These findings might be reconciled with those we obtained using the yeast two-hybrid interaction assay. The Nutlin 3a binding to VipB may simply be too weak to be revealed by this assay. Interestingly, the two-hybrid assay did detect binding between the N-terminus of ClpV and VipA as well as two VipA homologues encoded by P. aeruginosa and Y. pseudotuberculosis. This

may be a reflection of that the peptide library used by Pietrosiuk et al. may not be sufficient to reveal an interaction present between the ClpV N-terminus and intact VipA proteins, since there may be Cell Cycle inhibitor secondary structures of VipA that allow its binding to ClpV. Our finding also implies that the VipA-VipB interaction with ClpV may be more complicated than previously anticipated. Although the study by Pietrosiuk et al. did not detect VipA degradation in a cell-free context, levels were significantly reduced when intact V. cholerae bacteria were analyzed, indicating that there may be direct interaction between ClpV and VipA [9]. Altogether, our findings indicate that the VipA/VipB complex

has unique functional constraints and our previous findings indicate that the constraints are shared by the homologous complexes in other Gram-negative bacteria. Since VipA-VipB homologues are present in such a wide variety of pathogens, this interaction offers a unique and attractive target for the development of novel antibacterial agents. Future investigations to identify drugs that block the VipA-VipB interaction could lead to the development of therapeutics effective against a wide range of infectious diseases. Crenolanib Conclusions VipA and VipB homologues are known to interact in many Gram-negative pathogens. In V. cholerae, their essential role in the secretion of T6S substrates has been demonstrated previously. Using site-directed mutagenesis

Liothyronine Sodium within VipA, we demonstrated that a dramatically diminished interaction to VipB was shown to correlate with a decrease in VipB stability and a loss of Hcp secretion and rendered the bacterium unable to compete with Escherichia coli in a competition assay. This confirms the biological relevance of the VipA-VipB interaction, which is a prerequisite also for the T6S activity of intracellular pathogens like Francisella tularensis and Burkholderia cenocepacia. Thus, this conserved interaction offers an attractive target for the development of novel antibacterials. Methods Bacterial strains, plasmids and growth conditions Bacterial strains and plasmids used in this study are listed in a table [see Additional file 1]. E. coli and V. cholerae were cultivated on Luria Bertani (LB) agar or broth at 37°C unless stated otherwise. When necessary, carbenicillin (Cb; 100 μg/ml), kanamycin (Km; 50 μg/ml), chloramphenicol (Cm; 25 μg/ml), rifampicin (Rif; 100 μg/ml), streptomycin (Strp; 50 μg/ml) or tetracycline (Tet; 10 μg/ml) were used.