Chimeric Protein-Protein Interface Inhibitors Allow Efficient Inhibition of Type III Secretion Machinery and Pseudomonas aeruginosa Virulence.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen naturally resistant to many common antibiotics and acquires new resistance traits at an alarming pace. Targeting the bacterial virulence factors by an antivirulence strategy, therefore, represents a promising alternative approach besides antibiotic therapy. The Type III secretion system (T3SS) of P. aeruginosa is one of its main virulence factors. It consists of more than 20 proteins building a complex syringe-like machinery enabling the injection of toxin into host cells. Previous works showed that disrupting interactions between components of this machinery efficiently lowers the bacterial virulence. Using automated target-based screening of commercial and in-house libraries of small molecules, we identified compounds inhibiting the protein-protein interaction between PscE and PscG, the two cognate chaperones of the needle subunit PscF of P. aeruginosa T3SS. Two hits were selected and assembled using Split/Mix/Click chemistry to build larger hybrid analogues. Their efficacy and toxicity were evaluated using phenotypic analysis including automated microscopy and image analysis. Two nontoxic hybrid leads specifically inhibited the T3SS and reduced the ex vivo cytotoxicity of bacteria and their virulence in Galleria mellonella.

Cochaperone interactions in export of the type III needle component PscF of Pseudomonas aeruginosa.

Type III secretion (T3S) systems allow the export and translocation of bacterial effectors into the host cell cytoplasm. Secretion is accomplished by an 80-nm-long needle-like structure composed, in Pseudomonas aeruginosa, of the polymerized form of a 7-kDa protein, PscF. Two proteins, PscG and PscE, stabilize PscF within the bacterial cell before its export and polymerization. In this work we screened the 1,320-A(2) interface between the two chaperones, PscE and PscG, by site-directed mutagenesis and determined hot spot regions that are important for T3S function in vivo and complex formation in vitro. Three amino acids in PscE and five amino acids in PscG, found to be relevant for complex formation, map to the central part of the interacting surface. Stability assays on selected mutants performed both in vitro on purified PscE-PscG complexes and in vivo on P. aeruginosa revealed that PscE is a cochaperone that is essential for the stability of the main chaperone, PscG. Notably, when overexpressed from a bicistronic construct, PscG and PscF compensate for the absence of PscE in cytotoxic P. aeruginosa. These results show that all of the information needed for needle protein stabilization and folding, its presentation to the T3 secreton, and its export is present within the sequence of the PscG chaperone.

Structure of the heterotrimeric complex that regulates type III secretion needle formation.

Type III secretion systems (T3SS), found in several Gram-negative pathogens, are nanomachines involved in the transport of virulence effectors directly into the cytoplasm of target cells. T3SS are essentially composed of basal membrane-embedded ring-like structures and a hollow needle formed by a single polymerized protein. Within the bacterial cytoplasm, the T3SS needle protein requires two distinct chaperones for stabilization before its secretion, without which the entire T3SS is nonfunctional. The 2.0-A x-ray crystal structure of the PscE-PscF(55-85)-PscG heterotrimeric complex from Pseudomonas aeruginosa reveals that the C terminus of the needle protein PscF is engulfed within the hydrophobic groove of the tetratricopeptide-like molecule PscG, indicating that the macromolecular scaffold necessary to stabilize the T3SS needle is totally distinct from chaperoned complexes between pilus- or flagellum-forming molecules. Disruption of specific PscG-PscF interactions leads to impairment of bacterial cytotoxicity toward macrophages, indicating that this essential heterotrimer, which possesses homologs in a wide variety of pathogens, is a unique attractive target for the development of novel antibacterials.

An expeditious stereoselective synthesis of natural (-)-Cassine via cascade HWE [3 + 2]-cycloaddition process.

l-Rhamnose is transformed to (-)-Cassine via a remarkable four step one pot reaction. The Horner-Wadsworth-Emmons [3 + 2]-1,3-dipolar cycloaddition reaction cascade is the pivotal step in this reaction sequence and makes the synthesis highly efficient.