Type three secretion systems (TTSSs) are virulence factors found in many pathogenic Gram-negative species, including the family of pathogenic Yersinia spp. Yersinia pseudotuberculosis requires the translocation of a group of effector molecules, called Yops, to subvert the innate immune response and establish infection. Polarized transfer of Yops from bacteria to immune cells depends on several factors, including the presence of a functional TTSS, the successful attachment of Yersinia to the target cell, and translocon insertion into the target cell membrane. Here we employed a high-throughput screen to identify small molecules that block translocation of Yops into mammalian cells. We identified 6 compounds that inhibited translocation of effectors without affecting synthesis of TTSS components and secreted effectors, assembly of the TTSS, or secretion of effectors. One compound, C20, reduced adherence of Y. pseudotuberculosis to target cells. Additionally, the compounds caused leakage of Yops into the supernatant during infection and thus reduced polarized translocation. Furthermore, several molecules, namely, C20, C22, C24, C34, and C38, also inhibited ExoS-mediated cell rounding, suggesting that the compounds target factors that are conserved between Pseudomonas aeruginosa and Y. pseudotuberculosis. In summary, we have identified 6 compounds that specifically inhibit translocation of Yops into mammalian cells but not Yop synthesis or secretion.
Although identified as an early-diverged protozoan, Giardia lamblia shares many similarities with higher eukaryotic cells, including an internal membrane system and cytoskeleton, as well as secretory pathways. However, unlike many other eukaryotes, Giardia does not synthesize lipids de novo, but rather depends on exogenous sources for both energy production and organelle or membrane biogenesis. It is not known how lipid molecules are taken up by this parasite and if endocytic pathways are involved in this process. In this investigation, we tested the hypothesis that highly regulated and selective lipid transport machinery is present in Giardia and necessary for the efficient internalization and intracellular targeting of ceramide molecules, the major sphingolipid precursor. Using metabolic and pathway inhibitors, we demonstrate that ceramide is internalized through endocytic pathways and is primarily targeted into perinuclear/endoplasmic reticulum membranes. Further investigations suggested that Giardia uses both clathrin-dependent pathways and the actin cytoskeleton for ceramide uptake, as well as microtubule filaments for intracellular localization and targeting. We speculate that this parasitic protozoan has evolved cytoskeletal and clathrin-dependent endocytic mechanisms for importing ceramide molecules from the cell exterior for the synthesis of membranes and vesicles during growth and differentiation.
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