• 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.
    • Identification of an immune-regulated phagosomal Rab cascade in macrophages.

      Pei, Gang; Repnik, Urska; Griffiths, Gareth; Gutierrez, Maximiliano Gabriel; Helmholtz Centre for infection research, Inhoffenstr. 7 , D-38124 Braunschweig, Germany. (2014-05-01)
      Interferon-γ (IFN-γ) has been shown to regulate phagosome trafficking and function in macrophages, but the molecular mechanisms involved are poorly understood. Here, we identify Rab20 as part of the machinery by which IFN-γ controls phagosome maturation. We found that IFN-γ stimulates the association of Rab20 with early phagosomes in macrophages. By using imaging of single phagosomes in live cells, we found that Rab20 induces an early delay in phagosome maturation and extends the time for which Rab5a and phosphatidylinositol 3-phosphate (PI3P) remain associated with phagosomes. Moreover, Rab20 depletion in macrophages abrogates the delay in phagosome maturation induced by IFN-γ. Finally, we demonstrate that Rab20 interacts with the Rab5a guanine nucleotide exchange factor Rabex-5 (also known as RABGEF1) and that Rab20 knockdown impairs the IFN-γ-dependent recruitment of Rabex-5 and Rab5a into phagosomes. Taken together, here, we uncover Rab20 as a key player in the Rab cascade by which IFN-γ induces a delay in phagosome maturation in macrophages.
    • Identification of B- and T-Cell Epitopes within the Fibronectin-Binding Domain of the SfbI Protein of Streptococcus pyogenes

      Schulze, Kai; Medina, Eva; Chhatwal, Gursharan S.; Guzmán, Carlos A. (American Society for Microbiology, 2003-12)
    • Identification of endoribonuclease specific cleavage positions reveals novel targets of RNase III in Streptococcus pyogenes.

      Le Rhun, Anaïs; Lécrivain, Anne-Laure; Reimegård, Johan; Proux-Wéra, Estelle; Broglia, Laura; Della Beffa, Cristina; Charpentier, Emmanuelle; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-03-17)
      A better understanding of transcriptional and post-transcriptional regulation of gene expression in bacteria relies on studying their transcriptome. RNA sequencing methods are used not only to assess RNA abundance but also the exact boundaries of primary and processed transcripts. Here, we developed a method, called identification of specific cleavage position (ISCP), which enables the identification of direct endoribonuclease targets in vivo by comparing the 5΄ and 3΄ ends of processed transcripts between wild type and RNase deficient strains. To demonstrate the ISCP method, we used as a model the double-stranded specific RNase III in the human pathogen Streptococcus pyogenes. We mapped 92 specific cleavage positions (SCPs) among which, 48 were previously described and 44 are new, with the characteristic 2 nucleotides 3΄ overhang of RNase III. Most SCPs were located in untranslated regions of RNAs. We screened for RNase III targets using transcriptomic differential expression analysis (DEA) and compared those with the RNase III targets identified using the ISCP method. Our study shows that in S. pyogenes, under standard growth conditions, RNase III has a limited impact both on antisense transcripts and on global gene expression with the expression of most of the affected genes being downregulated in an RNase III deletion mutant.
    • Identification of new acceptor specificities of glycosyltransferase R with the aid of substrate microarrays.

      Seibel, Jürgen; Hellmuth, Hendrik; Hofer, Bernd; Kicinska, Anna-Maria; Schmalbruch, Bodo (2006-02-01)
      Finding opportunities to construct sugar motifs and to transfer them to targets of biological relevance and rapid identification of glycosylation events are important goals for glycobiology and a field of increasing interest. Here we have applied an enzyme microarray screening system for the identification of new acceptor specificities of the glycosyltransferase R (GTFR) from Streptococcus oralis (E.C. 2.4.1.5), which was able to effect the synthesis of sugar motifs in short times and with low amounts of substrate. These observations resulted in the development of a convenient alpha-glycosylation by the non-Leloir glycosyltransferase GTFR, with sucrose as substrate and with different alcohols and amino acid derivatives as acceptors, for the synthesis of glycoethers and glycosylated amino acids not observed with the use of familiar GTFs with high sequence homology.
    • 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.
    • Interaction of Listeria monocytogenes with mouse dendritic cells.

      Guzman, C A; Rohde, M; Chakraborty, T; Domann, E; Hudel, M; Wehland, J; Timmis, K N (1995-09)
    • Interferon-γ-inducible Rab20 regulates endosomal morphology and EGFR degradation in macrophages.

      Pei, Gang; Schnettger, Laura; Bronietzki, Marc; Repnik, Urska; Griffiths, Gareth; Gutierrez, Maximiliano Gabriel; Helmholtz Centre for infection research (HZI), 38124 Braunschweig, Germany. (2015-09-01)
      Little is known about the molecular players that regulate changes in the endocytic pathway during immune activation. Here we investigate the role of Rab20 in the endocytic pathway during activation of macrophages. Rab20 is associated with endocytic structures, but the function of this Rab GTPase in the endocytic pathway remains poorly characterized. We find that in macrophages, Rab20 expression and endosomal association significantly increase after interferon-γ (IFN-γ) treatment. Moreover, IFN-γ and Rab20 expression induce a dramatic enlargement of endosomes. These enlarged endosomes are the result of homotypic fusion promoted by Rab20 expression. The expression of Rab20 or the dominant-negative mutant Rab20T19N does not affect transferrin or dextran 70 kDa uptake. However, knockdown of Rab20 accelerates epidermal growth factor (EGF) trafficking to LAMP-2-positive compartments and EGF receptor degradation. Thus this work defines a function for Rab20 in the endocytic pathway during immune activation of macrophages.
    • Internalization, phagolysosomal biogenesis and killing of mycobacteria in enucleated epithelial cells.

      de Souza Carvalho, Cristiane; Kasmapour, Bahram; Gronow, Achim; Rohde, Manfred; Rabinovitch, Michel; Gutierrez, Maximiliano Gabriel; Department of Vaccinology and Applied Microbiology, Research Group Phagosome Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany. (2011-08)
      Bacterial and parasitic intracellular pathogens or their secreted products have been shown to induce host cell transcriptional responses, which may benefit the host, favour the microorganism or be unrelated to the infection. In most instances, however, it is not known if the host cell nucleus is proximately required for the development of an intracellular infection. This information can be obtained by the infection of artificially enucleated host cells (cytoplasts). This model, although rather extensively used in studies of viral infection, has only been applied to few bacterial pathogens, which do not include Mycobacterium spp. Here, we investigate the internalization, phagosome biogenesis and survival of M. smegmatis in enucleated type II alveolar epithelial cells. Cytoplasts were infected with M. smegmatis, but the percentage of infection was significantly lower than that of nucleated cells. Scanning electron microscopy indicated that in both cells and cytoplasts, bacteria were internalized by a phagocytosis-like mechanism. Interestingly, phagosome fusion with lysosomes and mycobacterial killing were both more efficient in enucleated than in nucleated cells, a finding that may be correlated with the increased number of autophagic vesicles developed in cytoplasts. We provide evidence that although quantitative changes were observed, the full development of the infection, as well as mycobacterial killing did not require the presence of the host cell nucleus.
    • Intracellular Survival of Streptococcus pyogenes in Polymorphonuclear Cells Results in Increased Bacterial Virulence

      Medina, Eva; Rohde, Manfred; Chhatwal, Gursharan S. (American Society for Microbiology, 2003-09)
    • 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.