Project leader: Professor SEPPO MERI,
Department of Bacteriology & Immunology, PO Box 21, Haartman Institute (Haartmaninkatu 3), FIN-00014 University of Helsinki, Helsinki, Finland, +58-9-191 26758, gsm 358-(0)50-581 2462, seppo.meri@helsinki.fi
Homepage: http://www.hi.helsinki.fi/complement/

Partners:
Sakari Jokiranta, Haartman Institute, Sakari.Jokiranta@helsinki.fi
Anna Blom, The Wallenberg Laboratory, Malmo University Hospital & University of Lund,
anna.blom@klkemi.mas.lu.se
Jens Hellwage, University of Hamburg/Jena, Germany

Researchers:
- Antti Alitalo, Haartman Institute,
- Hanne Amdahl, Haartman Institute,
- Zhu-zhu Cheng, Haartman Institute,
- Tong Chen, Haartman Institute,
- Per Comstedt, University of Umeå,
- Hanna Jarva, Haartman Institute,
- Sami Junnikkala, Haartman Institute,
- Vesa Kirjavainen Haartman Institute,
- Antti Lavikainen, Haartman Institute,
- Markus Lehtinen, Haartman Institute,
- Tamara Manuelian, University of Hamburg/Jena, Germany,
- Taru Meri, Haartman Institute,
- Rossella Murghia, University of Trieste,
- Mikko Seppänen, Helsinki University Central Hospital,
- Jari Suvilehto, Helsinki University Central Hospital,
- Paola Stephanel, University of Trieste,
- Jorma Tissari, Haartman Institute

Key words: Innate immunity, complement, borrelia, candida, yersinia, immunodeficiency

Results

This project has addressed innate immune escape by pathogenic microbes. Complement evasion by different mechanisms is important for microbial virulence and survival in the host. On the host side, i.e. in human beings, we have collected a large number of samples from patients (and their controls) with increased susceptibility to infections for studies on potential mechanisms to explain such sensitivity. In particular, we have analyzed the mutations and functions of the central complement inhibitor, factor H, in human disease. Mutations in factor H lead to atypical hemolytic uremic syndrome (aHUS) or membranoproliferative glomerulonephritis type II (MPGN II). The mutations are associated with a reduced ability of the protein to bind to cell surfaces and to complement C3b/d, and thereby a consequent inability to protect the cells from complement attack (111). Factor H binds to endothelial cells (131) and is found in vascular walls (110), joint synovia (114) and skin keratinocytes (115), where it functions to prevent complement-mediated inflammation. Binding to C3d and cell surfaces is mediated by the C-terminus of factor H, whose structure was solved by X-ray crystallography (141). The 3D structure of factor H C-terminus provides a basis for understanding the disease mechanism of aHUS and of the multiple interactions factor H has with microbes.

The focus of our MicMan project has been on complement evasion by streptococci, meningococci, Candida and spirochetes including borreliae and leptospires. A major strategy utilized by pathogenic bacteria to avoid opsonophagocytosis and direct complement killing is to bind the soluble inhibitor factor H to their surfaces. In group B streptococci and pneumococci factor H binding has been shown to be mediated by the surface proteins _ (Bac) and Hic, respectively (119). We showed earlier that Hic binds to the middle region of factor H and protects the pneumococcus from opsonophagocytosis. The _ protein and Hic are structurally closely related. Thus, their factor H-binding characteristics are very similar. By using direct binding assays with radiolabeled proteins and surface plasmon resonance analysis we observed that both _ and Hic bind to the short consensus repeats 8-11 and 12-14 in the middle region of factor H (120). Peptide mapping analysis suggested that the factor H-binding sites on _ and Hic are composed of discontinuous and partially homologous sequences. Thus, the bacterial virulence proteins utilize multiple binding sites on factor H to secure high avidity. Also, the functionally active sites on factor H are left free to inhibit C3b deposition and opsonophagocytosis. These results reveal the evolutionary conservation of an analogous immune evasion strategy in different types of pathogenic streptococci.

On Borrelia burgdorferi we discovered originally two types of complement regulators (Cregs) and identified the first one as the cp32-plasmid-encoded OspE protein family (class I factor H binding proteins; also called Erps; Alitalo et al, 2001 & 2002). The first member of other family (Bba68; class II) was cloned in Germany and named as CRASP-1. These proteins are encoded by genes in virulence plasmids and each single bacterium can express multiple OspE proteins (119, 133). OspE is important for inhibiting complement during infection. In collaboration with Darrin Akins (Univ of Oklahoma) we have shown that CRASP-1 is also essential for complement resistance, at least during early infections (132). The bacteria are carried by ticks and exist in nature in various animals, including rodents, deers and birds. It is likely that a repertoire of Cregs is required for this range of host specificity. At the protein level we have mapped the reciprocal binding sites on both OspE and factor H (119). OspE binding site is located at the C-terminus, where aHUS mutations are located. Different borrelial strains show somewhat different sensitivities to complement attack, and curiously the Borrelia garinii subspecies, spread by birds, has been sensitive to killing in vitro yet it can cause invasive disease, usually neuroborreliosis. Thanks to the MicMan collaboration with professor Sven Bergström in Umeå we have observed that B. garinii strains recently isolated directly from patients, in fact, have ospE genes and do express Cregs (133). This explains why the bacteria can resist immune attack and be invasive all the way to human brain. Apparently, during in vitro culture the bacteria lose some of their virulence plasmids.

During the project we have also found that serogroup B meningococci utilize another type of complement evasion mechanism by being able to bind the classical pathway inhibitor C4bp in a porin A-dependent manner (117, 129). Candida albicans yeast also binds C4bp in addition to factor H (123). Other microbes whose immune evasion mechanisms have been addressed include Yersinia sp. (in collaboration with Mikael Skurnik, manusript in preparation), Leptospira sp. (130) and relapsing fever spirochetes Borrelia recurrentis and B. duttonii (142). As yet another mechanism of potential immune evasion together with prof Ewert Linder (Stockholm, Sweden) we observed that the causative agent of bilharzia, Schistosoma mansoni binds immunoglobulin IgG into special hollow structures, called elongated bodies, on the worm surfaces (138).

In summary, we have identified new mechanisms and found new proteins responsible for immune evasion of microbes. This has opened many new research lines, and several other laboratories around the world have started working on similar issues. The new virulence factors are potential candidates for new vaccines, because immune responses against them would not only lead to recognition of microbes but to neutralization of their important virulence mechanisms.

Selected publications:

Jarva H, Hellwage J, Jokiranta TS, Lehtinen M, Zipfel PF, Meri S. The group B streptococcal beta and pneumococcal Hic proteins are structurally related immune evasion molecules that bind the complement inhibitor factor H in an analogous fashion. J Immunol, 172: 3111-8, 2004.

Alitalo A, Meri T, Chen T, Lankinen H, Cheng ZZ, Jokiranta S, Seppälä I, Lahdenne P, Hefty PS, Akins DR, and Meri S. Lysine-dependent Multi-point Binding of the Borrelia burgdorferi Virulence Factor OspE to the C-terminus of Factor H. J Immunol, 172: 6195-6201, 2004

Meri T, Blom AM, Hartmann A, Lenk D, Meri S, Zipfel PF. The hyphal and yeast forms of Candida albicansbind the complement inhibitor C4bp.  Infect Immun, 72: 6633-41, 2004
129. Jarva H, Ram S, Vogel U, Blom AM, Meri S. Binding of the complement inhibitor C4bp to serogroup B Neisseria meningitidis, J Immunol, 174:6299-6307, 2005.

Meri T, Murgia R, Stefanel P, Meri S, Cinco M. Regulation of complement activation at the C3-level by pathogenic leptospires. Microb Pathog, 9:139-47, 2005

Brooks CS, Vuppala SR, Jett AM, Alitalo A, Meri S, Akins DA. Complement regulator acquiring surface protein 1 (CRASP-1) imparts resistance to human serum in B. burgdorferi, J Immunol, 175: 3299-3308, 2005

Alitalo A, Meri T, Comstedt P, Jeffery L, Tornberg J, Strandin T, Lankinen H, Bergström S, Cinco M, Vuppula SR, Akins D, Meri S, Expression of complement factor H binding immunoevasion proteins in Borrelia garinii isolated from patients with neuroborreliosis. Eur J Immunol, 35: 3043-3053, 2005

Jokiranta TS, Jaakola VP, Lehtinen MJ, Pärepalo M, Meri S, and Goldman A. Structure of Complement Factor H C-terminus reveals molecular basis of atypical hemolytic uremic syndrome. EMBO J, 2006, in press

Meri T, Cutler SJ, Blom AM, Meri S, Jokiranta TS. Relapsing fever spirochetes Borrelia recurrentis and B. duttonii utilize C4b binding protein for complement evasion.  Infect Immun, 2006, in press