Anti-Virulence Strategy against the Honey Bee Pathogenic Bacterium Paenibacillus larvae via Small Molecule Inhibitors of the Bacterial Toxin Plx2A

Abstract

American Foulbrood, caused by Paenibacillus larvae, is the most devastating bacterial honey bee brood disease. Finding a treatment against American Foulbrood would be a huge breakthrough in the battle against the disease. Recently, small molecule inhibitors against virulence factors have been suggested as candidates for the development of anti-virulence strategies against bacterial infections. We therefore screened an in-house library of synthetic small molecules and a library of flavonoid natural products, identifying the synthetic compound M3 and two natural, plant-derived small molecules, Acacetin and Baicalein, as putative inhibitors of the recently identified P. larvae toxin Plx2A. All three inhibitors were potent in in vitro enzyme activity assays and two compounds were shown to protect insect cells against Plx2A intoxication. However, when tested in exposure bioassays with honey bee larvae, no effect on mortality could be observed for the synthetic or the plant-derived inhibitors, thus suggesting that the pathogenesis strategies of P. larvae are likely to be too complex to be disarmed in an anti-virulence strategy aimed at a single virulence factor. Our study also underscores the importance of not only testing substances in in vitro or cell culture assays, but also testing the compounds in P. larvae-infected honey bee larvae.

A Structural Approach to Anti-Virulence: A Discovery Pipeline

“…A fluorescence-based plate-reader enzyme assay is often used to determine the inhibitor properties (IC 50 , K i values) of the selected compounds (Figure 2). A fluorescence-based binding assay for lead compounds with the target mART is also established based on quenching of intrinsic fluorescence upon enzyme-inhibitor association to provide a measure of binding affinity (K D ) [9].…”

“…Lead compounds identified in virtual screens or from "directed libraries" that show promise in enzyme and mammalian cell assays are further developed with respect to binding potency and pharmacokinetic properties. These compounds are investigated initially through a The IC 50 values were calculated from the fit to a dose-response curve of Plx2A GH activity against the concentration of inhibitor/flavonoid compounds to produce a directed library of 20 compounds for experimental testing against Plx2A activity [9]. c ND, not determined.…”

“…These factors include two mART toxins, Plx1 and Plx2, shown to be important ERICI virulence factors [94]; Plx2A was recently characterized and crystallized [61]. However, to date, only a few of the P. larvae virulence factors have been targeted for an anti-virulence strategy [9,61,95].…”

“…A compelling, innovative, and alternative approach to antibiotic therapy is the antivirulence or anti-infective strategy that involves targeting virulence-associated rather than survival/fitness-relevant traits in the offending pathogen [8][9][10][11][12][13]. Thus, the "anti-infective" agent/drug interferes with the pathogen's ability to cause disease [14,15].…”

“…Thus, the "anti-infective" agent/drug interferes with the pathogen's ability to cause disease [14,15]. One avenue for this approach is to target bacterial toxins using this powerful method [9,[16][17][18][19]. This approach involves the use of patho-or virulence blockers developed specifically to bind bacterial virulence factors with high affinity, neutralizing or reducing the virulence of the offending pathogen [17,20,21].…”