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

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen naturally resistant to many common antibiotics that acquires new resistance traits at an alarming pace. Targeting the bacterial virulence factors by an anti-virulence 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.

Review of Potential Pseudomonas Weaponry, Relevant to the Pseudomonas–Aspergillus Interplay, for the Mycology Community

“…Recent studies have identified fungal-specific T6SS effector proteins (Tfe1 and Tfe2) in another Gram-negative opportunistic bacteria Serratia marcescens [154,155]. These effector proteins contributed to T6SS-mediated inhibition of fungal growth in the fungal species Candida albicans, Candida glabrata, and Saccharomyces cerevisiae [154,155]. Effector-based antifungal activity was also reported for P. aeruginosa T6SS protein Tse2, which showed toxic effects and inhibition of growth in the yeast Saccharomyces cerevisiae [156].…”

“…The HSI-I and HSI-II loci have been implicated in P. aeruginosa-mediated pathogenesis in progressive lung diseases [153]; HSI-III has also been identified as an essential component for bacterial virulence [148]. Recent studies have identified fungal-specific T6SS effector proteins (Tfe1 and Tfe2) in another Gram-negative opportunistic bacteria Serratia marcescens [154,155]. These effector proteins contributed to T6SS-mediated inhibition of fungal growth in the fungal species Candida albicans, Candida glabrata, and Saccharomyces cerevisiae [154,155].…”

Metabotypes of Pseudomonas aeruginosa Correlate with Antibiotic Resistance, Virulence and Clinical Outcome in Cystic Fibrosis Chronic Infections

“…Cytotoxic potential of the P.a clinical isolates on eukaryotic cells was tested according to previously described protocols [73,74], with slight modifications. Antibiotic resistance phenotypes were tested following the recommendations of the European Committee for Antimicrobial Susceptibility Testing 2017 criteria [75].…”

High-Throughput Approaches for the Identification of Pseudomonas aeruginosa Antivirulents

“…Finally, a different group used a clever set of ELISAs featuring PscE and PscG as "bait" and signal (106). Compounds that bound to the bait protein and prevented the formation of the heterodimer were then combined using synthetic chemistry approaches to develop larger, more complex molecules with increased affinity and efficacy, an approach that is increasingly commonly used in the development pipeline for drugs coming from fragment-based screening (34).…”