• Adherence and invasion of streptococci to eukaryotic cells and their role in disease pathogenesis.

      Rohde, Manfred; Chhatwal, G Singh; Department of Medical Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany. manfred.rohde@helmholtz-hzi.de (2013)
      Streptococcal adhesion, invasion, intracellular trafficking, dissemination, and persistence in eukaryotic cells have a variety of implications in the infection pathogenesis. While cell adhesion establishes the initial host contact, adhering bacteria exploit the host cell for their own benefit. Internalization into the host cell is an essential step for bacterial survival and subsequent dissemination and persistence, thus playing a key role in the course of infection. This chapter summarizes the current knowledge about the diverse mechanisms of streptococcal adhesion to and invasion into different eukaryotic cells and the impact on dissemination and persistence which is reflected by consequences for the pathogenesis of streptococcal infections.
    • Biological functions of GCS3, a novel plasminogen-binding protein of Streptococcus dysgalactiae ssp. equisimilis.

      Bergmann, René; Dinkla, Katrin; Nitsche-Schmitz, D Patric; Graham, Rikki M A; Lüttge, Melanie; Sanderson-Smith, Martina L; Nerlich, Andreas; Rohde, Manfred; Chhatwal, Gursharan S; Dept. of Medical Microbiology, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2011-02)
      Increasing awareness of the relevance of Streptococcus dysgalactiae ssp. equisimilis as a human pathogen motivates the analysis of its pathomechanisms. One of the mechanisms that increases infectivity and dissemination of several streptococcal species is the recruitment and subsequent activation of host plasminogen on the streptococcal surface. This study identified GCS3 as a novel plasminogen-binding M protein of S. dysgalactiae ssp. equisimilis and revealed a difference in the mode of binding as compared to the plasminogen-binding protein PAM of S. pyogenes. In contrast to PAM, GCS3 did not bind to the kringle 1-3 region of plasminogen. Despite this difference, GCS3 exerts the same function of recruiting plasminogen to the streptococcal surface, which can be activated by streptokinase and host plasminogen activators to serve as a spreading factor. Moreover, we demonstrate a role of GCS3 in plasminogen-dependent streptococcal adherence to human pharyngeal cells (cell line Detroit 562) that indicates an additional function of the protein as an adhesin in the oral cavity.
    • Contribution of plasminogen activation towards the pathogenic potential of oral streptococci.

      Itzek, Andreas; Gillen, Christine M; Fulde, Marcus; Friedrichs, Claudia; Rodloff, Arne C; Chhatwal, Gursharan S; Nitsche-Schmitz, Daniel Patric; Department of Medical Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2010)
      Oral streptococci are a heterogeneous group of human commensals, with a potential to cause serious infections. Activation of plasminogen has been shown to increase the virulence of typical human pathogenic streptococci such as S. pneumoniae. One important factor for plasminogen activation is the streptococcal α-enolase. Here we report that plasminogen activation is also common in oral streptococci species involved in clinical infection and that it depends on the action of human plasminogen activators. The ability to activate plasminogen did not require full conservation of the internal plasminogen binding sequence motif FYDKERKVY of α-enolase that was previously described as crucial for increased plasminogen binding, activation and virulence. Instead, experiments with recombinant α-enolase variants indicate that the naturally occurring variations do not impair plasminogen binding. In spite of these variations in the internal plasminogen binding motif oral streptococci showed similar activation of plasminogen. We conclude that the pathomechanism of plasminogen activation is conserved in oral streptococci that cause infections in human. This may contribute to their opportunistic pathogenic character that is unfurled in certain niches.
    • Contribution of Streptococcus anginosus to infections caused by groups C and G streptococci, southern India.

      Reissmann, Silvana; Friedrichs, Claudia; Rajkumari, Reena; Itzek, Andreas; Fulde, Marcus; Rodloff, Arne C; Brahmadathan, Kootallur N; Chhatwal, Gursharan S; Nitsche-Schmitz, D Patric; Helmholtz Centre for Infection Research, Braunschweig, Germany. (2010-04)
      Vellore, a region in southern India, has a high incidence of severe human infections with Beta-hemolytic group C and G streptococci (GCGS). To determine the causative species in these infections, we conducted 16S rRNA gene sequencing: Streptococcus dysgalactiae subsp. equisimilis (81%) and S. anginosus (19%) were the causative organisms in the 2-year study period (2006-2007). We used PCR to detect the virulence-related emm gene; results showed that it was restricted to S. dysgalactieae subsp. equisimilis isolates of 99.2% tested positive. Due to a novel marker, S. anginosus and S. constellatus can be quickly and accurately distinguished from other members of the genus. The notable contribution of the anginosus group to human infections suggests that this group of obligate pathogens deserves more attention in healthcare and research.
    • Cooperative plasminogen recruitment to the surface of Streptococcus canis via M protein and enolase enhances bacterial survival.

      Fulde, Marcus; Rohde, Manfred; Polok, Andy; Preissner, Klaus T; Chhatwal, Gursharan Singh; Bergmann, Simone; Helmholtz Centre for Infection Research (HZI), Department of Medical Microbiology, Braunschweig, Germany. marcus.fulde@web.de (2013)
      Streptococcus canis is a zoonotic pathogen capable of causing serious invasive diseases in domestic animals and humans. Surface-exposed M proteins and metabolic enzymes have been characterized as major virulence determinants in various streptococcal species. Recently, we have identified SCM, the M-like protein of S. canis, as the major receptor for miniplasminogen localized on the bacterial surface. The present study now characterizes the glycolytic enzyme enolase as an additional surface-exposed plasminogen-binding protein. According to its zoonotic properties, purified S. canis enolase binds to both human and canine plasminogen and facilitates degradation of aggregated fibrin matrices after activation with host-derived urokinase-type plasminogen activator (uPA). Unlike SCM, which binds to the C terminus of human plasminogen, the S. canis enolase interacts N terminally with the first four kringle domains of plasminogen, representing angiostatin. Radioactive binding analyses confirmed cooperative plasminogen recruitment to both surface-exposed enolase and SCM. Furthermore, despite the lack of surface protease activity via SpeB in S. canis, SCM is released and reassociated homophilically to surface-anchored SCM and heterophilically to surface-bound plasminogen. In addition to plasminogen-mediated antiphagocytic activity, reassociation of SCM to the bacterial surface significantly enhanced bacterial survival in phagocytosis analyses using human neutrophils.
    • Differences in the aromatic domain of homologous streptococcal fibronectin-binding proteins trigger different cell invasion mechanisms and survival rates.

      Rohde, Manfred; Graham, Rikki M; Branitzki-Heinemann, Katja; Borchers, Patricia; Preuss, Claudia; Schleicher, Ina; Zähner, Dorothea; Talay, Susanne R; Fulde, Marcus; Dinkla, Katrin; et al. (2011-03)
      Group A streptococci (GAS, Streptococcus pyogenes) and Group G streptococci (GGS, Streptococcus dysgalactiae ssp. equisimilis) adhere to and invade host cells by binding to fibronectin. The fibronectin-binding protein SfbI from GAS acts as an invasin by using a caveolae-mediated mechanism. In the present study we have identified a fibronectin-binding protein, GfbA, from GGS, which functions as an adhesin and invasin. Although there is a high degree of similarity in the C-terminal sequence of SfbI and GfbA, the invasion mechanisms are different. Unlike caveolae-mediated invasion by SfbI-expressing GAS, the GfbA-expressing GGS isolate trigger cytoskeleton rearrangements. Heterologous expression of GfbA on the surface of a commensal Streptococcus gordonii and purified recombinant protein also triggered actin rearrangements. Expression of a truncated GfbA (lacking the aromatic domain) and chimeric GfbA/SfbI protein (replacing the aromatic domain of SfbI with the GfbA aromatic domain) on S. gordonii or recombinant proteins alone showed that the aromatic domain of GfbA is responsible for different invasion mechanisms. This is the first evidence for a biological function of the aromatic domain of fibronectin-binding proteins. Furthermore, we show that streptococci invading via cytoskeleton rearrangements and intracellular trafficking along the classical endocytic pathway are less persistence than streptococci entering via caveolae.
    • Genetic variation in group A streptococci.

      McMillan, David J; Sriprakash, Kadaba S; Chhatwal, Gursharan S; Department of Microbial Pathogenesis, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. (2007-11)
      Group A streptococcus (GAS) is responsible for a range of human diseases that vary in their clinical manifestations and severity. While numerous virulence factors have been described, the way these factors interact to promote different streptococcal diseases is less clear. In order to identify multifactorial relationships between GAS and the human host, novel high-throughput techniques such as microarrays are necessary. We have performed comparative studies using custom-designed virulence arrays to enhance our understanding of the high degree of genotypic variation that occurs in streptococci. This study has pointed to mobile genetic elements as the major agents that promote variation. Our results show that multiple combinations of genes might bring about similar clinical pictures. This adds further complexity to the intricate relationship between pathogen and host.
    • 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.
    • 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.
    • 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.
    • SCM, a novel M-like protein from Streptococcus canis, binds (mini)-plasminogen with high affinity and facilitates bacterial transmigration.

      Fulde, Marcus; Rohde, Manfred; Hitzmann, Angela; Preissner, Klaus T; Nitsche-Schmitz, D Patric; Nerlich, Andreas; Chhatwal, Gursharan Singh; Bergmann, Simone; Department of Medical Microbiology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany. Marcus.Fulde@helmholtz-hzi.de (2011-02-24)
      Streptococcus canis is an important zoonotic pathogen capable of causing serious invasive diseases in domestic animals and humans. In the present paper we report the binding of human plasminogen to S. canis and the recruitment of proteolytically active plasmin on its surface. The binding receptor for plasminogen was identified as a novel M-like protein designated SCM (S. canis M-like protein). SPR (surface plasmon resonance) analyses, radioactive dot-blot analyses and heterologous expression on the surface of Streptococcus gordonii confirmed the plasminogen-binding capability of SCM. The binding domain was located within the N-terminus of SCM, which specifically bound to the C-terminal part of plasminogen (mini-plasminogen) comprising kringle domain 5 and the catalytic domain. In the presence of urokinase, SCM mediated plasminogen activation on the bacterial surface that was inhibited by serine protease inhibitors and lysine amino acid analogues. Surface-bound plasmin effectively degraded purified fibrinogen as well as fibrin clots, resulting in the dissolution of fibrin thrombi. Electron microscopic illustration and time-lapse imaging demonstrated bacterial transmigration through fibrinous thrombi. The present study has led, for the first time, to the identification of SCM as a novel receptor for (mini)-plasminogen mediating the fibrinolytic activity of S. canis.
    • Virulence gene pool detected in bovine group C Streptococcus dysgalactiae subsp. dysgalactiae isolates by use of a group A S. pyogenes virulence microarray.

      Rato, Márcia G; Nerlich, Andreas; Bergmann, René; Bexiga, Ricardo; Nunes, Sandro F; Vilela, Cristina L; Santos-Sanches, Ilda; Chhatwal, Gursharan S; Centro de Recursos Microbiológicos, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal. (2011-07)
      A custom-designed microarray containing 220 virulence genes of Streptococcus pyogenes (group A Streptococcus [GAS]) was used to test group C Streptococcus dysgalactiae subsp. dysgalactiae (GCS) field strains causing bovine mastitis and group C or group G Streptococcus dysgalactiae subsp. equisimilis (GCS/GGS) isolates from human infections, with the latter being used for comparative purposes, for the presence of virulence genes. All bovine and all human isolates carried a fraction of the 220 genes (23% and 39%, respectively). The virulence genes encoding streptolysin S, glyceraldehyde-3-phosphate dehydrogenase, the plasminogen-binding M-like protein PAM, and the collagen-like protein SclB were detected in the majority of both bovine and human isolates (94 to 100%). Virulence factors, usually carried by human beta-hemolytic streptococcal pathogens, such as streptokinase, laminin-binding protein, and the C5a peptidase precursor, were detected in all human isolates but not in bovine isolates. Additionally, GAS bacteriophage-associated virulence genes encoding superantigens, DNase, and/or streptodornase were detected in bovine isolates (72%) but not in the human isolates. Determinants located in non-bacteriophage-related mobile elements, such as the gene encoding R28, were detected in all bovine and human isolates. Several virulence genes, including genes of bacteriophage origin, were shown to be expressed by reverse transcriptase PCR (RT-PCR). Phylogenetic analysis of superantigen gene sequences revealed a high level (>98%) of identity among genes of bovine GCS, of the horse pathogen Streptococcus equi subsp. equi, and of the human pathogen GAS. Our findings indicate that alpha-hemolytic bovine GCS, an important mastitis pathogen and considered to be a nonhuman pathogen, carries important virulence factors responsible for virulence and pathogenesis in humans.