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Enterohemorrhagic Escherichia coli infection stimulates Shiga toxin 1 macropinocytosis and transcytosis across intestinal epithelial cells - PubMed

Enterohemorrhagic Escherichia coli infection stimulates Shiga toxin 1 macropinocytosis and transcytosis across intestinal epithelial cells

Valeriy Lukyanenko et al. Am J Physiol Cell Physiol. 2011 Nov.

Abstract

Gastrointestinal infection with Shiga toxins producing enterohemorrhagic Escherichia coli causes the spectrum of gastrointestinal and systemic complications, including hemorrhagic colitis and hemolytic uremic syndrome, which is fatal in ∼10% of patients. However, the molecular mechanisms of Stx endocytosis by enterocytes and the toxins cross the intestinal epithelium are largely uncharacterized. We have studied Shiga toxin 1 entry into enterohemorrhagic E. coli-infected intestinal epithelial cells and found that bacteria stimulate Shiga toxin 1 macropinocytosis through actin remodeling. This enterohemorrhagic E. coli-caused macropinocytosis occurs through a nonmuscle myosin II and cell division control 42 (Cdc42)-dependent mechanism. Macropinocytosis of Shiga toxin 1 is followed by its transcytosis to the basolateral environment, a step that is necessary for its systemic spread. Inhibition of Shiga toxin 1 macropinocytosis significantly decreases toxin uptake by intestinal epithelial cells and in this way provides an attractive, antibiotic-independent strategy for prevention of the harmful consequences of enterohemorrhagic E. coli infection.

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Figures

Fig. 1.
Fig. 1.

Infection of intestinal epithelial cells (IEC) with enterohemorrhagic Escherichia coli (EHEC) significantly increases Shiga toxin 1 (Stx1) uptake by stimulation of macropinocytosis (MPC). A: uptake of Stx1-Alexa680 by T84 cells was stimulated by increasing concentrations of EDL933. B: 104 EDL933/ml significantly increased Stx1-Alexa680 uptake over time. n ≥ 6. The amount of endocytosed toxin in A and B was measured in total cell lysates in triplicates by fluorescent plate reader and normalized to the protein concentrations (n ≥ 6). *Significant compared with uninfected cells; **significant compared with 105 EDL933/ml. C: scanning electron microscopy (SEM) of T84 cells infected with EDL933 shows apical bleb (arrow) in places without attached bacteria. D: SIM image of EDL933-infected T84 cells and immunostained with phalloidin-AlexaFluor 488 demonstrates the F-actin nature of the apical bleb (arrow). Image represents the superimposition of 6 optical sections with 1-μm step. E and F: transmission EM (TEM) images of cecal epithelium from rabbits infected for 3 days with RDEC-H19A bacteria show that the apical blebs (white arrows in E and F) appear in places without and with attached bacteria (black arrow in F).

Fig. 2.
Fig. 2.

Stx1 taken up by MPC is transcytosed across the T84 cells in holotoxin form. A: XZ projection through an EHEC-infected T84 cell shows apical F-actin bleb (red) that takes up Stx1 (green). B: XZ projection through EHEC-infected T84 cells with internalized Stx1 shows that Stx1 (green) is transported to the basolateral side of the cells inside the F-actin-coated macropinosomes (red). F-actin, red by phalloidin-AlexaFluor 568; Stx1, green by conjugation to AlexaFluor 488; nuclei, blue by Hoechst. White arrows in A and B indicate the green Stx1-containing vesicles coated with red F-actin. C: SDS-PAGE shows the relative distribution of Stx1A and cleaved Stx1A1 in total cell lysates (endocytosis) and in basal conditioned media (transcytosis) from T84 cells incubated with Stx1 over indicated time in the presence of 50 μM furin inhibitor decanoyl-RVKR-CMK or in EHEC-infected cells. M indicates the corresponding molecular mass marker on a gel. Stx1 taken up by MPC is not cleaved inside the T84 cells 4 h later. Transcytosed toxin collected from basal conditioned media 4 h after EHEC infection is also not cleaved.

Fig. 3.
Fig. 3.

Nonmuscle myosin II (NMII) plays a role in EHEC-stimulated Stx1 MPC. A: representative immunoblot of Stx1-Alexa 680 and GAPDH from infected and uninfected T84 cells that were treated or not with 50 μM blebbistatin or 5 nM calyculin A for 4 h. B: treatments of T84 cells with either 50 μM blebbistatin or 5 nM calyculin A for 4 h affect EHEC-induced Stx1 MPC; n ≥ 3 per each condition. C: immunoblot of T84 total cell lysates shows increase in RLC phosphorylation upon EHEC infection with corresponding quantification of RLC phosphorylation. *Significant compared with uninfected cells (P < 0.05; n = 5). D: representative immunoblot of MNIIA shows the increase in the NMIIA amount in cells infected for 4 h with EDL933 compared with control uninfected cells. E: representative immunoblot of MNIIB shows no changes in the NMIIB amount in cells infected for 4 h with EDL933 compared with control uninfected cells. F–G: NMIIA is present in F-actin apical blebs induced by EDL933. Representative XY optical section through the apical region with corresponding XZ projection of T84 cells uninfected (F) or infected (G) with EDL933 for 4 h and immunostained against NMIIA with F-actin; green by phalloidin-AlexaFluor488 and to visualize NMIIA. The pattern of NMIIA overlaps with the pattern of F-actin at the perimeter of the bleb.

Fig. 4.
Fig. 4.

Cdc42 is necessary for EHEC-induced MPC in vitro and in vivo. A: pharmacological inhibition of Cdc42 activity decreases EHEC-stimulated Stx1 uptake in T84 cells; n ≥ 3. NS, nonsignificant. B: Cdc42 KD virtually abolishes the EHEC stimulation of Stx1 uptake. Cdc42KD data represent the combined data from 3 different short hairpin RNA (shRNA) constructs. *Significant compared with control (uninfected T84 cells transduced with lentivirus containing scrambled shRNA; P < 0.05; n = 8). C: constitutively active Cdc42 enhances the EHEC effect on Stx1 endocytosis but is not sufficient to stimulate MPC itself. EV, empty vector; n ≥ 3 for each condition. D: Cdc42 was active (red by anti-Cdc42-GTP Abs, with secondary AlexaFluor 568) in subset of rabbit cecal epithelial cells that often did not have attached bacteria; attached RDEC-H19A bacteria and T84 cell nuclei, blue by Hoechst (33); apical F-actin, green by phalloidin-AlexaFluor 488.

Fig. 5.
Fig. 5.

Stx2 does not bind to normal human colonocytes and its uptake is stimulated by EHEC infection in T84 cells. A and B: representative projection through 5 confocal optical sections (step 0.5 μm) of normal human colonic tissue immunostained with Stx2-Alexa 568 (red) (A) and colabeled with CTB-Alexa 488 (green) (B); nuclei, blue by Hoechst. No Stx2 binding was detected in human colonocytes, but Stx2 (red) labeled some cells in lamina propria which its nature has to be identified. In contract, cholera toxin B (CTB) (green) binds to the human surface and crypt colonocytes. C: representative immunoblot of Stx2-Alexa 790 and GAPDH from infected (104 EDL933/ml) and uninfected T84 cells and exposed apically to different Stx2 concentrations for 4 h. D: corresponding quantification of Stx2 uptake by uninfected (control, open circles) T84 cells or infected for 4 h with 104 EDL933/ml (black squares); *Significant compared with corresponding control conditions, P < 0.01; n = 3 per each condition.

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