However, most putative drug hits fail at later stages of development and/or implementation, and few hits actually become leads. The growing technological capabilities of better and faster instrumentation coupled to assays both in vitro and ex vivo have had a huge impact on high-throughput screening (HTS) platforms that have propelled scientific and drug discoveries. We describe for the first time a cell-based assay that monitors DenV prM processing within the classical secretory pathway, which was exploited to identify a potential novel drug against DenV. A pilot screen against 1280 compounds was executed, leading to the identification of a potential active and corroborating the robustness of our assay for large-scale screening. The assay, miniaturized into a 96-well plate format, was multiplexed with the HIV-1 envelope boundary, also cleaved in the same pathway. The assay discriminates between a single cell-surface tag when prM is cleaved and two tags when it is not, as detected through fluorescent-coupled antibodies by flow cytometry. We have thus established a cell-based platform to monitor prM processing that relies on an engineered two-tag scaffold that travels to the cell surface through the secretory pathway. Blockade of prM cleavage would restrict fusion and represents a novel druggable opportunity against DenV. prM molecules in immature particles are cleaved by host proteases, leading to mature fusogenic virions. Our work shows that bacteria use stress responses to detect and respond to competition in a manner important for major phenotypes, including biofilm formation, virulence, and antibiotic tolerance.The DenV pre-membrane protein (prM) is a crucial chaperone for the viral envelope protein, preventing premature fusion with vesicles during viral export. Moreover, inactivation of the type VI secretion system (T6SS) of a competitor annuls the responses to competition, indicating that T6SS-derived cell damage activates these stress response systems. We further show that these responses arise via activation of major stress responses, providing direct support for the competition sensing hypothesis. We validate that these regulatory changes result in the predicted phenotypic changes in biofilm, mammalian cell invasion, and antibiotic tolerance. This screening reveals the presence of competing strains drives up the expression of genes associated with biofilm matrix production (CsgD pathway), epithelial invasion (SPI1 invasion system), and, finally, chemical efflux and antibiotic tolerance (TolC efflux pump and AadA aminoglycoside 3-adenyltransferase). Here, we solve this problem by using differential fluorescence induction to screen the Salmonella Typhimurium genome for loci that respond, at the single-cell level, to life in biofilms with competing strains of S.

The competition sensing hypothesis states that bacterial stress response systems can serve to detect ecological competition, but studying regulatory responses in diverse communities is challenging. Bacteria use complex regulatory networks to cope with stress, but the function of these networks in natural habitats is poorly understood.

Flowjo 7.6.5 free download