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Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

Tafesse FG, Rashidfarrokhi A, Schmidt FI, Freinkman E, Dougan S, Dougan M, Esteban A, Maruyama T, Strijbis K, Ploegh HL - PLoS Pathog. (2015)

Bottom Line: Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans.While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface.Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America; Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts, United States of America.

ABSTRACT
The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

No MeSH data available.


Related in: MedlinePlus

Myriocin and Fumonisin B1 block sphingolipid biosynthesis in RAW macrophages and dendritic cell lines.(A) Schematic representation of the sphingolipid biosynthetic pathway in mammalian cells. Myriocin, a serine palmitoyltransferase (SPT) inhibitor, and Fumonisin B1 (FB1), a ceramide synthase (CS) inhibitor, block sphingolipid biosynthesis (boxed). The salvage pathway is shown in broken arrows. (B) Myriocin- or FB1-treated cells were labeled with the sphingomyelin precursor N-methyl-[14C]-choline, and total lipids were extracted and analyzed by TLC and autoradiography. (C) Quantification of the [14C]-SM and [14C]-PC signals from [14C]-choline labeling experiment in B. Error bars display SD of three independent experiments. Unpaired t-test was used to analyze the significance of the observed differences. ** p < 0.001.
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ppat.1005188.g001: Myriocin and Fumonisin B1 block sphingolipid biosynthesis in RAW macrophages and dendritic cell lines.(A) Schematic representation of the sphingolipid biosynthetic pathway in mammalian cells. Myriocin, a serine palmitoyltransferase (SPT) inhibitor, and Fumonisin B1 (FB1), a ceramide synthase (CS) inhibitor, block sphingolipid biosynthesis (boxed). The salvage pathway is shown in broken arrows. (B) Myriocin- or FB1-treated cells were labeled with the sphingomyelin precursor N-methyl-[14C]-choline, and total lipids were extracted and analyzed by TLC and autoradiography. (C) Quantification of the [14C]-SM and [14C]-PC signals from [14C]-choline labeling experiment in B. Error bars display SD of three independent experiments. Unpaired t-test was used to analyze the significance of the observed differences. ** p < 0.001.

Mentions: The small molecule inhibitors myriocin or fumonisin B1 (FB1) inhibit production of sphingolipids in mammalian cells. While myriocin blocks the activity of SPT-the first and rate-limiting reaction of this pathway- FB1 inhibits ceramide synthase [26, 30–32], causing a blockade in the production of ceramides, the backbone of all sphingolipids (Fig 1A).


Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

Tafesse FG, Rashidfarrokhi A, Schmidt FI, Freinkman E, Dougan S, Dougan M, Esteban A, Maruyama T, Strijbis K, Ploegh HL - PLoS Pathog. (2015)

Myriocin and Fumonisin B1 block sphingolipid biosynthesis in RAW macrophages and dendritic cell lines.(A) Schematic representation of the sphingolipid biosynthetic pathway in mammalian cells. Myriocin, a serine palmitoyltransferase (SPT) inhibitor, and Fumonisin B1 (FB1), a ceramide synthase (CS) inhibitor, block sphingolipid biosynthesis (boxed). The salvage pathway is shown in broken arrows. (B) Myriocin- or FB1-treated cells were labeled with the sphingomyelin precursor N-methyl-[14C]-choline, and total lipids were extracted and analyzed by TLC and autoradiography. (C) Quantification of the [14C]-SM and [14C]-PC signals from [14C]-choline labeling experiment in B. Error bars display SD of three independent experiments. Unpaired t-test was used to analyze the significance of the observed differences. ** p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4592247&req=5

ppat.1005188.g001: Myriocin and Fumonisin B1 block sphingolipid biosynthesis in RAW macrophages and dendritic cell lines.(A) Schematic representation of the sphingolipid biosynthetic pathway in mammalian cells. Myriocin, a serine palmitoyltransferase (SPT) inhibitor, and Fumonisin B1 (FB1), a ceramide synthase (CS) inhibitor, block sphingolipid biosynthesis (boxed). The salvage pathway is shown in broken arrows. (B) Myriocin- or FB1-treated cells were labeled with the sphingomyelin precursor N-methyl-[14C]-choline, and total lipids were extracted and analyzed by TLC and autoradiography. (C) Quantification of the [14C]-SM and [14C]-PC signals from [14C]-choline labeling experiment in B. Error bars display SD of three independent experiments. Unpaired t-test was used to analyze the significance of the observed differences. ** p < 0.001.
Mentions: The small molecule inhibitors myriocin or fumonisin B1 (FB1) inhibit production of sphingolipids in mammalian cells. While myriocin blocks the activity of SPT-the first and rate-limiting reaction of this pathway- FB1 inhibits ceramide synthase [26, 30–32], causing a blockade in the production of ceramides, the backbone of all sphingolipids (Fig 1A).

Bottom Line: Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans.While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface.Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America; Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts, United States of America.

ABSTRACT
The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

No MeSH data available.


Related in: MedlinePlus