David E. Saslowsky

Intoxication of zebrafish and mammalian cells by cholera toxin depends on the flotillin/reggie proteins but not Derlin-1 or -2

Cholera toxin (CT) causes the massive secretory diarrhea associated with epidemic cholera. To induce disease, CT enters the cytosol of host cells by co-opting a lipid-based sorting pathway from the plasma membrane, through the trans-Golgi network (TGN), and into the endoplasmic reticulum (ER). In the ER, a portion of the toxin is unfolded and retro- translocated to the cytosol. Here, we established zebrafish as a genetic model of intoxication and examined the Derlin and flotillin proteins, which are thought to be usurped by CT for retro-translocation and lipid sorting, respectively. Using antisense morpholino oligomers and siRNA, we found that depletion of Derlin-1, a component of the Hrd-1 retro-translocation complex, was dispensable for CT-induced toxicity. In contrast, the lipid raft-associated proteins flotillin-1 and -2 were required. We found that in mammalian cells, CT intoxication was dependent on the flotillins for trafficking between plasma membrane/endosomes and two pathways into the ER, only one of which appears to intersect the TGN. These results revise current models for CT intoxication and implicate protein scaffolding of lipid rafts in the endo-somal sorting of the toxin-GM1 complex.

Attenuated Endocytosis and Toxicity of a Mutant Cholera Toxin with Decreased Ability To Cluster Ganglioside GM1 Molecules

ABSTRACT Cholera toxin (CT) moves from the plasma membrane (PM) of host cells to the endoplasmic reticulum (ER) by binding to the lipid raft ganglioside GM1. The homopentomeric B-subunit of the toxin can bind up to five GM1 molecules at once. Here, we examined the role of polyvalent binding of GM1 in CT action by producing chimeric CTs that had B-subunits with only one or two normal binding pockets for GM1. The chimeric toxins had attenuated affinity for binding to host cell PM, as expected. Nevertheless, like wild-type (wt) CT, the CT chimeras induced toxicity, fractionated with detergent-resistant membranes extracted from toxin-treated cells, displayed restricted diffusion in the plane of the PM in intact cells, and remained bound to GM1 when they were immunoprecipitated. Thus, binding normally to two or perhaps only one GM1 molecule is sufficient for association with lipid rafts in the PM and toxin action. The chimeric toxins, however, were much less potent than wt toxin, and they entered the cell by endocytosis more slowly, suggesting that clustering of GM1 molecules by the B-subunit enhances the efficiency of toxin uptake and perhaps also trafficking to the ER.

Functional Analysis of VopF Activity Required for Colonization in Vibrio cholerae

ABSTRACT Vibrio cholerae, a Gram-negative facultative pathogen, is the etiologic agent for the diarrheal disease cholera. We previously characterized a clinical isolate, AM-19226, that translocates a type III secretion system (T3SS) effector protein with actin-nucleating activity, VopF, into the host cells. From comparative genomic studies, we identified a divergent T3SS island in additional isolates which possess a VopF homolog, VopN. Unlike the VopF-mediated protrusion formation, VopN localizes to stress fiber in host cells similarly to VopL, which is present in the pandemic strain of Vibrio parahaemolyticus. Chimera and yeast two-hybrid studies indicated that the amino-terminal regions of VopF and VopN proteins interact with distinct host cell factors. We determined that AM-19226-infected cells are arrested at S phase of the cell cycle and that VopF/VopN are antiapoptotic factors. To understand how VopF may contribute to the pathogenesis of AM-19226, we examined the effect of VopF in an in vitro polarized-epithelial model and an in vivo adult rabbit diarrheal model. Within the T3SS pathogenicity island is VopE, a homolog of YopE from Yersinia, which has been shown to loosen tight junctions. In polarized intestinal epithelia, VopF and VopE compromised the integrity of tight junctions by inducing cortical actin depolymerization and aberrant localization of the tight-junction protein ZO-1. An assay for pathogenicity in the adult rabbit diarrhea model suggested that these effectors are involved in eliciting the diarrheal response in infected rabbits. IMPORTANCE Vibrio cholerae is a bacterial pathogen that causes the diarrheal disease cholera, which remains a major public health problem in many developing countries. While the major virulence factors of the pandemic V. cholerae strains have been characterized, new clinical strains of V. cholerae have arisen, causing sporadic cholera-like diseases using unknown pathogenic mechanisms. Previously, we discovered the type III secretion system in a new clinical strain of V. cholerae and also identified an effector protein, VopF, which is injected into the host cells and induces changes in the actin cytoskeleton. In this work, we identified a homolog of VopF that causes a distinct cellular phenotype and interactions between the effectors and host proteins. We also discovered that both effectors prevent bacterium-induced cell death in infected cells. In our tissue culture and animal models, we showed that VopF contributes to the disruption of epithelial integrity and the diarrheal response. Vibrio cholerae is a bacterial pathogen that causes the diarrheal disease cholera, which remains a major public health problem in many developing countries. While the major virulence factors of the pandemic V. cholerae strains have been characterized, new clinical strains of V. cholerae have arisen, causing sporadic cholera-like diseases using unknown pathogenic mechanisms. Previously, we discovered the type III secretion system in a new clinical strain of V. cholerae and also identified an effector protein, VopF, which is injected into the host cells and induces changes in the actin cytoskeleton. In this work, we identified a homolog of VopF that causes a distinct cellular phenotype and interactions between the effectors and host proteins. We also discovered that both effectors prevent bacterium-induced cell death in infected cells. In our tissue culture and animal models, we showed that VopF contributes to the disruption of epithelial integrity and the diarrheal response.

Ganglioside GM1-mediated Transcytosis of Cholera Toxin Bypasses the Retrograde Pathway and Depends on the Structure of the Ceramide Domain

Background: Mechanisms for intracellular lipid sorting remain poorly understood. Results: Polarized epithelial cells sort ganglioside GM1, the receptor for cholera toxin, into distinct retrograde and transcytotic pathways, provided that GM1 contains ceramide domains with short or unsaturated fatty acid chains. Conclusion: Sphingolipid sorting depends on ceramide structure, implicating a mechanism for lipid sorting by lipid shape. Significance: The results identify a lipid-sorting pathway across epithelial barriers with clinical applications. Cholera toxin causes diarrheal disease by binding ganglioside GM1 on the apical membrane of polarized intestinal epithelial cells and trafficking retrograde through sorting endosomes, the trans-Golgi network (TGN), and into the endoplasmic reticulum. A fraction of toxin also moves from endosomes across the cell to the basolateral plasma membrane by transcytosis, thus breeching the intestinal barrier. Here we find that sorting of cholera toxin into this transcytotic pathway bypasses retrograde transport to the TGN. We also find that GM1 sphingolipids can traffic from apical to basolateral membranes by transcytosis in the absence of toxin binding but only if the GM1 species contain cis-unsaturated or short acyl chains in the ceramide domain. We found previously that the same GM1 species are needed to efficiently traffic retrograde into the TGN and endoplasmic reticulum and into the recycling endosome, implicating a shared mechanism of action for sorting by lipid shape among these pathways.

Conversion of apical plasma membrane sphingomyelin to ceramide attenuates the intoxication of host cells by cholera toxin

Cholera toxin (CT) enters host cells by binding to ganglioside GM1 in the apical plasma membrane (PM). GM1 carries CT retrograde from the PM to the endoplasmic reticulum (ER), where a portion of the toxin, the A1‐chain, retro‐translocates to the cytosol, causing disease. Trafficking in this pathway appears to depend on the association of CT–GM1 complexes with sphingomyelin (SM)‐ and cholesterol‐rich membrane microdomains termed lipid rafts. Here, we find that in polarized intestinal epithelia, the conversion of apical membrane SM to ceramide by bacterial sphingomyelinase attenuates CT toxicity, consistent with the lipid raft hypothesis. The effect is reversible, specific to toxin entry via the apical membrane, and recapitulated by the addition of exogenous long‐chain ceramides. Conversion of apical membrane SM to ceramide inhibits the efficiency of toxin endocytosis, but retrograde trafficking from the apical PM to the Golgi and ER is not affected. This result suggests that the cause for toxin resistance occurs at steps required for retro‐translocation of the CT A1‐chain to the cytosol.

Mechanism for Adhesion G Protein-Coupled Receptor GPR56-Mediated RhoA Activation Induced By Collagen III Stimulation

GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) family. Despite the importance of GPR56 in brain development, where mutations cause a devastating human brain malformation called bilateral frontoparietal polymicrogyria (BFPP), the signaling mechanism(s) remain largely unknown. Like many other adhesion GPCRs, GPR56 is cleaved via a GPCR autoproteolysis-inducing (GAIN) domain into N- and C-terminal fragments (GPR56N and GPR56C); however, the biological significance of this cleavage is elusive. Taking advantage of the recent identification of a GPR56 ligand and the presence of BFPP-associated mutations, we investigated the molecular mechanism of GPR56 signaling. We demonstrate that ligand binding releases GPR56N from the membrane-bound GPR56C and triggers the association of GPR56C with lipid rafts and RhoA activation. Furthermore, one of the BFPP-associated mutations, L640R, does not affect collagen III-induced lipid raft association of GPR56. Instead, it specifically abolishes collagen III-mediated RhoA activation. Together, these findings reveal a novel signaling mechanism that may apply to other members of the adhesion GPCR family.

Staphylococcus aureus Colonization of the Mouse Gastrointestinal Tract Is Modulated by Wall Teichoic Acid, Capsule, and Surface Proteins

Staphylococcus aureus colonizes the nose, throat, skin, and gastrointestinal (GI) tract of humans. GI carriage of S. aureus is difficult to eradicate and has been shown to facilitate the transmission of the bacterium among individuals. Although staphylococcal colonization of the GI tract is asymptomatic, it increases the likelihood of infection, particularly skin and soft tissue infections caused by USA300 isolates. We established a mouse model of persistent S. aureus GI colonization and characterized the impact of selected surface antigens on colonization. In competition experiments, an acapsular mutant colonized better than the parental strain Newman, whereas mutants defective in sortase A and clumping factor A showed impaired ability to colonize the GI tract. Mutants lacking protein A, clumping factor B, poly-N-acetyl glucosamine, or SdrCDE showed no defect in colonization. An S. aureus wall teichoic acid (WTA) mutant (ΔtagO) failed to colonize the mouse nose or GI tract, and the tagO and clfA mutants showed reduced adherence in vitro to intestinal epithelial cells. The tagO mutant was recovered in lower numbers than the wild type strain in the murine stomach and duodenum 1 h after inoculation. This reduced fitness correlated with the in vitro susceptibility of the tagO mutant to bile salts, proteases, and a gut-associated defensin. Newman ΔtagO showed enhanced susceptibility to autolysis, and an autolysin (atl) tagO double mutant abrogated this phenotype. However, the atl tagO mutant did not survive better in the mouse GI tract than the tagO mutant. Our results indicate that the failure of the tagO mutant to colonize the GI tract correlates with its poor adherence and susceptibility to bactericidal factors within the mouse gut, but not to enhanced activity of its major autolysin.

Ceramide activates JNK to inhibit a cAMP-gated K+ conductance and Cl– secretion in intestinal epithelia

Sphingomyelinases (SMases) hydrolyze membrane sphingomyelin to ceramide and are ex‐pressed by diverse host and microbial cell types popu‐lating mucosal surfaces. Exogenous bacterial SMase acts on the basolateral membrane of polarized human intestinal epithelial cells to repress the cAMP‐induced Cl‐ secretory response, but how this occurs is un‐known. We show here that SMase acts by down‐regulating a cAMP‐gated basolateral membrane K+ conduc‐tance. Neither phosphocholine, ceramide‐1‐phosphate, nor sphingosine‐1‐phosphate recapitulates this effect, indicating that ceramide production is the decisive factor. Basolaterally applied SMase induced the phos‐phorylation of c‐Jun NH2‐terminal kinase (JNK), and inhibition of JNK rescued the effect of SMase on cAMP‐dependant secretion. SMase secreted by normal human fibroblasts specifically recapitulated the effect on cAMP‐induced Cl‐ secretion, indicating that cell types inhabiting the subepithelial space can provide such an activity to the basolateral membrane of intesti‐nal enterocytes in trans. Thus, conversion of sphingo‐myelin to ceramide in basolateral membranes of intes‐tinal cells rapidly activates JNK to inhibit a cAMP‐gated K+ conductance and thereby attenuates Cl‐ secretion. These results define a novel lipid‐mediated pathway for regulation of salt and water homeostasis at mucosal surfaces.—Saslowsky, D. E., Tanaka, N., Reddy, K. P., Lencer, W. I. Ceramide activates JNK to inhibit a cAMP‐gated K+ conductance and Cl‐ secretion in intestinal epithelia. FASEBJ. 23, 259‐270 (2009)

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

Both commensal and pathogenic microbes that colonize the GI tract can synthesize and secrete spingomyelinase enzymes that cleave membrane sphingomyelin, leaving the ceramide component intact in the cell membrane. This study examines how this reaction affects the structure and function of host enterocytes and mucosal defense.

Congenital chloride-losing diarrhea in a Mexican child with the novel homozygous SLC26A3 mutation G393W

Congenital chloride diarrhea is an autosomal recessive disease caused by mutations in the intestinal lumenal membrane Cl−/HCO−3 exchanger, SLC26A3. We report here the novel SLC26A3 mutation G393W in a Mexican child, the first such report in a patient from Central America. SLC26A3 G393W expression in Xenopus oocytes exhibits a mild hypomorphic phenotype, with normal surface expression and moderately reduced anion transport function. However, expression of HA-SLC26A3 in HEK-293 cells reveals intracellular retention and greatly decreased steady-state levels of the mutant polypeptide, in contrast to peripheral membrane expression of the wildtype protein. Whereas wildtype HA-SLC26A3 is apically localized in polarized monolayers of filter-grown MDCK cells and Caco2 cells, mutant HA-SLC26A3 G393W exhibits decreased total polypeptide abundance, with reduced or absent surface expression and sparse punctate (or absent) intracellular distribution. The WT protein is similarly localized in LLC-PK1 cells, but the mutant fails to accumulate to detectable levels. We conclude that the chloride-losing diarrhea phenotype associated with homozygous expression of SLC26A3 G393W likely reflects lack of apical surface expression in enterocytes, secondary to combined abnormalities in polypeptide trafficking and stability. Future progress in development of general or target-specific folding chaperonins and correctors may hold promise for pharmacological rescue of this and similar genetic defects in membrane protein targeting.