TY  - JOUR
A1  - Dembek, Marcin
A1  - Barquist, Lars
A1  - Boinett, Christine J.
A1  - Cain, Amy K.
A1  - Mayho, Matthew
A1  - Lawley, Trevor D.
A1  - Fairweather, Neil F.
A1  - Fagan, Robert P.
T1  - High-throughput analysis of gene essentiality and sporulation in Clostridium difficile
JF  - mBio
N2  - Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen.
KW  - Bacillus subtilis
KW  - expression
KW  - spores
KW  - toxin
KW  - transcription
KW  - germination
KW  - transposition
KW  - metabolism
KW  - infection
KW  - in vitro
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143745
VL  - 6
IS  - 2
ER  - 
TY  - JOUR
A1  - Piteau, Marianne
A1  - Papatheodorou, Panagiotis
A1  - Schwan, Carsten
A1  - Schlosser, Andreas
A1  - Aktories, Klaus
A1  - Schmidt, Gudula
T1  - Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)
JF  - PLoS Pathogens
N2  - The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which is a major virulence factor of pathogenic Escherichia coli strains. Here, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as cellular receptor for CNF1 by co-precipitation of cell surface molecules with tagged toxin. The CNF1-Lu/BCAM interaction was verified by direct protein-protein interaction analysis and competition studies. These studies revealed amino acids 720 to 1014 of CNF1 as the binding site for Lu/BCAM. We suggest two cell interaction sites in CNF1: first the N-terminus, which binds to p37LRP as postulated before. Binding of CNF1 to p37LRP seems to be crucial for the toxin's action. However, it is not sufficient for the binding of CNF1 to the cell surface. A region directly adjacent to the catalytic domain is a high affinity interaction 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 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 Escherichia coli 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 (UPEC) strains. We and others elucidated the molecular mechanism of the E. coli 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 interaction site to the C-terminal part of the toxin. We performed direct protein-protein interaction 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 for the generation of immunotoxins.
KW  - laminin receptor
KW  - factor-I
KW  - toxin
KW  - RHO
KW  - cells
KW  - activation
KW  - protein
KW  - domain
KW  - translocation
KW  - membrane
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117987
SN  - 1553-7374
VL  - 10
IS  - 1
ER  -