Cytolethal distending toxin family members are differentially affected by alterations in host glycans and membrane cholesterol

Abstract

Cytolethal distending toxins (CDTs) are tripartite protein exotoxins produced by a diverse group of pathogenic Gram-negative bacteria. Based on their ability to induce DNA damage, cell cycle arrest, and apoptosis of cultured cells, CDTs are proposed to enhance virulence by blocking cellular division and/or directly killing epithelial and immune cells. Despite the widespread distribution of CDTs among several important human pathogens, our understanding of how these toxins interact with host cells is limited. Here we demonstrate that CDTs from Haemophilus ducreyi, Aggregatibacter actinomycetemcomitans, Escherichia coli, and Campylobacter jejuni differ in their abilities to intoxicate host cells with defined defects in host factors previously implicated in CDT binding, including glycoproteins, and glycosphingolipids. The absence of cell surface sialic acid sensitized cells to intoxication by three of the four CDTs tested. Surprisingly, fucosylated N-linked glycans and glycolipids, previously implicated in CDT-host interactions, were not required for intoxication by any of the CDTs tested. Finally, altering host-cellular cholesterol, also previously implicated in CDT binding, affected intoxication by only a subset of CDTs tested. The findings presented here provide insight into the molecular and cellular basis of CDT-host interactions.

Figures

FIGURE 1.

Dendogram of CdtA and CdtC proteins. CdtA and -C protein sequences were aligned with ClustalW using BLOSUM 30 similarity matrix, and a midpoint rooted dendogram was constructed using MacVector version 7.2. Numbers indicate relative evolutionary distance.

FIGURE 2.

Differential sensitivity of cell lines to divergent CDTs. Cells from indicated lines were seeded on 384-well plates, intoxicated for 24 h, fixed, permeabilized, and probed with anti-phospho H2AX antibodies followed by Alexa Fluor 488-labeled goat anti-rabbit antibodies. Nuclei were identified by staining with Hoechst. The relative level of activated H2AX per cell nucleus was determined by measuring Alexa Fluor 488 and Hoechst fluorescence intensity by laser scanning cytometry. Results are plotted as mean Alexa Fluor 488 fluorescence intensity per nucleus from triplicate samples ± S.E. Results are representative of three independent experiments.

FIGURE 3.

CDT intoxication of tunicamycin-treated and cholesterol-loaded CHO-K1 cells. A, CHO-K1 cells were treated with 0.5 μg/ml tunicamycin for 2 days and/or incubated with 2 mg/ml methyl-β-cyclodextrin conjugated cholesterol for 30 min prior to intoxication. Cells were incubated with toxin for 10 min, then washed and incubated for a further 24 h. DNA content was determined by PI staining and flow cytometry. Histograms indicate the number of cells (y axis) at a given PI fluorescence intensity (x axis), with the left hand peak representing cells in G0/G1 phase of cell cycle, the peak on the right representing cells in G2/M, and the area between peaks representing cells in S phase as indicated in the top left histogram. B, total cellular cholesterol was quantified and normalized to total protein in CHO-K1 cells treated with tunicamycin. Average values calculated from triplicate samples ± S.E. are shown. A and B, results are representative of at least three independent experiments.

FIGURE 4.

CDT intoxication of CHO glycosylation mutants. A, predicted N- and O-linked glycan and glycolipid structures (adapted from Ref. 45). GM3 is the predominant glycolipid in CHO cells; therefore, the mutant structures are based on GM3. B, cells were intoxicated for 24 h, stained with PI, and subjected to flow cytometry. Unshaded histograms represent intoxicated loss of function mutants, and gray shaded histograms represent the intoxicated parental ProCHO controls. Results are representative of at least three independent experiments.

FIGURE 5.

Role of fucose in CDT intoxication. A, CDT intoxication of the fucose biosynthetic mutant (Lec13) cells. Cells were grown in media containing dialyzed serum for 2 days, intoxicated with CDT for 24 h, stained with PI, and subjected to flow cytometry. Unshaded histograms represent intoxicated Lec13 cells, and gray shaded histograms are intoxicated parental ProCHO cells. B, UEA binding of Fut1-expressing CHO-K1 cells. Cells transduced with Fut1-encoding or empty vectors were detached with EDTA, washed, and bound with FITC-conjugated UEA lectin for 15 min at room temperature. After three successive washes, the cells were resuspended in PBS plus 1% formaldehyde and subjected to flow cytometry. Data represent geometric mean fluorescence of 104 cells. C, CHO-K1 cells were transduced with a retroviral vector encoding Fut1 (unshaded histograms) or empty vector (gray histrograms), intoxicated for 24 h, stained with PI, and subjected to flow cytometry. D and E, ProCHO and Lec2 cells transduced with Fut1-encoding or empty vectors were bound with FITC-conjugated wheat germ agglutinin (D) or FITC-conjugated UEA (E), washed, and subjected to flow cytometry, as in B. F, CDT intoxication of parental (ProCHO) or sialic acid transporter mutant (Lec2) cells transduced with Fut1 encoding (unshaded histograms) or empty (gray histograms) retroviral vectors. Transduced cells were intoxicated for 24 h, stained with PI, and subjected to flow cytometry. Results are representative of at least three independent experiments.

FIGURE 6.

Role of glycolipids in CDT intoxication. A, cells were treated with 5 μm PPMP for 10 days, intoxicated with CDT for 24 h, stained with PI, and subjected to flow cytometry. Unshaded histograms are intoxicated PPMP-treated cells, and gray shaded histograms are untreated controls. B, lipid glycosylation mutant cells (LY-B, gray shaded histograms) or the cDNA-complemented counterpart (Ly-BcLCB1, unshaded histograms) were intoxicated with CDT for 24 h, stained with PI, and subjected to flow cytometry. Results are representative of at least three independent experiments.