The B Subunit of an AB5 Toxin Produced by Salmonella enterica Serovar Typhi Up-Regulates Chemokines, Cytokines, and Adhesion Molecules in Human Macrophage, Colonic Epithelial, and Brain Microvascular Endothelial Cell Lines

Abstract

bThe principal function of bacterial AB5 toxin B subunits is to interact with glycan receptors on the surfaces of target cells and mediate the internalization of holotoxin. However, B subunit-receptor interactions also have the potential to impact cell signaling pathways and, in so doing, contribute to pathogenesis independently of the catalytic (toxic) A subunits. Various Salmonella enterica serovars, including Salmonella enterica serovar Typhi, encode an AB5 toxin (ArtAB), the A subunit of which is an ADPribosyltransferase 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. We therefore examined the capacity of the purified B subunit (ArtB) from S. Bacterial AB5 toxins are so termed because they comprise a catalytic A subunit noncovalently linked to a pentameric B subunit. They exert their effects in a two-step process: first, the B subunit pentamer binds to specific glycan receptors on the cell surface, triggering uptake of the holotoxin; this is followed by inhibition or corruption of essential host functions, mediated by the enzymatic activity of the A subunit. AB5 toxins are critical weapons in the armory of virulence factors deployed by major bacterial pathogens, which collectively kill over a million people each year (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) family (comprising Ctx and the enterotoxigenic Escherichia coli labile enterotoxin [LT]) and the pertussis toxin (Ptx) family catalyze the ADP-ribosylation of 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 have RNA N-glycosidase activity and inhibit eukaryotic protein synthesis by cleaving a specific adenine base from 28S rRNA. The fourth and most recently discovered AB5 toxin family is subtilase cytotoxin (SubAB), produced by a subset of Shiga-toxigenic E. coli (STEC) strains (2). Its A subunit is a highly specific subtilase-like serine protease that cleaves the essential endoplasmic reticulum (ER) chaperone BiP/GRP78, thereby inducing a massive ER stress response and triggering cellular apoptosis (3-6).The B subunits of the Ctx and Stx families (CtxB and StxB, respectively) recognize glycans displayed by host glycolipids (gangliosides, including GM1, and glycosphingolipids, such as Gb3 and Gb4) (1). The Ptx family is unique among AB5 toxins, as its B subunit is not a homopentamer, comprising 4 nonidentical subunits (S2, S3, S4, and S5) in a 1:1:2:1 stoichiometry. The S2 and S3 subunits both bind to sialylated glycoproteins rather than glycolipids (1). The B subunit of SubAB (SubB) is a homopentamer, like CtxB and StxB, but it binds to glycoproteins like Ptx. SubB and the C-terminal portion of Ptx S2 ex...

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

“…At present, the role of ArtAB in the pathogenesis of salmonellosis remains obscure, apart from its role as a delivery vehicle for CdtB in S. Typhi (10). However, in addition to the vacuolation phenotype reported in this study, we have shown that similar concentrations of ArtB trigger the secretion of chemokines, proinflammatory cytokines, and adhesion molecules in diverse cell types (12). Thus, ArtB may also contribute to pathogenesis independently of the A subunit by promoting and maintaining a strong inflammatory response at the site of infection as well as by inducing vacuolation.…”

“…using Ni-nitrilotriacetic acid (NTA) chromatography, as previously described (12). Where required, proteins were fluorescently labeled by using a DyLight 488 antibody labeling kit (Thermo Scientific) according to the manufacturer's instructions.…”

“…Thus, the B subunits may directly contribute to pathogenesis by triggering host responses independently of their respective catalytic ("toxic") A subunits. Indeed, we have shown that purified S. Typhi ArtB elicits cytokine, chemokine, and adhesion molecule responses in human monocyte, colonic epithelial, and brain microvascular endothelial cell lines (12). In the present study, we have investigated the uptake and intracellular trafficking of ArtB and demonstrate its capacity to induce a severe vacuolation phenotype.…”

Differential Effects of Escherichia coli Subtilase Cytotoxin and Shiga Toxin 2 on Chemokine and Proinflammatory Cytokine Expression in Human Macrophage, Colonic Epithelial, and Brain Microvascular Endothelial Cell Lines

“…These findings are most likely attributable to signaling events triggered by interactions of the mutant toxin's B-subunit pentamer with receptors on the target cell surface. Indeed, we recently showed that ArtB, a homologue with ϳ50% identity to SubB produced by Salmonella enterica serovar Typhi, upregulates a range of chemokines, cytokines, and adhesion molecules in the same cell lines used in the present study (23). Thus, the A and B subunits of SubAB may have distinct, perhaps even opposing effects on chemokine and cytokine responses during infection with SubAB-producing STEC.…”

“…The examined proinflammatory cytokines were TNF-␣ and IL-6. Secreted chemokines and cytokines in cell culture supernatants were assayed using cytometric bead array kits (Bender Medsystems) according to the manufacturer's instructions, as described elsewhere (23). During incubation of the beads with culture supernatant, different analytes were captured by their corresponding beads.…”