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Microbial sphingomyelinase induces RhoA-mediated reorganization of the apical brush border membrane and is protective against invasion.

Saslowsky DE, Thiagarajah JR, McCormick BA, Lee JC, Lencer WI - Mol. Biol. Cell (2016)

Bottom Line: How the BBM dynamically responds to pathogenic and commensal bacterial signals can define intestinal homeostasis and immune function.We previously found that in model intestinal epithelium, the conversion of apical membrane sphingomyelin to ceramide by exogenous bacterial sphingomyelinase (SMase) protected against the endocytosis and toxicity of cholera toxin.Here we elucidate a mechanism of action by showing that SMase induces a dramatic, reversible, RhoA-dependent alteration of the apical cortical F-actin network.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology and Nutrition, Boston Children's Hospital, Boston, MA 02115 Harvard Digestive Diseases Center, Boston Children's Hospital, Boston, MA 02115 Harvard Medical School, Boston, MA 02115 david.saslowsky@nih.gov.

No MeSH data available.


Related in: MedlinePlus

SMase secretion by live bacteria induces cortical F-actin reorganization. T84 monolayers were incubated apically with wild-type S. aureus or a mutant devoid of SMase activity (MOI = 20) for 1 h, fixed, and F-actin imaged as in Figure 1. Bar, 10 μm.
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Figure 2: SMase secretion by live bacteria induces cortical F-actin reorganization. T84 monolayers were incubated apically with wild-type S. aureus or a mutant devoid of SMase activity (MOI = 20) for 1 h, fixed, and F-actin imaged as in Figure 1. Bar, 10 μm.

Mentions: To demonstrate that the SMase-induced phenotype can be elicited via host–microbe interactions, we performed coculture experiments with human T84 monolayers and S. aureus bacteria that synthesize and secrete a SMase enzyme (Hlb/β-hemolysin). T84 monolayers incubated apically for 75 min with wild-type, SMase-secreting S. aureus (multiplicity of infection [MOI] = 20; strain 8325-4) displayed a similar F-actin phenotype as when exposed to purified SMase alone (Figure 2, top left). Coculture of monolayers with an isogenic S. aureus strain harboring a viral-inactivating insertion in the β-hemolysin gene (MOI = 20; strain DU5719; Patel et al., 1987) resulted in a completely normal actin cytoskeleton (Figure 2, bottom left), consistent with the F-actin phenotype being induced by SMase enzymatic activity. As with the purified enzyme, apical exposure of monolayers to S. aureus had no effect on basolateral stress fibers (Figure 2, right) or TER (unpublished data).


Microbial sphingomyelinase induces RhoA-mediated reorganization of the apical brush border membrane and is protective against invasion.

Saslowsky DE, Thiagarajah JR, McCormick BA, Lee JC, Lencer WI - Mol. Biol. Cell (2016)

SMase secretion by live bacteria induces cortical F-actin reorganization. T84 monolayers were incubated apically with wild-type S. aureus or a mutant devoid of SMase activity (MOI = 20) for 1 h, fixed, and F-actin imaged as in Figure 1. Bar, 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4814219&req=5

Figure 2: SMase secretion by live bacteria induces cortical F-actin reorganization. T84 monolayers were incubated apically with wild-type S. aureus or a mutant devoid of SMase activity (MOI = 20) for 1 h, fixed, and F-actin imaged as in Figure 1. Bar, 10 μm.
Mentions: To demonstrate that the SMase-induced phenotype can be elicited via host–microbe interactions, we performed coculture experiments with human T84 monolayers and S. aureus bacteria that synthesize and secrete a SMase enzyme (Hlb/β-hemolysin). T84 monolayers incubated apically for 75 min with wild-type, SMase-secreting S. aureus (multiplicity of infection [MOI] = 20; strain 8325-4) displayed a similar F-actin phenotype as when exposed to purified SMase alone (Figure 2, top left). Coculture of monolayers with an isogenic S. aureus strain harboring a viral-inactivating insertion in the β-hemolysin gene (MOI = 20; strain DU5719; Patel et al., 1987) resulted in a completely normal actin cytoskeleton (Figure 2, bottom left), consistent with the F-actin phenotype being induced by SMase enzymatic activity. As with the purified enzyme, apical exposure of monolayers to S. aureus had no effect on basolateral stress fibers (Figure 2, right) or TER (unpublished data).

Bottom Line: How the BBM dynamically responds to pathogenic and commensal bacterial signals can define intestinal homeostasis and immune function.We previously found that in model intestinal epithelium, the conversion of apical membrane sphingomyelin to ceramide by exogenous bacterial sphingomyelinase (SMase) protected against the endocytosis and toxicity of cholera toxin.Here we elucidate a mechanism of action by showing that SMase induces a dramatic, reversible, RhoA-dependent alteration of the apical cortical F-actin network.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology and Nutrition, Boston Children's Hospital, Boston, MA 02115 Harvard Digestive Diseases Center, Boston Children's Hospital, Boston, MA 02115 Harvard Medical School, Boston, MA 02115 david.saslowsky@nih.gov.

No MeSH data available.


Related in: MedlinePlus