Clathrin-dependent trafficking of subtilase cytotoxin, a novel AB<sub>5</sub>toxin that targets the endoplasmic reticulum chaperone BiP

Chong, D.C.; Paton, James C.; Thorpe, Cheleste M.; Paton, Adrienne W.

doi:10.1111/j.1462-5822.2007.01085.x

Cited by 60 publications

(63 citation statements)

References 39 publications

(65 reference statements)

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“…At high doses, only a brief exposure to ArtB was required to elicit near-maximal vacuolation, as washing of the treated cells after as little as 5 min of incubation did not prevent subsequent vacuole development. This is consistent with the rapid binding and internalization of ArtB, as is known to occur for other AB5 holotoxins (2,24). Treatment with neuraminidase significantly inhibited vacuolation, indicating that sialylated glycans on the Vero cell surface are functional receptors for ArtB.…”

Section: Discussionsupporting

confidence: 85%

Vacuolation Activity and Intracellular Trafficking of ArtB, the Binding Subunit of an AB5 Toxin Produced by Salmonella enterica Serovar Typhi

et al. 2017

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Various Salmonella enterica serovars, including S. enterica serovar Typhi, encode an AB5 toxin (ArtAB), the A subunit of which is an ADP-ribosyltransferase related to the S1 subunit of pertussis toxin. However, although the A subunit is able to catalyze ADP-ribosylation of host G proteins, a cytotoxic phenotype has yet to be identified for the holotoxin. Here we show that its B subunit pentamer (ArtB) binds to receptors on the surface of Vero (African green monkey kidney) cell, CHO (Chinese hamster ovary) cell, U937 (human monocyte) cell, and HBMEC (human brain microvascular endothelial cell) lines. Moreover, ArtB induced marked vacuolation in all cell lines after 4 h of incubation. Further studies in Vero cells showed that vacuolation was inhibited by bafilomycin A1 and was dependent on the clathrin-mediated uptake of ArtB. Vacuolation was also inhibited by treatment of cells with neuraminidase, indicating that sialylated glycans are functional receptors for ArtB. Confocal colocalization studies indicated that after cell binding and internalization, ArtB undergoes retrograde transport via early endosomes, the trans-Golgi network, and the Golgi apparatus, reaching the endoplasmic reticulum (ER) after approximately 2 h. The onset of vacuolation also coincided with gross cytoskeletal reorganization. At later time points, ArtB colocalized with ERTracker Red in the vacuolar membrane, implying that vacuolation is a consequence of ER disorganization. Thus, the isolated B subunit of this cryptic AB5 toxin has significant effects on target cells with the potential to contribute directly to pathogenesis independently of the catalytic A subunit. KEYWORDS AB5 toxins, B subunit, Salmonella, vacuolation A B5 toxins are critical weapons in the armory of virulence factors deployed by several major bacterial pathogens. They comprise a catalytic A subunit noncovalently linked to a pentameric B subunit. Their mechanism of action commences with the binding of the B subunit pentamer to specific glycan receptors on the cell surface, triggering the uptake of the holotoxin. The A subunit is then able to covalently modify intracellular substrates, inhibiting or corrupting essential host functions (1). The AB5 toxins characterized to date are classified into four families according to A subunit sequence homology and catalytic activity as well as the structural organization of the holotoxin (1). The A subunits of both the cholera toxin (Ctx) and pertussis toxin (Ptx) families are ADP-ribosyltransferases that respectively modify G s ␣ and G i ␣ proteins in the host cell cytosol, disrupting their signal transduction pathways. The A subunits of the Shiga toxin (Stx) family are RNA N-glycosidases that cleave a specific adenine base from 28S rRNA, inhibiting eukaryotic protein synthesis. The fourth and most recently discovered AB5 toxin family is Escherichia coli subtilase cytotoxin (SubAB), produced by a subset of strains that also produce Stx (2). Its A subunit

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Section: Discussionsupporting

confidence: 85%

Vacuolation Activity and Intracellular Trafficking of ArtB, the Binding Subunit of an AB5 Toxin Produced by Salmonella enterica Serovar Typhi

et al. 2017

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“…Cells were fixed with formalin (10% in PBS) for 10 min and washed with PBS and water. Samples were mounted onto glass slides using Prolong Gold antifade and cured for 2 h. All samples were viewed using a 60ϫ 1.4-numerical-aperture oil objective in an Olympus IMT-2 inverted microscope coupled to a Bio-Rad MRC-600 dual-laser confocal microscope, as described previously (6).…”

Section: Methodsmentioning

confidence: 99%

Sab, a Novel Autotransporter of Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli O113:H21, Contributes to Adherence and Biofilm Formation

Herold

Paton

2009

Infect Immun

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Shiga-toxigenic Escherichia coli (STEC) strains cause serious gastrointestinal disease, which can lead to potentially life-threatening systemic complications such as hemolytic-uremic syndrome. Although the production of Shiga toxin has been considered to be the main virulence trait of STEC for many years, the capacity to colonize the host intestinal epithelium is a crucial step in pathogenesis. In this study, we have characterized a novel megaplasmid-encoded outer membrane protein in locus of enterocyte effacement (LEE)-negative O113:H21 STEC strain 98NK2, termed Sab (for STEC autotransporter [AT] contributing to biofilm formation). The 4,296-bp sab gene encodes a 1,431-amino-acid protein with the features of members of the AT protein family. When expressed in E. coli JM109, Sab contributed to the diffuse adherence to human epithelial (HEp-2) cells and promoted biofilm formation on polystyrene surfaces. A 98NK2 sab deletion mutant was also defective in biofilm formation relative to its otherwise isogenic wild-type parent, and this was complemented by transformation with a sab-carrying plasmid. Interestingly, an unrelated O113:H21 STEC isolate that had a naturally occurring deletion in sab was similarly defective in biofilm formation. PCR analysis indicated that sab is present in LEE-negative STEC strains belonging to serotypes/groups O113:H21, O23, and O82:H8. These findings raise the possibility that Sab may contribute to colonization in a subset of LEE-negative STEC strains.Shiga-toxigenic Escherichia coli (STEC) strains are prominent food-borne pathogens that cause watery or bloody diarrhea and hemorrhagic colitis, which can progress to the lifethreatening hemolytic-uremic syndrome (HUS) (15,21,29). In order to establish and maintain an infection, STEC strains are equipped with a diverse array of virulence factors. Among these factors, Shiga toxin has been considered to be a sine qua non of virulence, as reviewed previously (21, 29). However, attachment of STEC to the human intestinal mucosa is a critical first step in pathogenesis. Many STEC strains, including those of the highly prevalent O157:H7 serotype, carry the locus of enterocyte effacement (LEE) pathogenicity island, which encodes the capacity to produce attaching and effacing (A/E) lesions on the intestinal epithelium, similarly to those produced by enteropathogenic E. coli strains (11, 35). These STEC strains are often referred to as enterohemorrhagic E. coli (EHEC), although this classification is ill defined. A/E lesions are characterized by ultrastructural changes including the remodeling of the host cell cytoskeleton and intimate attachment of the bacteria to the cell surface (11,35). The process of the generation of A/E lesions involves the expression of the eae gene, which encodes intimin, an outer membrane surface adhesin, and the delivery of the intimin receptor Tir and several other effector proteins into host cells via the LEE-encoded type III secretion apparatus (reviewed in references 5 and 11).However, many STEC isolates from cases of ...

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“…The name subtilase cytotoxin emanates from the similarity of the 35 kDa subunit A to members of the Subtilase S8 family of serine proteases, and is most similar to a protease produced by Bacillus anthracis; the B subunit of SubAB mediates binding of toxin to the surface of eukaryotic cells and bears similarity with putative exported proteins from Salmonella typhi and Yersinia pestis (Paton et al, 2004). SubB also directs internalization of the toxin and retrograde trafficking to the ER in a clathrin-dependent fashion (Chong et al, 2007). HUS is a life-threatening complication of STEC infection characterized by microangipathic hemolytic anemia, thrombocytopenia and renal failure, which can be reproduced in experimental mice by injection of purified SubAB (Wang et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Decreased ER-associated degradation of α-TCR induced by Grp78 depletion with the SubAB cytotoxin

Lass

Kujawa

McConnell

et al. 2008

The International Journal of Biochemistry & Cell Biology

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HeLa cells stably expressing the α chain of T-cell receptor (αTCR), a model substrate of ERAD (ERassociated degradation), were used to analyze the effects of BiP/Grp78 depletion by the SubAB cytotoxin. SubAB induced XBP1 splicing, followed by JNK phosphorylation, eIF2α phosphorylation, upregulation of ATF3/4 and partial ATF6 cleavage. Other markers of ER stress, including elements of ER-associated degradation (ERAD) pathway, as well as markers of cytoplasmic stress, were not induced. SubAB treatment decreased absolute levels of αTCR, which was caused by inhibition of protein synthesis. At the same time, the half-life of αTCR was extended almost fourfold from 70 min to 210 min, suggesting that BiP normally facilitates ERAD. Depletion ofp97/VCP partially rescued SubAB-induced depletion of αTCR, confirming the role of VCP in ERAD of αTCR. It therefore appears that ERAD of αTCR is driven by at least two different ATPase systems located at two sides of the ER membrane, BiP located on the lumenal side, while p97/ VCP on the cytoplasmic side. While SubAB altered cell morphology by inducing cytoplasm vacuolization and accumulation of lipid droplets, caspase activation was partial and subsided after prolonged incubation. Expression of CHOP/GADD153 occurred only after prolonged incubation and was not associated with apoptosis.

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Clathrin-dependent trafficking of subtilase cytotoxin, a novel AB₅toxin that targets the endoplasmic reticulum chaperone BiP

Cited by 60 publications

References 39 publications

Vacuolation Activity and Intracellular Trafficking of ArtB, the Binding Subunit of an AB5 Toxin Produced by Salmonella enterica Serovar Typhi

Vacuolation Activity and Intracellular Trafficking of ArtB, the Binding Subunit of an AB5 Toxin Produced by Salmonella enterica Serovar Typhi

Sab, a Novel Autotransporter of Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli O113:H21, Contributes to Adherence and Biofilm Formation

Decreased ER-associated degradation of α-TCR induced by Grp78 depletion with the SubAB cytotoxin

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Clathrin-dependent trafficking of subtilase cytotoxin, a novel AB5toxin that targets the endoplasmic reticulum chaperone BiP

Cited by 60 publications

References 39 publications

Vacuolation Activity and Intracellular Trafficking of ArtB, the Binding Subunit of an AB5 Toxin Produced by Salmonella enterica Serovar Typhi

Vacuolation Activity and Intracellular Trafficking of ArtB, the Binding Subunit of an AB5 Toxin Produced by Salmonella enterica Serovar Typhi

Sab, a Novel Autotransporter of Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli O113:H21, Contributes to Adherence and Biofilm Formation

Decreased ER-associated degradation of α-TCR induced by Grp78 depletion with the SubAB cytotoxin

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Clathrin-dependent trafficking of subtilase cytotoxin, a novel AB₅toxin that targets the endoplasmic reticulum chaperone BiP