ABSTRACT
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 ER-Tracker 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.
Subject(s)
Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , Salmonella typhi/physiology , Vacuoles/metabolism , Animals , Biological Transport , CHO Cells , Cell Line , Chlorocebus aethiops , Cricetinae , Cricetulus , Endoplasmic Reticulum/metabolism , Endothelial Cells/metabolism , Golgi Apparatus/metabolism , Humans , Macrolides/pharmacology , Neuraminidase/pharmacology , Protein Transport , Salmonella typhi/chemistry , Salmonella typhi/pathogenicity , U937 Cells , Vacuoles/drug effects , Vero CellsABSTRACT
Pathogenic strains of Escherichia coli produce a number of toxins that belong to the AB5 toxin family, which comprise a catalytic A-subunit that induces cellular dysfunction and a B-pentamer that recognizes host glycans. Although the molecular actions of many of the individual subunits of AB5 toxins are well understood, how they self-associate and the effect of this association on cytotoxicity are poorly understood. Here we have solved the structure of the holo-SubAB toxin that, in contrast to other AB5 toxins whose molecular targets are located in the cytosol, cleaves the endoplasmic reticulum chaperone BiP. SubA interacts with SubB in a similar manner to other AB5 toxins via the A2 helix and a conserved disulfide bond that joins the A1 domain with the A2 helix. The structure revealed that the active site of SubA is not occluded by the B-pentamer, and the B-pentamer does not enhance or inhibit the activity of SubA. Structure-based sequence comparisons with other AB5 toxin family members, combined with extensive mutagenesis studies on SubB, show how the hydrophobic patch on top of the B-pentamer plays a dominant role in binding the A-subunit. The structure of SubAB and the accompanying functional characterization of various mutants of SubAB provide a framework for understanding the important role of the B-pentamer in the assembly and the intracellular trafficking of this AB5 toxin.
Subject(s)
Bacterial Toxins/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Subtilisins/chemistry , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Disulfides , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli/pathogenicity , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Mutagenesis , Protein Structure, Quaternary , Protein Structure, Tertiary , Protein Transport , Structure-Activity Relationship , Subtilisins/genetics , Subtilisins/metabolismABSTRACT
The 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 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. We therefore examined the capacity of the purified B subunit (ArtB) from S. Typhi to elicit cytokine, chemokine, and adhesion molecule responses in human macrophage (U937), colonic epithelial (HCT-8) cell, and brain microvascular endothelial cell (HBMEC) lines. Secretion of the chemokines monocyte chemotactic protein 1 (MCP-1) and interleukin 8 (IL-8) was increased in all three tested cell lines, with macrophage inflammatory protein 1α (MIP-1α), MIP-1ß, and granulocyte colony-stimulating factor (G-CSF) also significantly increased in U937 cells. ArtB also upregulated the cytokines tumor necrosis factor alpha (TNF-α) and IL-6 in HBMECs and HCT-8 cells, but not in U937 cells, while intercellular adhesion molecule 1 (ICAM-1) was upregulated in HCT-8 and U937 cells and vascular cell adhesion molecule 1 (VCAM-1) was upregulated in HBMECs. Thus, ArtB may contribute to pathogenesis independently of the A subunit by promoting and maintaining a strong inflammatory response at the site of infection.
Subject(s)
Bacterial Toxins/toxicity , Cytokines/metabolism , Endothelial Cells/drug effects , Epithelial Cells/drug effects , Macrophages/drug effects , Salmonella typhi/metabolism , Bacterial Toxins/chemistry , Brain/blood supply , Cell Adhesion/physiology , Cell Line , Cytokines/genetics , Dose-Response Relationship, Drug , Endothelial Cells/metabolism , Epithelial Cells/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Humans , Intestinal Mucosa/cytology , Macrophages/metabolism , Protein Subunits/chemistry , Protein Subunits/metabolismABSTRACT
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 [Formula: see text] 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.