemolytic streptococcal species. Mice immunized with streptococcal proteins that contain the collagen binding octapeptide motif developed high serum titers of anti-collagen antibodies. In sera of rheumatic fever patients such a collagen autoimmune response was accompanied by specific reactivity against the collagen-binding proteins, linking the observed effect to clinical cases. Taken together, the data demonstrate that the identified octapeptide motif through its action on collagen plays a crucial role in the pathogenesis of rheumatic fever. Eradication of streptococci that express proteins with the collagen binding motif appears advisable for controlling rheumatic fever.
    • Impact of glutamine transporters on pneumococcal fitness under infection-related conditions.

      Härtel, Tobias; Klein, Matthias; Koedel, Uwe; Rohde, Manfred; Petruschka, Lothar; Hammerschmidt, Sven; Department of Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, Ernst Moritz Arndt Universität Greifswald, Friedrich-Ludwig-Jahn-Str. 15a, D-17487 Greifswald, Germany. (2011-01)
      The genomic analysis of Streptococcus pneumoniae predicted six putative glutamine uptake systems, which are expressed under in vitro conditions, as shown here by reverse transcription-PCR. Four of these operons consist of glnHPQ, while two lack glnH, which encodes a soluble glutamine-binding protein. Here, we studied the impact of two of these glutamine ATP-binding cassette transporters on S. pneumoniae D39 virulence and phagocytosis, which consist of GlnQ and a translationally fused protein of GlnH and GlnP. Mice infected intranasally with D39Δgln0411/0412 showed significantly increased survival times and a significant delay in the development of pneumococcal pneumonia compared to those infected with D39, as observed in real time using bioluminescent pneumococci. In a mouse sepsis model, the mutant D39Δgln0411/0412 showed only moderate but significant attenuation. In contrast, the D39Δgln1098/1099 knockout strain was massively attenuated in the pneumonia and septicemia mouse infection model. To cause pneumonia or sepsis with D39Δgln1098/1099, infection doses 100- to 10,000-fold higher than those used for wild-type strain D39 were required. In an experimental mouse meningitis model, D39Δgln1098/1099 produced decreased levels of white blood cells in cerebrospinal fluid and showed decreased numbers of bacteria in the bloodstream compared to D39 and D39Δgln0411/0412. Phagocytosis experiments revealed significantly decreased intracellular survival rates of mutants D39Δgln1098/1099 and D39Δgln0411/0412 compared to wild-type D39, suggesting that the deficiency of Gln uptake systems impairs resistance to oxidative stress. Taken together, our results demonstrate that both glutamine uptake systems are required for full virulence of pneumococci but exhibit different impacts on the pathogenesis of pneumococci under in vivo conditions.
    • In situ analysis of sulfur species in sulfur globules produced from thiosulfate by Thermoanaerobacter sulfurigignens and Thermoanaerobacterium thermosulfurigenes.

      Lee, Yong-Jin; Prange, Alexander; Lichtenberg, Henning; Rohde, Manfred; Dashti, Mona; Wiegel, Juergen; Department of Microbiology, The University of Georgia, Athens, GA 30602-2605, USA. (2007-10)
      The Firmicutes Thermoanaerobacter sulfurigignens and Thermoanaerobacterium thermosulfurigenes convert thiosulfate, forming sulfur globules inside and outside cells. X-ray absorption near-edge structure analysis revealed that the sulfur consisted mainly of sulfur chains with organic end groups similar to sulfur formed in purple sulfur bacteria, suggesting the possibility that the process of sulfur globule formation by bacteria is an ancient feature.
    • Increased neutrophil extracellular trap-mediated Staphylococcus aureus clearance through inhibition of nuclease activity by clindamycin and immunoglobulin.

      Schilcher, Katrin; Andreoni, Federica; Uchiyama, Satoshi; Ogawa, Taiji; Schuepbach, Reto A; Zinkernagel, Annelies S; Helmholtz Centre for infection reseach, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014-08-01)
      The Gram-positive human pathogen Staphylococcus aureus causes a variety of human diseases such as skin infections, pneumonia, and endocarditis. The micrococcal nuclease Nuc1 is one of the major S. aureus virulence factors and allows the bacterium to avoid neutrophil extracellular trap (NET)-mediated killing. We found that addition of the protein synthesis inhibitor clindamycin to S. aureus LAC cultures decreased nuc1 transcription and subsequently blunted nuclease activity in a molecular beacon-based fluorescence assay. We also observed reduced NET degradation through Nuc1 inhibition translating into increased NET-mediated clearance. Similarly, pooled human immunoglobulin specifically inhibited nuclease activity in a concentration-dependent manner. Inhibition of nuclease activity by clindamycin and immunoglobulin enhanced S. aureus clearance and should be considered in the treatment of S. aureus infections.
    • Influence of internalin a murinisation on host resistance to orally acquired listeriosis in mice.

      Bergmann, Silke; Beard, Philippa M; Pasche, Bastian; Lienenklaus, Stefan; Weiss, Siegfried; Gahan, Cormac G M; Schughart, Klaus; Lengeling, Andreas; Infection and Immunity Division, The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush Veterinary Campus, Edinburgh EH25 9RG, UK. andreas.lengeling@roslin.ed.ac.uk. (2013)
      The bacterial surface protein internalin (InlA) is a major virulence factor of the food-born pathogen Listeria monocytogenes. It plays a critical role in the bacteria crossing the host intestinal barrier by a species-specific interaction with the cell adhesion molecule E-cadherin. In mice, the interaction of InlA with murine E-cadherin is impaired due to sequence-specific binding incompatibilities. We have previously used the approach of 'murinisation' to establish an oral listeriosis infection model in mice by exchanging two amino acid residues in InlA. This dramatically increases binding to mouse E-cadherin. In the present study, we have used bioluminescent murinised and non-murinised Listeria strains to examine the spatiotemporal dissemination of Listeria in four diverse mouse genetic backgrounds after oral inoculation.
    • Intranasal Vaccination with Streptococcal Fibronectin Binding Protein Sfb1 Fails To Prevent Growth and Dissemination of Streptococcus pyogenes in a Murine Skin Infection Model

      McArthur, J.; Medina, Eva; Mueller, A.; Chin, J.; Currie, B. J.; Sriprakash, K. S.; Talay, S. R.; Chhatwal, G. S.; Walker, M. J. (American Society for Microbiology, 2004-12)
    • Invasion mechanisms of Gram-positive pathogenic cocci.

      Nitsche-Schmitz, D Patric; Rohde, Manfred; Chhatwal, Gursharan S; Helmholtz Centre for Infection Research, Microbial Pathogenesis, Braunschweig, Germany. (2007-09)
      Gram-positive cocci are important human pathogens. Streptococci and staphylococci in particular are a major threat to human health, since they cause a variety of serious invasive infections. Their invasion into normally sterile sites of the host depends on elaborated bacterial mechanisms that involve adhesion to the host tissue, its degradation, internalisation by host cells, and passage through epithelia and endothelia. Interactions of bacterial surface proteins with proteins of the host's extracellular matrix as well as with cell surface receptors are crucial factors in these processes, and some of the key mechanisms are similar in many pathogenic Gram-positive cocci. Therapies that interfere with these mechanisms may become efficient alternatives to today's antibiotic treatments.
    • Kroppenstedtia eburnea gen. nov., sp. nov., a thermoactinomycete isolated by environmental screening, and emended description of the family Thermoactinomycetaceae Matsuo et al. 2006 emend. Yassin et al. 2009.

      von Jan, Mathias; Riegger, Nicole; Pötter, Gabriele; Schumann, Peter; Verbarg, Susanne; Spröer, Cathrin; Rohde, Manfred; Lauer, Bettina; Labeda, David P; Klenk, Hans-Peter (2011-09)
      A Gram-positive, spore-forming, aerobic, filamentous bacterium, strain JFMB-ATE(T), was isolated in 2008 during environmental screening of a plastic surface in grade C in a contract manufacturing organization in southern Germany. The isolate grew at temperatures of 25-50 °C and at pH 5.0-8.5, forming ivory-coloured colonies with sparse white aerial mycelia. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the family Thermoactinomycetaceae, except that the cell-wall peptidoglycan contained LL-diaminopimelic acid, while all previously described members of this family display this diagnostic diamino acid in meso-conformation. The DNA G+C content of the novel strain was 54.6 mol%, the main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol, and the major menaquinone was MK-7. The major fatty acids had saturated C₁₄-C₁₆ branched chains. No diagnostic sugars were detected. Based on the chemotaxonomic results and 16S rRNA gene sequence analysis, the isolate is proposed to represent a novel genus and species, Kroppenstedtia eburnea gen. nov. sp. nov. The type strain is JFMB-ATE(T) ( = DSM 45196(T)  = NRRL B-24804(T)  = CCUG 59226(T)).
    • Live Helicobacter pylori in the root canal of endodontic-infected deciduous teeth.

      Hirsch, Christian; Tegtmeyer, Nicole; Rohde, Manfred; Rowland, Marion; Oyarzabal, Omar A; Backert, Steffen; Department of Paediatric Dentistry, University School of Dental Medicine, University of Leipzig, Nuernberger Straße 57, 04103, Leipzig, Germany. Christian.Hirsch@medizin.uni-leipzig.de (2012-08)
      Many polymerase chain reaction (PCR)-based studies have shown that Helicobacter pylori DNA is prevalent in the oral cavity, but reports on the isolation of live bacteria are extremely rare. Thus, it is still unclear whether H. pylori can indeed survive in the oral environment.
    • Localization of the C3-Like ADP-ribosyltransferase from Staphylococcus aureus during bacterial invasion of mammalian cells.

      Molinari, Gabriella; Rohde, Manfred; Wilde, Christian; Just, Ingo; Aktories, Klaus; Chhatwal, Gursharan S; Department of Microbial Pathogenesis, GBF-German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany. (2006-06)
      The C3stau2 exoenzyme from Staphylococcus aureus is a C3-like ADP-ribosyltransferase which possesses no specific receptor-binding domain or translocation unit required for entry in target cells where its substrate is located. Here we show that C3stau2 can reach its target after invasion of staphylococci in eukaryotic cells without needing translocation.
    • M protein-mediated plasminogen binding is essential for the virulence of an invasive Streptococcus pyogenes isolate.

      Dinkla, K; Cole, J N; Cork, A J; Maamary, P G; McArthur, J D; Chhatwal, G S; Walker, M J; School of Biological Sciences, University of Wollongong, Wollongong, NSW, 2522, Australia. (2008-08)
      The human protease plasmin plays a crucial role in the capacity of the group A streptococcus (GAS; Streptococcus pyogenes) to initiate invasive disease. The GAS strain NS88.2 was isolated from a case of bacteremia from the Northern Territory of Australia, a region with high rates of GAS invasive disease. Mutagenesis of the NS88.2 plasminogen binding M protein Prp was undertaken to examine the contribution of plasminogen binding and cell surface plasmin acquisition to virulence. The isogenic mutant NS88.2prp was engineered whereby four amino acid residues critical for plasminogen binding were converted to alanine codons in the GAS genome sequence. The mutated residues were reverse complemented to the wild-type sequence to construct GAS strain NS88.2prpRC. In comparison to NS88.2 and NS88.2prpRC, the NS88.2prp mutant exhibited significantly reduced ability to bind human plasminogen and accumulate cell surface plasmin activity during growth in human plasma. Utilizing a humanized plasminogen mouse model of invasive infection, we demonstrate that the capacity to bind plasminogen and accumulate surface plasmin activity plays an essential role in GAS virulence.
    • The M1 protein of Streptococcus pyogenes triggers an innate uptake mechanism into polarized human endothelial cells.

      Ochel, Anja; Rohde, Manfred; Chhatwal, Gursharan S; Talay, Susanne R; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweíg, Germany. (2014)
      Serotype M1 Streptococcus pyogenes is a major human pathogen associated with severe invasive diseases causing high morbidity and mortality. In a substantial number of cases, invasive disease develops in previously healthy individuals with no obvious port of entry. This has led to the hypothesis that the source of streptococci in these cases is a transient bacteraemia. This study focuses on the analysis of interaction of tissue-invasive serotype M1 S. pyogenes with human endothelial cells (EC) of the vascular system. We identify the M1 surface protein of S. pyogenes as the EC invasin which is recognised by polarized human blood EC, thereby triggering rapid, phagocytosis-like uptake of streptococci into polarized EC layers. Upon internalization, the M1 S. pyogenes serotype is incorporated into phagosomes which traffic via the endosomal/lysosomal pathway. However, some of the streptococci successfully evade this innate killing process and hereby mediate their escape into the cytoplasm of the host cell. The results of this study demonstrate that blood EC possess an efficient uptake mechanism for serotype M1 S. pyogenes. Despite efficient phagocytosis, streptococcal survival within EC constitutes one potential mechanism which favours intracellular persistence and thus facilitates continuous infection and dissemination from the primary side of infection into deep tissue.
    • Microevolution of group A streptococci in vivo: capturing regulatory networks engaged in sociomicrobiology, niche adaptation, and hypervirulence.

      Aziz, Ramy K; Kansal, Rita; Aronow, Bruce J; Taylor, William L; Rowe, Sarah L; Kubal, Michael; Chhatwal, Gursharan S; Walker, Mark J; Kotb, Malak; Research Services, Veterans Affairs Medical Center, Memphis, Tennessee, United States of America. ramy.aziz@salmonella.org (2010)
      The onset of infection and the switch from primary to secondary niches are dramatic environmental changes that not only alter bacterial transcriptional programs, but also perturb their sociomicrobiology, often driving minor subpopulations with mutant phenotypes to prevail in specific niches. Having previously reported that M1T1 Streptococcus pyogenes become hypervirulent in mice due to selection of mutants in the covRS regulatory genes, we set out to dissect the impact of these mutations in vitro and in vivo from the impact of other adaptive events. Using a murine subcutaneous chamber model to sample the bacteria prior to selection or expansion of mutants, we compared gene expression dynamics of wild type (WT) and previously isolated animal-passaged (AP) covS mutant bacteria both in vitro and in vivo, and we found extensive transcriptional alterations of pathoadaptive and metabolic gene sets associated with invasion, immune evasion, tissue-dissemination, and metabolic reprogramming. In contrast to the virulence-associated differences between WT and AP bacteria, Phenotype Microarray analysis showed minor in vitro phenotypic differences between the two isogenic variants. Additionally, our results reflect that WT bacteria's rapid host-adaptive transcriptional reprogramming was not sufficient for their survival, and they were outnumbered by hypervirulent covS mutants with SpeB(-)/Sda(high) phenotype, which survived up to 14 days in mice chambers. Our findings demonstrate the engagement of unique regulatory modules in niche adaptation, implicate a critical role for bacterial genetic heterogeneity that surpasses transcriptional in vivo adaptation, and portray the dynamics underlying the selection of hypervirulent covS mutants over their parental WT cells.
    • microscopy

      Rohde, Manfred (Elsevier, 2011)