Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane

Abstract

Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC) hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG) with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-γ (EYFP-C1AB). DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase Cγ-1 (PLCγ-1), a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLCγ-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step) and activation of endogenous PLCγ-1 (second step) leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA.

Clostridium perfringens Epsilon-Toxin Impairs the Barrier Function in MDCK Cell Monolayers in a Ca2+-Dependent Manner

“…In contrast, U73343 did not block the toxin-induced responses. To determine DAG formation in the cytoplasmic membrane of MDCK cells, we utilized an indicator, enhanced yellow fluorescent protein (EYFP)-tagged C1A and C1B regions of protein kinase C-γ (EYFP-C1AB), which permits the binding of DAG [39]. The transfection of MDCK cells with EYFP-C1AB resulted in homogeneous fluorescence signals throughout the cells ( Figure 2E).…”

“…Plasmid for the enhanced yellow fluorescent protein-fused C1AB domain of protein kinase C-γ (EYFP-C1AB) was constructed [39]. For DG staining on the outer leaflet of the plasma membrane, the plasmid was electroporated into MDCK cells utilizing a Neon Transfection System (ThermoFisher).…”

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…perfringens alpha-toxin induces the respiratory burst in neutrophils by inducing the activation of PKC via two separate pathways: elevation of DAG generated by activation of an endogenous PLC through a pertussis toxin-sensitive GTP-binding protein, and activation of a tropomyosin-related kinase receptor (TrkA receptor) that leads to PDK1 phosphorylation (225,226). In epithelial cells, alpha-toxin binding to GM1a ganglioside induces TNF-␣ and IL-8 production through the activation of the TrkA receptor and the p38 mitogen-activated protein kinase (MAPK) pathway, as well as a PLC-␥1, ERK1/2-NF-B-dependent pathway (232)(233)(234). TNF-␣ plays an important role in the toxic effect of alpha-toxin, since the administration of specific antibodies against TNF-␣ protects mice from its lethal effect (235).…”

Membrane-Binding Mechanism of Clostridium perfringens Alpha-Toxin

“…Ichikawa et al [ 33 ] reported that GM1 clustering promotes the enrichment of TrkA in the lipid raft and activation of downstream signal transduction pathways. We also found that alpha-toxin induced diacylglycerol formation at the plasma membrane [ 34 ]. The ensuing flip-flop motion of diacylglycerol influences membrane dynamics, thereby promoting GM1a clustering and activation of endogenous phospholipase C-γ1 via TrkA [ 34 , 35 ].…”

“…We also found that alpha-toxin induced diacylglycerol formation at the plasma membrane [ 34 ]. The ensuing flip-flop motion of diacylglycerol influences membrane dynamics, thereby promoting GM1a clustering and activation of endogenous phospholipase C-γ1 via TrkA [ 34 , 35 ]. These results suggest that the binding of alpha-toxin to GM1a plays an important role in the clustering and activation of TrkA ( Figure 2 ).…”