ABSTRACT
Bacterial pore-forming toxin aerolysin-like proteins (ALPs) are widely distributed in animals and plants. However, functional studies on these ALPs remain in their infancy. ßγ-CAT is the first example of a secreted pore-forming protein that functions to modulate the endolysosome pathway via endocytosis and pore formation on endolysosomes. However, the specific cell surface molecules mediating the action of ßγ-CAT remain elusive. Here, the actions of ßγ-CAT were largely attenuated by either addition or elimination of acidic glycosphingolipids (AGSLs). Further study revealed that the ALP and trefoil factor (TFF) subunits of ßγ-CAT bind to gangliosides and sulfatides, respectively. Additionally, disruption of lipid rafts largely impaired the actions of ßγ-CAT. Finally, the ability of ßγ-CAT to clear pathogens was attenuated in AGSL-eliminated frogs. These findings revealed a previously unknown double binding pattern of an animal-secreted ALP in complex with TFF that initiates ALP-induced endolysosomal pathway regulation, ultimately leading to effective antimicrobial responses.
Subject(s)
Acidic Glycosphingolipids/chemistry , Amphibian Proteins/immunology , Bacterial Toxins/immunology , Gram-Negative Bacterial Infections/immunology , Lysosomes/immunology , Multiprotein Complexes/immunology , Pore Forming Cytotoxic Proteins/immunology , Trefoil Factor-3/immunology , Acidic Glycosphingolipids/antagonists & inhibitors , Acidic Glycosphingolipids/biosynthesis , Aeromonas hydrophila/growth & development , Aeromonas hydrophila/pathogenicity , Amphibian Proteins/genetics , Amphibian Proteins/metabolism , Animals , Anura , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Ceramides/antagonists & inhibitors , Ceramides/biosynthesis , Ceramides/chemistry , Cerebrosides/antagonists & inhibitors , Cerebrosides/biosynthesis , Cerebrosides/chemistry , Gangliosides/antagonists & inhibitors , Gangliosides/biosynthesis , Gangliosides/chemistry , Gene Expression , Gram-Negative Bacterial Infections/genetics , Gram-Negative Bacterial Infections/microbiology , Humans , Interleukin-1beta/biosynthesis , Lysosomes/drug effects , Lysosomes/microbiology , Membrane Microdomains/drug effects , Membrane Microdomains/immunology , Membrane Microdomains/microbiology , Meperidine/analogs & derivatives , Meperidine/pharmacology , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Pore Forming Cytotoxic Proteins/genetics , Pore Forming Cytotoxic Proteins/metabolism , Sphingosine/antagonists & inhibitors , Sphingosine/biosynthesis , Sphingosine/chemistry , THP-1 Cells , Trefoil Factor-3/genetics , Trefoil Factor-3/metabolismABSTRACT
Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex (Lala, P., Ito, S., and Lingwood, C. A. (2000) J. Biol. Chem. 275, 6246-6251). We now show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral glycosphingolipid biosynthesis in 11 of 12 cell lines tested. The exception, HeLa cells, do not express MDR1. Microsomal lactosyl ceramide and globotriaosyl ceramide synthesis from endogenous or exogenously added liposomal glucosyl ceramide was inhibited by cyclosporin A, consistent with a direct role for MDR1/glucosyl ceramide translocase activity in their synthesis. In contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition, suggesting neutral and acid glycosphingolipids are synthesized from distinct precursor glycosphingolipid pools. Metabolic labeling in wild type and knock-out (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid glycosphingolipid synthesis.