disk diffusion assay, Yersinia pestis, type III secretion systems, type III secretion system inhibitors, magnesium oxalate agar, imidocarb dipropionate, small molecule inhibitors
Current methods for screening small molecules that inhibit the plasmid pCD1-encoded Yersinia pestis type III secretion system (T3SS) include lengthy growth curves followed by multistep luminescence assays or Western blot assays to detect secretion, or lack thereof, of effector proteins. The goal of this research was to develop a novel disk diffusion assay on magnesium oxalate (MOX) agar as a simple way to evaluate the susceptibility of Y. pestis to type III secretion system inhibitors. MOX agar produces distinct Y. pestis growth characteristics based on the bacteria’s ability or inability to secrete effector proteins; small, barely visible colonies are observed when secretion is activated versus larger, readily visible colonies when secretion is inhibited. Wild-type Y. pestis was diluted and spread onto a MOX agar plate. Disks containing 20ml of various concentrations of imidocarb dipropionate, a known Y. pestis T3SS inhibitor, or distilled water (dH2O) were placed on the plate. After incubation at 37°C for 48 h, visible colonies of Y. pestis were observed surrounding the disks with imidocarb dipropionate, suggesting that T3S was inhibited. The diameter of the growth of colonies surrounding the disks increased as the concentration of the T3SS inhibitor increased. Imidocarb dipropionate was also able to inhibit Y. pestis strains lacking effector Yops and Yop chaperones, suggesting that they are not necessary for T3S inhibition. This disk diffusion assay is a feasible and useful method for testing the susceptibility of Y. pestis to type III secretion system inhibitors and has the potential to be used in a clinical setting.
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Prashar, Sukriti; Miguel Portales Guemes; Poorandai Shivbaran; Eugenia Jimenez Alvarez; Christopher Soha; Samir Nacer; Michael McDonough; Gregory V. Plano; and Julie Torruellas Garcia. 2021. "Novel Disk Diffusion Assay on Magnesium Oxalate Agar To Evaluate the Susceptibility of Yersinia pestis to Type III Secretion System Inhibitors." Microbiology Spectrum 9, (1): e00005-21. doi:https://doi.org/10.1128/spectrum.00005-21.