• 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.
    • 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.
    • The FbaB-type fibronectin-binding protein of Streptococcus pyogenes promotes specific invasion into endothelial cells.

      Amelung, Silva; Nerlich, Andreas; Rohde, Manfred; Spellerberg, Barbara; Cole, Jason N; Nizet, Victor; Chhatwal, Gursharan S; Talay, Susanne R; Department of Medical Microbiology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany. (2011-08)
      Invasive serotype M3 Streptococcus pyogenes are among the most frequently isolated organisms from patients suffering from invasive streptococcal disease and have the potential to invade primary human endothelial cells (EC) via a rapid and efficient mechanism. FbaB protein, the fibronectin-binding protein expressed by M3 S. pyogenes, was herein identified as a potent invasin for EC. By combining heterologous gene expression with allelic replacement, we demonstrate that FbaB is essential and sufficient to trigger EC invasion via a Rac1-dependent phagocytosis-like uptake. FbaB-mediated uptake follows the classical endocytic pathway with lysosomal destination. FbaB is demonstrated to be a streptococcal invasin exhibiting EC tropism. FbaB thus initiates a process that may contribute to the deep tissue tropism and spread of invasive S. pyogenes isolates into the vascular EC lining.
    • 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.
    • 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.
    • 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.
    • 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.
    • 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.