5-Fluorouracil blocks quorum-sensing of biofilm-embedded methicillin-resistant Staphylococcus aureus in mice.

Ferdinand Sedlmayer,Daniel Baeschlin,Vincent Unterreiner,Nina Khanna,Anne-Kathrin Woischnig,Martin Fussenegger,Florian Fuchs

doi:10.1093/nar/gkab251

Abstract

Antibiotic-resistant pathogens often escape antimicrobial treatment by forming protective biofilms in response to quorum-sensing communication via diffusible autoinducers. Biofilm formation by the nosocomial pathogen methicillin-resistant Staphylococcus aureus (MRSA) is triggered by the quorum-sensor autoinducer-2 (AI-2), whose biosynthesis is mediated by methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) and S-ribosylhomocysteine lyase (LuxS). Here, we present a high-throughput screening platform for small-molecular inhibitors of either enzyme. This platform employs a cell-based assay to report non-toxic, bioavailable and cell-penetrating inhibitors of AI-2 production, utilizing engineered human cells programmed to constitutively secrete AI-2 by tapping into the endogenous methylation cycle via ectopic expression of codon-optimized MTAN and LuxS. Screening of a library of over 5000 commercial compounds yielded 66 hits, including the FDA-licensed cytostatic anti-cancer drug 5-fluorouracil (5-FU). Secondary screening and validation studies showed that 5-FU is a potent quorum-quencher, inhibiting AI-2 production and release by MRSA, Staphylococcus epidermidis, Escherichia coli and Vibrio harveyi. 5-FU efficiently reduced adherence and blocked biofilm formation of MRSA in vitro at an order-of-magnitude-lower concentration than that clinically relevant for anti-cancer therapy. Furthermore, 5-FU reestablished antibiotic susceptibility and enabled daptomycin-mediated prevention and clearance of MRSA infection in a mouse model of human implant-associated infection.

Highlights

Molecular communication among bacteria by means of small diffusible signaling molecules, known as quorum sensing, serves to coordinate inter- and intra-population behavior [1]
We previously showed in a proof-of-concept study [31] that the modular biosynthetic AI-2 production platform can function in HEK cells, and AI-2 production is significantly decreased by the addition of known methylthioadenosine/Sadenosylhomocysteine nucleosidase (MTAN) or LuxS inhibitors such as immunicillin-A [32] and Sribosylhomocysteine analogues [33]
The functional combination of HEKAI-2-mediated AI-2 production with V. harveyi (MM32)-based AI-2 quantification provides a potent mammalian cell-based assay platform for the detection of bioavailable, non-cytotoxic and target-specific smallmolecular drugs quenching the production of AI-2 (Figure 2A)

Summary

Introduction

Molecular communication among bacteria by means of small diffusible signaling molecules, known as quorum sensing, serves to coordinate inter- and intra-population behavior [1]. Molecular interference with the pathogens’ quorum sensing, i.e., quorum quenching [10], by using degradative enzymes [11] or small-molecular inhibitors to block the synthesis and release of quorum-sensing signaling compounds, is one possible approach to switch off biofilm formation [12,13] and reduce or eliminate antibiotic resistance [14]. In this context, AI-2 is synthesized in two sequential steps:

Methods

Results

Conclusion