Complex sphingolipid profiling and identification of an inositol-phosphorylceramide synthase in Dictyostelium discoideum.

Dictyostelium discoideum is a professional phagocyte frequently used to study cellular processes underlying the recognition, engulfment, and infection course of microbial pathogens. Sphingolipids are abundant components of the plasma membrane that bind cholesterol, control membrane properties, participate in signal transmission, and serve as adhesion molecules in recognition processes relevant to immunity and infection. By combining lipidomics with a bioinformatics-based cloning strategy, we show here that D. discoideum produces phosphoinositol-containing sphingolipids with predominantly phytoceramide backbones. Cell-free expression of candidate inositol-phosphorylceramide (IPC) synthases from D. discoideum enabled identification of an enzyme that selectively catalyzes the transfer of phosphoinositol from phosphatidylinositol onto ceramide. The IPC synthase, DdIPCS1, shares multiple sequence motifs with yeast IPC and human sphingomyelin synthases and localizes to the Golgi apparatus as well as the contractile vacuole of D. discoideum. These findings open up important opportunities for exploring a role of sphingolipids in phagocytosis and infection across major evolutionary boundaries.

ER-dependent membrane repair of mycobacteria-induced vacuole damage.

IMPORTANCE: Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. Mycobacterium tuberculosis, the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the endoplasmic reticulum (ER)-Golgi lipid transfer protein OSBP8 in the Dictyostelium discoideum/Mycobacterium marinum system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defense mechanism against intracellular pathogens such as M. tuberculosis.

Fungal hyphae colonization by Bacillus subtilis relies on biofilm matrix components.

Bacteria interact with their environment including microbes and higher eukaryotes. The ability of bacteria and fungi to affect each other are defined by various chemical, physical and biological factors. During physical association, bacterial cells can directly attach and settle on the hyphae of various fungal species. Such colonization of mycelia was proposed to be dependent on biofilm formation by the bacteria, but the essentiality of the biofilm matrix was not represented before. Here, we demonstrate that secreted biofilm matrix components of the soil-dwelling bacterium, Bacillus subtilis are essential for the establishment of a dense bacterial population on the hyphae of the filamentous black mold fungus, Aspergillus niger and the basidiomycete mushroom, Agaricus bisporus. We further illustrate that these matrix components can be shared among various mutants highlighting the community shaping impact of biofilm formers on bacteria-fungi interactions.