title: Development of a quantitative assay amenable for high-throughput screening to target the type II secretion system for new treatments against plant-pathogenic bacteria.
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 for their control, particularly because phytopathogenic bacteria are often resistant to available treatments. The type II secretion (T2S) system is a key virulence factor used 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. 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. This assay uses 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. The assay was evaluated by screening natural extracts, culture filtrates isolated from rhizosphere bacteria, and a collection of pharmaceutically active compounds in LOPAC(1280). The calculated Z' values of 0.63, 0.63, and 0.58, respectively, strongly suggest that the assay is applicable for a high-throughput screening platform.
authors.affiliation: Department of Pharmaceutical Sciences, Oregon State University, Corvallis, OR, USA.
authors.forename: Nini
authors.initials: N
authors.lastname: Tran
authors.forename: Ryszard A
authors.initials: RA
authors.lastname: Zielke
authors.forename: Oliver B
authors.initials: OB
authors.lastname: Vining
authors.forename: Mark D
authors.initials: MD
authors.lastname: Azevedo
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Armstrong
authors.forename: Gary M
authors.initials: GM
authors.lastname: Banowetz
authors.forename: Kerry L
authors.initials: KL
authors.lastname: McPhail
authors.forename: Aleksandra E
authors.initials: AE
authors.lastname: Sikora
mesh_terms: D001419:Bacteria / Q000187:drug effects / Q000254:growth & development / Q000472:pathogenicity; D058947:Bacterial Secretion Systems / Q000187:drug effects*; D002480:Cellulase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D055808:Drug Discovery; D004755:Enterobacteriaceae / Q000187:drug effects*; D057166:High-Throughput Screening Assays / Q000379:methods*; D008826:Microbial Sensitivity Tests; D010935:Plant Diseases / Q000382:microbiology / Q000628:therapy*; D010944:Plants / Q000382:microbiology; D058441:Rhizosphere
chemical_list: D058947:Bacterial Secretion Systems; D002480:Cellulase
keywords: Dickeya dadantii; cellulase activity; high-throughput assay; phytopathogenic bacteria; type II secretion system