The Clostridium difficile Cell Wall Protein CwpV is Antigenically Variable between Strains, but Exhibits Conserved Aggregation-Promoting Function

Clostridium difficile is the main cause of antibiotic-associated diarrhea, leading to significant morbidity and mortality and putting considerable economic pressure on healthcare systems. Current knowledge of the molecular basis of pathogenesis is limited primarily to the activities and regulation of two major toxins. In contrast, little is known of mechanisms used in colonization of the enteric system. C. difficile expresses a proteinaceous array on its cell surface known as the S-layer, consisting primarily of the major S-layer protein SlpA and a family of SlpA homologues, the cell wall protein (CWP) family. CwpV is the largest member of this family and is expressed in a phase variable manner. Here we show CwpV promotes C. difficile aggregation, mediated by the C-terminal repetitive domain. This domain varies markedly between strains; five distinct repeat types were identified and were shown to be antigenically distinct. Other aspects of CwpV are, however, conserved. All CwpV types are expressed in a phase variable manner. Using targeted gene knock-out, we show that a single site-specific recombinase RecV is required for CwpV phase variation. CwpV is post-translationally cleaved at a conserved site leading to formation of a complex of cleavage products. The highly conserved N-terminus anchors the CwpV complex to the cell surface. Therefore CwpV function, regulation and processing are highly conserved across C. difficile strains, whilst the functional domain exists in at least five antigenically distinct forms. This hints at a complex evolutionary history for CwpV

Bordetella pertussis adenylate cyclase toxin: a versatile screening tool

International audienceThe calmodulin-activated adenylate cyclase (AC) toxin is an essential virulence factor of Bordetella pertussis, the causative agent of whooping cough. This toxin has been exploited to devise screening techniques for investigating diverse biological processes. This mini-review describes several such applications. First, AC has been utilized as a selective reporter for protein translocation from bacteria to eukaryotic cells, in particular to study protein targeting by type III secretion machinery. More recently, AC has been used as a signal transducer in Escherichia coli to elaborate genetic screens for protein-protein interactions ("bacterial two-hybrid system") or site-specific proteolytic activities

Corrosion des alliages d'aluminium et des aciers inoxydables par les produits de nettoyage et de desinfection.

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Construction of Pseudomonas aeruginosa two-hybrid libraries for high-throughput assays.

International audienceIn Pseudomonas aeruginosa, identification of new partners of a protein of interest could give precious clues to decipher a biological process in which this protein is involved. However, genes encoding for partners of a protein of interest are unknown and frequently scattered throughout the genome. We describe herein the construction and the use of pan-genomic bacterial two-hybrid libraries to identify new partners of a protein of interest encoded by P. aeruginosa

Autoantibodies specific for the phospholipase A2 receptor in recurrent and De Novo membranous nephropathy.

International audienceRecent findings in idiopathic membranous nephropathy (MN) suggest that in most patients, the disease is because of anti-phospholipase A(2) receptor (PLA(2) R1) autoantibodies. Our aim was to analyze the prevalence and significance of anti-PLA(2) R1 antibodies in recurrent and de novo MN after transplantation. We assessed circulating PLA(2) R1 autoantibodies by a direct immunofluorescence assay based on human embryonic kidney cells transfected with a PLA(2) R1 cDNA, and the presence of PLA(2) R1 antigen in immune deposits. We showed that PLA(2) R1 was involved in 5 of 10 patients with recurrent MN, but in none of the 9 patients with de novo MN. We also showed a marked heterogeneity in the kinetics and titers of anti-PLA(2) R1, which may relate to different pathogenic potential. We provide evidence that some patients with PLA(2) R1-related idiopathic MN and anti-PLA(2) R1 antibodies at the time of transplantation will not develop recurrence. Because PLA(2) R1 autoantibody was not always associated with recurrence, its predictive value should be carefully analyzed in prospective studies

A Bacterial Adenylate Cyclase-Based Two-Hybrid System Compatible with Gateway® Cloning

International audienceThe bacterial adenylate cyclase two-hybrid system (BACTH) is a genetic approach used to test protein interactions in vivo in E. coli. This system takes advantage of the two catalytic domains of Bordetella pertussis adenylate cyclase (CyaA) toxin, which can be fused separately to proteins of interest. If the proteins of interest interact, then the adenylate cyclase domains will be brought in close proximity to each other, reconstituting cyclic AMP (cAMP) production. Interacting proteins can be both qualitatively and quantitatively assessed by the expression of chromosomal genes of the E. coli lac or mal operon, which are positively regulated by cAMP production. Because cAMP is diffusible, the proteins of interest do not need to interact near the transcriptional machinery. Consequently, both cytosolic and membrane protein-protein interactions can be tested. The BACTH system has recently been modified to be compatible with Gateway® recombinational cloning, BACTHGW. This chapter explains the principle of the BACTH, its Gateway® modified system, and details of the general procedure