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- A1
A: Structural- and functional analysis of Yersinia adhesin YadA
The enteropathogenic bacteria Yersinia enterocolitica and Yersinia pseudo-tuberculosis mediate gastrointestinal infections like enteritis and enterocolitis, which in later stages are often accompanied by immunological sequelae like arthritis. Many Yersinia pathogenicity factors are encoded on a 70 kb virulence plasmid, as the outer membrane protein YadA, which is an essential pathogenicity factor for Y. enterocolitica. Its virulence associated properties are mediation of autoagglutination, binding to extracellular matrix molecules, to epithelial cells and granulocytes, and its involvement in serum resistance. Electron microscopical analysis and sequence comparisons helped to understand the structure of the adhesin.
Picture 1 shows an electron microscopical picture of YadA proteins (arrow) on the surface of Y. enterocolitica. It shows the lollipop-like structure of the proteins with head- and stalk domains (A1).
- A2
Structural analysis and protein crystals showed that YadA is an oligomeric lollipop-like outer membrane protein with different domains: N-terminal signalling sequence, head, neck, stalk, linker-region and C-terminal membrane anchoring region (A2).
Sequence homology comparisons showed that YadA is the prototype of a novel class of non-fimbrial adhesins present in many bacterial species. In over 40 additional gram-negative bacterial species YadA-like proteins with clear sequence homologies could be identified. Because of their characteristic oligomeric structure with an N-terminal head- (H), neck-, coiled-coil-stalk- (S) and C-terminal membrane anchoring (A)-region we termed this novel group “Oca-family” (short for: oligomeric coiled-coil adhesins). The highest homology region lies in the C-terminal pore, in the so-called membrane anchoring-region. The human pathogens of the respiratory tract Moraxella catarrhalis and Haemophilus influenzae each harbour at least two homologous proteins (UspA1 and UspA2 in M. catarrhalis, Hsf and Hia in H. influenzae), which mediate cell adherence like YadA. Previously it could also be shown that Neisseria meningitidis, a human pathogen causing bacterial meningitis, expresses at least one Oca-family protein (NadA), which is also able to mediate cell adherence. Until now only sequence comparisons and pathogen-specific analyses of individual Oca-proteins demonstrated their structural and functional relatedness. Our work group tries to compare different Oca-family genes in the Yersinia enterocolitica model. Focus of our previous studies was the characterization of the C-terminal membrane anchoring region. It could be demonstrated that the exchange of the C-terminal YadA-region with sequence-homologous anchoring regions of other Oca-proteins (YadAHS-XA-hybrids, XA=UspA1 from M. catarrhalis, EibA from E. coli strain ECOR-9, Hia from H. influenzae) led to differences in oligomeric stability but not to changes in adhesion to collagen or epithelial cells. Moreover, YadAHS-XA-hybrids showed a markedly reduced resistance to human serum, implying that the stability of the membrane anchoring region is important for this phenomenon.
Crystal structures of trimeric YadA and Hia head regions do not possess any major sequence homologies. Therefore, it is also interesting to compare the non homologous Oca head domains in our Yersinia enterocolitica model and analyze differences in their adhesive and invasive capabilities. Furthermore, we are also interested in YadA-mediated adherence, serum resistance, and virulence mechanisms and their contribution to Yersinia pathogenicity.
Projects related to YadA are executed by Dipl. Biol. Virginie Nägele, cand. med. Sarah Untiet, Gisela Anding and Dr. med. Nikolaus Ackermann, and supported by the Deutsche Forschungsgemeinschaft (DFG), Friedrich Baur-Stiftung and the Förderprogramm für Forschung und Lehre der LMU München (FöFoLe).
- B1
B: Role of cellular RhoGTPases in infection biology
The genome of many pathogenic enterobacteria encodes a specialized secretion system (type III), which acts as an injectisome. The type III secretion system (T3SS) plays a major role during infection, as with its help a multiplicity of different effectors can be channeled into the host cell. These effectors interact with cellular proteins, which are often essential components of major signal transduction cascades, thereby eliciting modulations or changes in the host cell. It was formerly demonstrated that RhoGTPases and the actin cytoskeleton represent the primary target for many bacterial species harbouring a T3SS, e.g. for enteropathogenic E. coli, Salmonella, Shigella und Yersinia spp. For instance the Yersinia T3SS effectors YopE and YopT interact with the RhoGTPases RhoA, Rac-1, and Cdc-42 by inactivating their cytoskeletal signalling functions.
RhoGTPases like RhoA, Rac-1, and Cdc-42 are central protein switches with on-off-function by interacting with GTP in their active and GDP in their inactive state, thereby influencing a multiplicity of different cellular functions. They control the dynamics of the actin cytoskeleton, take part in the organization of focal adhesion complexes, and modulate cell migration and phagocytosis. Furthermore they regulate the oxidative burst in professional phagocytic cells and have a massive influence on gene regulation (B1).
- B2
The most central function of RhoGTPases is probably the structuring and modulation of the actin cytoskeleton, with specific effects elicited by different RhoGTPase family members. In this way activation of RhoA, Rac1, or Cdc-42 causes the formation of actin stress fibres, lamellipodia, or filopodia, respectively (B2).
The coordination of this complex intracellular signalling network is critical for normal cellular function.
To transfer bacterial T3SS effectors in a RhoGTPase model system that matches in vivo like conditions, we use our established Yersinia enterocolitica mutant toolbox together with RhoA-, Rac-1, or Cdc-42-knockout mouse macrophages and granulocytes. With this strategy it should be possible to analyze the effects of T3SS effectors in a RhoA-, Rac-1, or Cdc-42 negative cellular context. This should reveal novel insights into mechanisms of action of T3SS effectors and RhoGTPases.
This project is executed by Dipl. Biol. Stefan Wölke and supported by the Deutsche Forschungsgemeinschaft (DFG) and Münchner Medizinische Wochenschrift.
