The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which

The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which is a major virulence factor of pathogenic strains. affinity conversation site for Lu/BCAM. We found Lu/BCAM to be essential for the binding of CNF1 to cells. Cells deficient in Lu/BCAM but expressing p37LRP could not bind labeled CNF1. Therefore we conclude Tandutinib that LRP and Lu/BCAM are both required for toxin action but with different functions. Author Summary We study a crucial virulence factor produced by pathogenic strains the Cytotoxic Necrotizing Factor 1 (CNF1). More than 80% of urinary tract infections (UTIs) which are counted among the most common bacterial infections of humans are caused by Uropathogenic Escherichia coli Rabbit Polyclonal to Cox2. (UPEC) strains. We and others elucidated the molecular mechanism of the toxin CNF1. It constitutively activates Rho GTPases by a direct covalent modification. The toxin enters mammalian cells by receptor-mediated endocytosis. Here we identified the protein receptor for CNF1 by co-precipitation of cell surface molecules with the tagged toxin and subsequent Maldi-TOF analysis. We identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as receptor for CNF1 and located its conversation site to the C-terminal part of the toxin. We performed direct protein-protein conversation analysis and competition studies. Moreover cells deficient in Lu/BCAM could not bind labeled CNF1. The identification of a toxin’s cellular receptor and receptor binding region is an important task for understanding the pathogenic function of the toxin and moreover to make the toxin accessible for its use as a cellbiological and pharmacological tool for example Tandutinib for the generation of immunotoxins. Tandutinib Introduction Urinary tract infections (UTIs) are among the most common bacterial infections of humans. More than 80% of UTIs are caused by Uropathogenic (UPEC) strains [1]. Many pathogenic strains including UPEC and strains inducing meningitis or soft tissue infections produce Cytotoxic Necrotizing Factor 1 (CNF1) a protein toxin which contributes to virulence [2]. Of major importance for its role as a virulence factor is the effect of CNF1 on epithelial barrier- and immune cell functions [3]. Both features are controlled by Rho GTPases which are directly targeted by the toxin. CNF1 deamidates a specific glutamine (Gln63/61) of Rho proteins which is crucial for GTP hydrolysis and therefore the Rho proteins are arrested in a constitutively activated state [4] [5]. Rho family GTPases are regulated in a GTPase cycle by the following cellular proteins: GEFs (toxin CNFY). This toxin is known to interact with a different yet unknown receptor on mammalian cells [17]. Following binding we lysed the cells and precipitated the toxin together with associated molecules using anti-GST magnetic beads. Eluates were separated on SDS-PAGE and the eluted proteins were subsequently identified by nanoLC-MS/MS. The only hit unique to the CNF1-precipitate was the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) (Fig. S1). This surface protein has a large extracellular Ig-like structure and is widely expressed. Interestingly Lu/BCAM like the proposed CNF1 receptor 67LR interacts with laminin suggesting that this receptor-binding domain name of CNF1 could interact with both laminin binding structures around the cell surface. To verify the CNF1-Lu/BCAM conversation we repeated the precipitation assay with HEK293 (Fig. 1A) and HeLa cells (Fig. 1B) and analyzed the presence of Lu/BCAM in the precipitate by Western-blotting with a specific antibody against Lu/BCAM. As shown in Fig. Tandutinib 1 Lu/BCAM was exclusively co-precipitated with GST-CNF1-GST but not with GST-CNFY-GST or GST alone. Notably we could not detect 37LRP/67LR in any lane by Tandutinib Western-blotting although the protein was expressed in HeLa and in HEK293 (human embryonic kidney) cells (Fig. S2). Physique 1 Lu/BCAM is usually co-precipitated with CNF1 but not with CNFY. We asked whether Lu/BCAM is an alternative receptor in the absence of 67LR or Tandutinib whether binding to Lu/BCAM is generally crucial for toxin uptake. In the latter case blocking the conversation of CNF1 with Lu/BCAM should inhibit.