Arbeitsgruppe: Molekulare MikrobiologieLeitung: PD Dr. rer. nat. Christine HeilmannStaphylokokken zählen zu den häufigsten Infektionserregern beim Menschen. Die Koagulase-positive Spezies Staphylococcus aureus verursacht eine Vielzahl von Infektionen, die ein breites Spektrum von harmloseren Wund- und Weichteilinfektionen bis hin zu schwerwiegenden, oft lebensbedrohlichen Infektionen wie die infektiöse Endokarditis und Sepsis umfassen und Fremdkörper-assoziiert sein können. Die Koagulase-negative Spezies Staphylococcus epidermidis ist zwar ein wesentlicher Bestandteil der gesunden Hautflora des Menschen, hat aber in den letzten drei Jahrzehnten auch als Infektionserreger zunehmend an Bedeutung gewonnen. Infektionen, die durch S. epidermidis verursacht werden, sind typischerweise Fremdkörper-assoziiert und verlaufen überwiegend subakut. Durch den zunehmenden Gebrauch von implantierten Fremdkörpern, wie intravasale Katheter, künstliche Herzklappen, Brustprothesen, Liquorableitungssysteme, Gelenkprothesen, künstliche Herzschrittmacher und Dialysesysteme aus unterschiedlichen Polymermaterialien ist in den letzten Jahren die Anzahl der Polymer-assoziierten Infektionen mit Staphylokokken sprunghaft angestiegen. Die Pathogenese Fremdkörper-assoziierter Infektionen ist vor allem durch die Fähigkeit der Staphylokokken, einen Biofilm auszubilden, gekennzeichnet. Die Kenntnis der molekularen Mechanismen der Biofilmbildung und Kolonisierung des Wirtsgewebes durch die Staphylokokken ist erforderlich, um neue Konzepte für die Therapie und Prophylaxe dieser Infektionen zu entwickeln. Wir haben in den letzten Jahren in verschiedenen Projekten eine Reihe der an diesen Prozessen beteiligten Staphylokokken-Faktoren identifiziert und charakterisiert.  

Molecular mechanisms of Staphylococcus epidermidis and Staphylococcus aureus biofilm formation on biotic and abiotic surfaces

Förderung: DFG GRK1409 S. epidermidis and S. aureus belong to the most important pathogens causing life-threatening infections, such as endocarditis and sepsis, which may be foreign-body associated. The ability of the staphylococci to form a biofilm, is considered to be the most critical pathogenicity factor involved. Biofilm formation is a two-step process. First, the bacteria attach to a biotic or abiotic surface mediated by Atl/AtlE. Then, the bacteria proliferate and accumulate into multilayered biofilms. The accumulation phase depends on intercellular adhesion, which is mediated by a polysaccharide, namely the ß-1,6 linked N-acetylglucosamin PIA (polysaccharide intercellular adhesin) or proteins, such as the accumulation-associated protein Aap from S. epidermidis and the unrelated, newly discovered S. aureus surface protein SasC. Recently, a new domain (G5) has been identified as a potential N-acetylglucosamin recognition domain. The G5 domain is part of Aap from S. epidermidis as well as of the Aap-homologous proteins Pls and SasG from S. aureus. Our recent results suggest that posttranslational protein glycosylation of Pls might enhance biofilm formation of S. aureus. We now seek to further characterize the molecular mechanisms involved in staphylococcal biofilm formation. Especially, we want to analyze whether the recently identified G5 domain is involved in binding of PIA or of carbohydrate-modified proteins at the staphylococcal surface. Moreover, we want to characterize the influence of posttranslational protein glycosylation on biofilm formation in detail. Upon biofilm formation, single bacteria or cell clusters can detach from the biofilm and hematogeneously spread to distant sites. This may lead to the seeding of the infection and/or the development of sepsis. We are also interested in the characterization of the molecular mechanisms involved in biofilm detachment. Characterization of the importance of the autolysin/adhesins Aaa and Atl in the Staphylococcus aureus colonization of human tissueFörderung: DFG-Normalverfahren HE 3546/2-1 (01.06. -12.08.)Staphylococcus aureus is a serious pathogen that causes life-threatening chronic and recurrent infections due to its ability to persist inside the host cells. Thus, the pathogens capability to adhere to host tissue and to be internalized by human host cells is considered the most critical pathogenicity factors in these infections leading to escape from the host immune response and antibiotic therapy. Furthermore, the coagulase-negative Staphylococcus epidermidis has also been reported to cause chronic and recurrent bone diseases. A novel class of staphylococcal adhesins has been reported from staphylococci: the autolysin/adhesins that include Atl and Aaa from S. aureus and AtlE and Aae from the coagulase-negative S. epidermidis. The main mechanism of S. aureus internalization is thought to depend on the fibronectin-binding proteins (FnBPs). In this project, we found a novel mechanism of staphylococcal internalization that involves the major autolysin/adhesin Atl and AtlE. Moreover, we identified the human heat shock cognate protein Hsc70 as putative host cell receptor in Atl-mediated uptake of staphylococcal cells. This novel mechanism of internalization may represent a “backup”-mechanism in S. aureus internalization, while it may represent the major mechanism involved in internalization of coagulase-negative staphylococci. The Atl-mediated staphylococcal internalization may play an important role in the pathogenesis of chronic and relapsing infections with these serious pathogens. Molecular characterization of adhesive interactions between staphylococci and CandidaFörderung: DFG-Normalverfahren HE 3546/3-1 (07.07. – 06.10.) Interaction between staphylococci and Candida yeasts can occur either directly or mediated by bridging molecules, such as the extracellular matrix and plasma proteins fibrinogen and fibronectin, as suggested by adhesion and coaggreagtion assays. To identify staphylococcal factors involved in this interaction, we used the phage display technique. Panning of the phage display library of the clinical isolate S. aureus 4074 against Candida biofilms revealed domains of the extracellular fibrinogen-binding proteins coagulase and Efb. The coagulase leads to the conversion of fibrinogen to fibrin, which may shield the Candida cells against the host immune defense. Indeed, phagocytosis assays demonstrate a reduced phagocytosis rate of Candida in the presence of coagulase. However, further analyses are necessary to elucidate the exact role of the coagulase in modulating the immune response to Candida infections. Further panning experiments affinity-selected portions of the mostly uncharacterized S. aureus surface proteins SdrE and SasA, which needs further investigations.


Molecular characterization of the SasC-mediated mechanism of cell aggregation and biofilm formation in Staphylococcus aureus

Förderung: IZKF Münster Hei2/022/09

Staphylococci are serious pathogens that can cause implant-associated infections leading to significant morbidity and mortality. Colonization of the implanted medical devices by the formation of a three-dimensional structure made of bacteria and host material called biofilm is considered the most critical factor in these infections. To form a biofilm, bacteria first attach to the surface of the medical device, and then proliferate and accumulate into multilayered cell clusters. Recently, we identified and characterized the 238 kDa S. aureus surface protein C (SasC) that has features typical of surface proteins from Gram-positive bacteria, such as an N-terminal signal peptide, a C-terminal LPXTG cell wall-anchorage motif, and a repeat region. SasC mediated the formation of huge cell aggregates indicative of intercellular adhesion, higher attachment to polystyrene, and enhanced biofilm formation to S. carnosus and S. aureus. We localized the domain conferring these traits in the N-terminal domain that included three repeats of approx. 40 amino acids each and two repeats of approx. 140 amino acids each that are followed by a motif found in various architectures (FIVAR), which is thought to have a general carbohydrate-binding function. Far-Western blot analysis identified homotypic interactions mediated by domains of the two 140-amino acid repeats. In future experiments, we plan to characterize this homotypic interaction in more detail to exactly characterize the molecular mechanism underlying the SasC-mediated cell aggregation and biofilm formation of S. aureus, which may play an important role in colonization during infection with this important pathogen.

 Mitglieder der Arbeitsgruppe:Dipl.-Biol. Isabelle BleizifferM. Sc. Tim SchlesierM. Sc. Nithya Babu RajendranCand. med. Mario JularicCand. med. Sarah Deppenkemper Publikationen: PD Dr. Christine Heilmann  

1.    Horn M, Bertling A, Brodde MF, Müller A, Roth J, Van Aken H, Jurk K, Heilmann C, Peters G, and Kehrel BE. 2012. Human neutrophil alpha-defensins induce formation of fibrinogen and thrombospondin-1 amyloid-like structures and activate platelets via GPIIbIIIa. J Thromb Haemost: doi:10.1111/j.1538-7836.2012.04640.x.

2.    Heilmann C. Adhesion mechanisms of staphylococci. 2011. In: Bacterial adhesion. Adv Exp Med Biol 715. D. Linke, A. Goldman, eds. Springer Science + Business Media, B.V., p. 105-23.

3.    Hirschhausen N, Schlesier T, Schmidt MA, Götz F, Peters G, and Heilmann C. 2010. A novel staphylococcal internalization mechanism involves the major autolysin Atl and heat shock cognate protein Hsc70 as host cell receptor. Cell Microbiol 12:1746-64.

4.    Heilmann C and Götz F. 2010. Cell-cell communication and biofilm formation in Gram-positive bacteria. In: Bacterial signaling. R. Krämer, K. Jung, eds. Wiley-VCH Verlag, Weinheim, p. 7-22.

5.    Schroeder K, Jularic M, Horsburgh SM, Hirschhausen N, Neumann C, Bertling A, Schulte A, Foster S, Kehrel BE, Peters G, and Heilmann C. 2009. Molecular characterization of a novel Staphylococcus aureus surface protein (SasC) involved in cell aggregation and biofilm accumulation. PLoS One 4: e7567.

6.    Niemann S, Kehrel BE, Heilmann C, Rennemeier C, Peters G, and Hammerschmidt S. 2009. Pneumococcal association to platelets is mediated by soluble fibrin and supported by thrombospondin-1. Thromb Haemost 102: 735-42.

7.    Al Laham N, Rohde H, Sander G, Fischer A, Hussain M, Heilmann C, Mack D, Proctor R, Peters G, Becker K, and von Eiff C. 2007. Augmented expression of polysaccharide intercellular adhesin in a defined Staphylococcus epidermidis mutant with the small-colony-variant phenotype. J Bacteriol 189(12):4494-501.

8.    Becker K, Almasri AS, von Eiff C, Peters G, Heilmann C, Fegeler W. 2007. Systematic survey of nonspecific agglutination by Candida spp. in latex assays. J Clin Microbiol 45(4):1315-8.

9.    Chatterjee I, Somerville GA, Heilmann C, Sahl HG, Maurer HH, and Herrmann M. 2006. Very low ethanol concentrations affect the viability and growth recovery in post-stationary-phase Staphylococcus aureus populations. Appl Environ Microbiol 72(4):2627-36.

10.Heilmann C and Peters G. 2006. Biology and Pathogenicity of Staphylococcus epidermidis. In: Gram-Positive Pathogens. 2nd edition. V.A. Fischetti et al. eds. American Society for Microbiology, Washington, D.C., USA, p. 560-571.

11.Heilmann C, Hartleib J, Hussain M, and Peters G. 2005. The multifunctional Staphylococcus aureus autolysin Aaa mediates adherence to immobilized fibrinogen and fibronectin. Infect Immun 73: 4793-802.

12.Rohde H, Burdelski C, Bartscht K, Hussain M, Buck F, Horstkotte MA, Knobloch JK, Heilmann C, Herrmann M, and Mack D. 2005. Induction of Staphylococcus epidermidis biofilm formation via proteolytic processing of the accumulation-associated protein by staphylococcal and host proteases. Mol Microbiol 55: 1883-95.

13.Grundmeier M, Hussain M, Becker P, Heilmann C, Peters G, Sinha B. 2004. Truncation of fibronectin-binding proteins in Staphylococcus aureus strain Newman leads to deficient adherence and host cell invasion due to loss of the cell wall anchor function. Infect Immun 72:7155-63.

14.Heilmann C, Niemann S, Sinha B, Herrmann M, Kehrel BE, and Peters G. 2004. Staphylococcus aureus Fibronectin-binding proteins (FnBPs) mediate adherence to platelets and FnBPA, but not FnBPB induces platelet aggregation. J Infect Dis 190: 321-9.

15.Heilmann C, Thumm G, Chhatwal GS, Hartleib J, Uekötter A, and Peters G. 2003. Identification and characterization of a novel autolysin (Aae) with adhesive properties from Staphylococcus epidermidis. Microbiology 149:2769-78.

16.Von Eiff C, Peters G, and Heilmann C. 2002. Pathogenesis of infections due to coagulase-negative staphylococci. Lancet Infect Dis 2:677-85. Review.

17.Hussain M, Haggar A, Heilmann C, Peters G, Flock JI, and Herrmann M. 2002. Insertional inactivation of Eap in Staphylococcus aureus strain Newman confers reduced staphylococcal binding to fibroblasts. Infect Immun 70:2933-40.

18.Heilmann C, Herrmann M, Kehrel BE, and Peters G. 2002. Platelet-binding domains in 2 fibrinogen-binding proteins of Staphylococcus aureus identified by phage display. J Infect Dis 186:32-9.

19.Hussain M, Heilmann C, Peters G, and Herrmann M. 2001.Teichoic acid enhances adhesion of Staphylococcus epidermidis to immobilized fibronectin. Microb Pathog 31: 261-70.

20.Rupp ME, Fey PD, Heilmann C, and Götz F. 2001. Characterization of the importance of Staphylococcus epidermidis autolysin and polysaccharide intercellular adhesin in the pathogenesis of intravascular catheter-associated infection in a rat model. J Infect Dis 183: 1038-42.

21.Götz F, Heilmann C, and Cramton SE. 2000. Molecular basis of catheter-associated infections by staphylococci. Adv Exp Med Biol 485:103-11. Review.

22.Sinha B, Francois P, Que YA, Hussain M, Heilmann C, Moreillon P, LEw D, Krause KH, Peters G, and Hermann M. 2000. Heterologously expressed Staphylococcus aureus febronectin-binding proteins are sufficient for invasion of host cells. Infect Immun 68: 6871-8.

23.Von Eiff C, Heilmann C, and Peters G. 1999. New aspects in the molecular basis of polymer-associated infections due to staphylococci. Eur J Clin Microbiol Infect Dis 18:843-846. Review.

24.Von Eiff C, Heilmann C, Herrmann M, and Peters G. 1999. Basic aspects of the pathogenesis of staphylococcal polymer-associated infections. Infection 27 Suppl 1: 7-10. Review.

25.Fey PD, Ulphani JS, Götz F, Heilmann C, Mack D, and Rupp ME. 1999. Characterization of the relationship between polysaccharide intercellular adhesin and hemagglutination in Staphylococcus epidermidis. J Infect Dis 179: 1561-4.

26.Heilmann C, and Götz F. 1998. Further characterization of Staphylococcus epidermidis transposon mutants deficient in primary attachment or intercellular adhesion. Zbl Bakteriol 7:69-83.

27.Von Eiff C, Heilmann C, Proctor RA, Wolz C, Peters G, and Götz F. 1997. A site-directed Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly. J Bacteriol 179: 4706-12.

28.Ziebuhr W, Heilmann C, Götz F, Meyer P, Wilms K, Straube E, and Hacker J. 1997. Detection of the intercellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates. Infect Immun 65: 890-6.

29.Heilmann C, Hussain M, Peters G, and Götz F. 1997. Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface. Mol Microbiol 24: 1013-24.

30.Heilmann C, Schweitzer O, Gerke C, Vanittanakom N, Mack D, and Götz F. 1996. Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis. Mol Microbiol 20: 1083-91.

31.Heilmann C, Gerke C, Perdreau-Remington F, and Götz F. 1996. Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation. Infect Immun 64: 277-82.

32.Schumacher-Perdreau F, Heilmann C, Peters G, Götz F, and Pulverer G. 1994. Comparative analysis of a biofilm-forming Staphylococcus epidermidis strain and its adhesion-positive, accumulation-negative mutant M7. FEMS Microbiol Letters 117: 71-8.