|Tran, Nini -|
|Zielkel, Ryszard -|
|Vining, Oliver -|
|Armstrong, Donald -|
|Mcphail, Kerry -|
|Sikora, Aleksandra -|
Submitted to: Journal of Biomolecular Screening
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 31, 2013
Publication Date: November 30, 2013
Citation: Tran, N., Zielkel, R.A., Vining, O.B., Azevedo, M.D., Armstrong, D.J., Banowetz, G.M., Mcphail, K.L., Sikora, A.E. 2013. Development and application of a quantitative assay amenable for high-throughput screening to target the type II secretion system for new treatments against plant-pathogenic bacteria. Journal of Biomolecular Screening. 18(8):921-929. Interpretive Summary: One factor which affects how readily a plant pathogen can infect and damage a crop plant is their Type II secretion system. The Type II secretion system helps the pathogen enter, damage, and spread through the plant. Finding approaches to inactivate the Type II secretion system of bacterial pathogens would provide a tool for reducing the impact of diseases on crop productivity. This study describes the development of a new assay that is able to screen large numbers of potential sources of compounds that inhibit the function of the Type II secretion system. The new assay was validated by utilizing substances with known activity against the Type II system and showed excellent precision and accuracy. The assay will be useful for subsequent for screening new sources of agents that inhibit the Type II secretion system.
Technical Abstract: Plant-pathogenic bacteria are the causative agents of diseases in important agricultural crops and ornamental plants. The severe economic burden of these diseases requires seeking new approaches, particularly because phytopathogenic bacteria are often resistant to currently available treatments. The type II secretion (T2S) system is a key virulence factor utilized by major groups of phytopathogenic bacteria. The T2S machinery transports many hydrolytic enzymes responsible for degradation of the plant cell-wall, thus enabling successful colonization and dissemination of the bacteria in the plant host. Importantly, the genetic inactivation of the T2S system leads to loss of virulence, which strongly suggests that targeting the T2S could enable new treatments against plant-pathogenic bacteria. Accordingly, we have designed and optimized an assay to identify small-molecule inhibitors of the T2S system. The assay utilizes a double parametric output: measurement of bacterial growth and the enzymatic activity of cellulase, which is secreted via the T2S pathway in our model organism Dickeya dadantii (formerly Erwinia chrysantemi). The assay was evaluated by screening natural extracts, culture filtrates isolated from rhizosphere bacteria, and a collection of pharmaceutically active compounds LOPAC1280TM. The calculated Z’-value of 0.61 strongly suggest that the assay is applicable for a full scale, high-throughput screening platform.