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Published on: June 30, 2016
Discovery and characterization of inhibitors of Pseudomonas aeruginosa type III secretion
Daniel Aiello1, John D Williams, Helena Majgier-Baranowska
1Microbiotix, Inc., Worcester, MA 01605, USA.
Abstract:
The type III secretion system (T3SS) is a clinically important virulence mechanism in Pseudomonas aeruginosa that secretes and translocates up to four protein toxin effectors into human cells, facilitating the establishment and dissemination of infections. To discover inhibitors of this important virulence mechanism, we developed two cellular reporter assays and applied them to a library of 80,000 compounds. The primary screen was based on the dependence of the transcription of T3SS operons on the T3SS-mediated secretion of a negative regulator and consisted of a transcriptional fusion of the Photorhabdus luminescens luxCDABE operon to the P. aeruginosa exoT effector gene. Secondary assays included direct measurements of the T3SS-mediated secretion of a P. aeruginosa ExoS effector-beta-lactamase fusion protein as well as the detection of the secretion of native ExoS by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of culture supernatants. Five inhibitors in three chemical classes were demonstrated to inhibit type III secretion selectively with minimal cytotoxicity and with no effects on bacterial growth or on the type II-mediated secretion of elastase. These inhibitors also block the T3SS-mediated secretion of a YopE effector-beta-lactamase fusion protein from an attenuated Yersinia pestis strain. The most promising of the inhibitors is a phenoxyacetamide that also blocks the T3SS-mediated translocation of effectors into mammalian cells in culture. Preliminary studies of structure-activity relationships in this phenoxyacetamide series demonstrated a strict requirement for the R-enantiomer at its stereocenter and indicated tolerance for a variety of substituents on one of its two aromatic rings.
Insights
Researchers identified novel inhibitors of the Pseudomonas aeruginosa type III secretion system (T3SS), a key bacterial virulence factor. These compounds selectively block T3SS activity, offering potential new treatments for infections.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Drug Discovery
Background:
- The type III secretion system (T3SS) is a critical virulence factor in Pseudomonas aeruginosa, enabling pathogen invasion and infection dissemination.
- T3SS facilitates the delivery of effector proteins into host cells, contributing to disease establishment.
Purpose of the Study:
- To discover and characterize novel inhibitors targeting the T3SS of Pseudomonas aeruginosa.
- To identify compounds that selectively disrupt T3SS function with minimal impact on bacterial viability or other secretion systems.
Main Methods:
- Development and application of two cellular reporter assays for high-throughput screening of an 80,000-compound library.
- Assays included transcriptional fusions and direct measurement of effector protein secretion (ExoS-beta-lactamase).
- Validation using SDS-PAGE analysis of culture supernatants and testing against Yersinia pestis T3SS.
Main Results:
- Identified five selective inhibitors of type III secretion across three chemical classes.
- Inhibitors demonstrated minimal cytotoxicity and no effect on bacterial growth or type II secretion.
- The most potent inhibitor, a phenoxyacetamide, blocked effector translocation into mammalian cells and showed specific structure-activity relationships.
Conclusions:
- Novel chemical classes of T3SS inhibitors were discovered, offering promising leads for anti-virulence therapies.
- The identified phenoxyacetamide derivatives represent a potential new avenue for combating Pseudomonas aeruginosa infections by targeting a crucial virulence mechanism.
