Background: AB 5 toxins consist of a pentameric B-subunit and a catalytic A-subunit. Results: Crystallographic data, dissociation, and intracellular trafficking of SubAB toxin are reported. Conclusion: SubAB architecture is similar to other AB 5 toxins, whereas the B-pentamer plays an important role in assembly and intracellular trafficking. Significance: The conserved hydrophobic ring in the B-pentamer supports the view that A-and B-subunits have evolved independently.

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...

Subtilase cytotoxin (SubAB) of Escherichia coli is an AB5 class bacterial toxin. The pentameric B subunit (SubB) binds the cellular carbohydrate receptor, α2–3-linked N-glycolylneuraminic acid (Neu5Gc). Neu5Gc is not expressed on normal human cells, but is expressed by cancer cells. Elevated Neu5Gc has been observed in breast, ovarian, prostate, colon and lung cancer. The presence of Neu5Gc is prognostically important, and correlates with invasiveness, metastasis and tumour grade. Neu5Gc binding by SubB suggests that it may have utility as a diagnostic tool for the detection Neu5Gc tumor antigens. Native SubB has 20-fold less binding to N-acetlylneuraminic acid (Neu5Ac); over 30-fold less if the Neu5Gc linkage was changed from α2–3 to α2–6. Using molecular modeling approaches, site directed mutations were made to reduce the α2–3  α2–6-linkage preference, while maintaining or enhancing the selectivity of SubB for Neu5Gc over Neu5Ac. Surface plasmon resonance and glycan array analysis showed that the SubBΔS106/ΔT107 mutant displayed improved specificity towards Neu5Gc and bound to α2–6-linked Neu5Gc. SubBΔS106/ΔT107 could discriminate NeuGc- over Neu5Ac-glycoconjugates in ELISA. These data suggest that improved SubB mutants offer a new tool for the testing of biological samples, particularly serum and other fluids from individuals with cancer or suspected of having cancer.

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