Receptor-Mediated Sorting of Typhoid Toxin during Its Export from Salmonella Typhi-Infected Cells - PubMed (original) (raw)
Receptor-Mediated Sorting of Typhoid Toxin during Its Export from Salmonella Typhi-Infected Cells
Shu-Jung Chang et al. Cell Host Microbe. 2016.
Abstract
Typhoid toxin is an essential virulence factor of Salmonella Typhi, the cause of typhoid fever. Typhoid toxin is secreted into the lumen of Salmonella-containing vacuole (SCV), after which it is packaged into vesicle carrier intermediates and released extracellularly through incompletely understood mechanisms. Following export, the toxin targets cells by interacting with human-specific Neu5Ac-terminated glycan receptors. We show that typhoid toxin is sorted from the SCV into vesicle carrier intermediates via interactions of its B subunit, PltB, with specific lumenal sialylated glycan packaging receptors. Cells deficient in N-glycosylation or the synthesis of specific gangliosides or displaying Neu5Gc-terminated, as opposed to Neu5Ac-terminated, glycans do not support typhoid toxin export. Additionally, typhoid toxin packaging requires the specific SCV environment, as toxin produced by an S. Typhi mutant with impaired trafficking is not properly sorted into vesicles. These results reveal how the exotoxin of an intracellular pathogen engages host pathways for packaging and release.
Keywords: Salmonella Typhi pathogenesis; bacterial pathogenesis; bacterial toxins; host-pathogen interactions; sialoglycans; vesicle traffic.
Copyright © 2016 Elsevier Inc. All rights reserved.
Figures
Figure 1. PltB is required for the packaging of typhoid toxin into vesicle-carrier intermediates
(A) Immunostaining of typhoid toxin in infected cells. Infected cells were stained with antibodies against the FLAG epitope (green) and S. Typhi LPS (red). Scale bar, 5 μm. (B) Quantification of the intensity of typhoid toxin-associated fluorescent puncta, a measure of typhoid toxin carrier intermediates, in infected cells. Values represent relative fluorescense intensity and are the mean ± SEM of three independent experiments in which at least 70 images were analyzed. ****: p < 0.0001. (C) Western immunoblot analysis of CdtB-3xFLAG expression in _Salmonella_-infected cells lysates. The levels of the S. Typhi protein RecA were used as loading control. See also Fig. S1.
Figure 2. Contribution of _N_-glycosylation and gangliosides to typhoid toxin sorting into export carriers
(A) Simplified schematic representation of the _N_-linked protein glycosylation pathway in mammalian cells. Asn: Asparagine; ManI: Mannosidase I; GnT1: N- acetylglucosaminyl transferase I; MsnII: Mannosidase II; GalT: Galactosyltransferase ; ST: Siayltransferase; Neu5Ac: N-Acetylneuraminic acid; GlcNac: _N_-acetylglucosamine. (B) Analysis of protein glycosylation. Lysates from HEK293T and MGAT1-deficient cells were examined by Western immunoblot for the mobility of the glycoprotein LAMP-1 in SDS-PAGE. (C) S. Typhi invasion of HEK293T and MGAT1-deficient cells determined by the gentamicin protection assay. Results represent the % of the inoculum that survived the gentamicin treatment and are the mean ± SD of three independent determinations. (D) Western immunoblot analysis of CdtB-3xFLAG expression in the indicated uninfected (mock) or _Salmonella_-infected cells lysates. (E) Quantification of the intensity of typhoid toxin-associated fluorescent puncta, a measure of typhoid toxin carrier intermediates, in infected cells. Values represent relative fluorescense intensity and are the mean ± SEM of three independent experiments in which at least 70 images were analyzed. ****: p < 0.0001. (F and G) Quantification of typhoid toxin export into the infection medium. Infection media obtained from the indicated S. Typhi-infected cells were diluted as indicated and applied to HEK293T cells. Forty-eight hours after treatment the cell cycle profile of the different cells was analyzed by flow cytometry and the percentage of cells in G2M (a measure of typhoid toxin toxicity) was determined. Values are mean ± SD of three independent determinations. (F) A measure of the relative toxicity (G) was obtained by comparing the dilutions of the infection media preparations (marked with dashed rectangles) that resulted in a ratio between the number of cells in G0G1 and G2M that when compared across samples showed statistically insignificant differences ****: p < 0.0001. (H) Simplified schematic representation of the major ganglioside synthesis pathway (Svennerholm, 1964). Cer: ceramide; Gal: galactose; Glu: glucose. (I) Binding of fluorescently-labeled cholera toxin B subunit to the indicated cells was measured by flow cytometry. Values represent relative fluorescence and are the mean ± SEM of three independente determinations. **: p < 0.01 (J) S. Typhi invasion of HEK 293T and SLC35A2-deficient cells determined by the gentamicin protection assay. Results represent the % of the inoculum that survived the gentamicin treatment and are the mean ± SD of three independent determinations. (K) Western immunoblot analysis of CdtB- 3xFLAG expression in the indicated uninfected (mock) or _Salmonella_-infected cells lysates. (L) Quantification of the intensity of typhoid toxin-associated fluorescent puncta in the indicated infected cells. Values represent relative fluorescense intensity and are the mean ± SEM of three independent experiments in which at least 90 images were analyzed. ****: p < 0.0001. (M and N) Quantification of typhoid toxin export into the infection medium (see above). Values are mean ± SD of three independent determinations. (N) A measure of the relative toxicity determined as indicated in (G). **: p < 0.01; ***: p < 0.001. See also Fig. S1, S2, S3, and S4.
Figure 3. Metabolic incorporation of Neu5Gc into human cells prevents the formation of typhoid toxin export carrier intermediates
(A) Immunostaining of typhoid toxin in Neu5Ac or Neu5Gc treated cells. Cells were infected with S. Typhi strains expressing 3xFLAG epitope-tagged CdtB and stained with antibodies against the FLAG epitope (green) and S. Typhi LPS (red) and visualized by fluorescence microscopy. Scale bar, 5 μm. (B) Quantification of the intensity of typhoid toxin-associated fluorescent puncta, a measure of typhoid toxin carrier intermediates, in infected cells treated as indicated. Values represent relative fluorescense intensity and are the mean ± SEM of three independent experiments in which at least 90 images were analyzed. **: p < 0.01 vs cells that received Neu5Ac (C) Western immunoblot analysis of CdtB-3xFLAG expression in the indicated uninfected (mock) or _Salmonella_-infected cell lysates. The levels of the S. Typhi protein RecA were used as loading control. See also Fig. S1.
Figure 4. Typhoid toxin packaging into vesicle transport intermediates requires a specific vacuolar environment
(A) Intracellular survival of S. Typhi wild type (WT) and the SPI-2 TTSS-defective spiA mutant in HEK293T cells. Values represent % of CFU 24 hours post infection relative to the CFU 2 hs after infection and have been standardized to the CFU of wild type, which was considered to be 100%. Data in (A) are mean ± SEM from three independent experiments. (B) Western immunoblot analysis of CdtB-3xFLAG expression in HEK293T cells infected with the indicated S. Typhi strains. The levels of the S. Typhi protein RecA were used as loading control. (C) Quantification of the intensity of typhoid toxin-associated fluorescent puncta, a measure of typhoid toxin carrier intermediates, in HEK293T cells infected with the indicated S. Typhi strains. Values represent relative fluorescense intensity and are the mean ± SEM of three independent experiments in which at least 60 images were analyzed. **: p < 0.01 (D and E) Quantification of typhoid toxin export into the infection medium. Infection media obtained from HEK293T cells infected with the indicated S. Typhi strains were diluted as indicated and applied to uninfected HEK293T cells. Forty-eight hours after treatment the cell cycle profile of the different cells was analyzed by flow cytometry and the percentage of cells in G2M (a measure of typhoid toxin toxicity) was determined. Values are mean ± SD of three independent determinations (D). A measure of the relative toxicity (E) was obtained by comparing the dilutions of the infection media preparations (marked with dashed rectangles) that resulted in a ratio between the number of cells in G0G1 and G2M that when compared across samples showed statistically insignificant differences. ****: p < 0.0001. See also Fig. S1 and S4.
References
- Baker S, Karkey A, Parry C. Are we adequately prepared for the emergence of Salmonella enterica serovar Paratyphi A? Lancet Glob Health. 2014;2:e195– 196. - PubMed
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