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rho, a small GTP-binding protein, is essential for Shigella invasion of epithelial cells.

Watarai M, Kamata Y, Kozaki S, Sasakawa C - J. Exp. Med. (1997)

Bottom Line: Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with alpha5beta1 integrin.Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA.Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology, Institute of Medical Science, University of Tokyo, Minato-ku, Japan.

ABSTRACT
Shigella, the causative agents of bacillary dysentery, are capable of invading mammalian cells that are not normally phagocytic. Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with alpha5beta1 integrin. We report here that Shigella invasion of epithelial cells requires rho activity, a ras-related GTP-binding protein. The invasive capacity of Shigella flexneri for Chinese hamister ovary (CHO) cells and other epithelial cells were greatly reduced when treated with Clostridium botulinum exoenzyme C3 transferase. Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA. Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase. Our results indicate that cellular signal transduction regulated by rho is essential for Shigella invasion of epithelial cells.

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Inhibitory effect of C3 on the rearrangement of F-actin and vinculin at the site of S. flexneri attachment to CHO cells. The CHO cells were  treated with C3 (1.25 μg/ml) (C, F, and I) or left untreated (A, D, and G) for 24 h and then infected by YSH6000T for 20 min. B, E, and H are the enlarged inset of A, D, and G, respectively. A–C represent CHO cells infected with YSH6000T and stained by rhodamine-labeled phalloidin. D–F represent the same CHO cells but stained by mouse FITC-labeled anti-vinculin. G–I represent the same CHO cells but stained by Cy5-labeled anti–S. flexneri  2a LPS. Arrowheads indicate F-actin and vinculin accumulation induced upon attachment of YSH6000T to CHO cells (A, D, and G). Bars: (H) 5 μm;  and (I) 10 μm.
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Figure 5: Inhibitory effect of C3 on the rearrangement of F-actin and vinculin at the site of S. flexneri attachment to CHO cells. The CHO cells were treated with C3 (1.25 μg/ml) (C, F, and I) or left untreated (A, D, and G) for 24 h and then infected by YSH6000T for 20 min. B, E, and H are the enlarged inset of A, D, and G, respectively. A–C represent CHO cells infected with YSH6000T and stained by rhodamine-labeled phalloidin. D–F represent the same CHO cells but stained by mouse FITC-labeled anti-vinculin. G–I represent the same CHO cells but stained by Cy5-labeled anti–S. flexneri 2a LPS. Arrowheads indicate F-actin and vinculin accumulation induced upon attachment of YSH6000T to CHO cells (A, D, and G). Bars: (H) 5 μm; and (I) 10 μm.

Mentions: To visualize the protein tyrosine phosphorylation elicited by Shigella invasion of CHO cells and the effect of C3 treatment, the semiconfluent CHO cell monolayers pretreated with or without C3 were infected with YSH6000T for 20 min and examined by triple fluorescence staining with rhodamine-labeled phalloidin, FITC-labeled anti-phosphotyrosine antibody and Cy5-labeled anti–S. flexneri LPS. As shown in Fig. 4, actin polymerization and foci of protein tyrosine phosphorylation was concentrated around the bacteria accumulated at the periphery of the untreated CHO cells (Fig. 4, A, B, D, E, G, and H). On the other hand, the CHO cells treated with C3 (at 1.25 μl/ml) for 1 d had an altered cell shape and became surrounded with an F-actin sash (Fig. 4 C). As has been indicated above, only a few bacteria were associated with the CHO cells in which accumulation of foci of protein tyrosine phosphorylation had disappeared (Fig. 4, F and I). Indeed, the bacteria associated with the C3-treated CHO cells (as counted in 20 different fields of the immunofluorescence micrograpy) were less than one tenth of those associated with the nontreated CHO cells. Furthermore, examination of the infected CHO cells using triple fluorescence staining with rhodamine-labeled phalloidin, FITC-labeled anti-vinculin, and Cy5-labeled anti–S. flexneri LPS revealed that localized actin polymerization (Fig. 5, A and B) and accumulation of vinculin (Fig. 5, D and E) were also associated with the infecting bacteria (Fig. 5, G and H). None of those events were observed in C3-treated CHO cells (Fig. 5, C, F and I), suggesting that localized rearrangement of the host cytoskeleton elements such as actin, vinculin, and protein tyrosine phosphorylation elicited around invading Shigella is regulated by rho.


rho, a small GTP-binding protein, is essential for Shigella invasion of epithelial cells.

Watarai M, Kamata Y, Kozaki S, Sasakawa C - J. Exp. Med. (1997)

Inhibitory effect of C3 on the rearrangement of F-actin and vinculin at the site of S. flexneri attachment to CHO cells. The CHO cells were  treated with C3 (1.25 μg/ml) (C, F, and I) or left untreated (A, D, and G) for 24 h and then infected by YSH6000T for 20 min. B, E, and H are the enlarged inset of A, D, and G, respectively. A–C represent CHO cells infected with YSH6000T and stained by rhodamine-labeled phalloidin. D–F represent the same CHO cells but stained by mouse FITC-labeled anti-vinculin. G–I represent the same CHO cells but stained by Cy5-labeled anti–S. flexneri  2a LPS. Arrowheads indicate F-actin and vinculin accumulation induced upon attachment of YSH6000T to CHO cells (A, D, and G). Bars: (H) 5 μm;  and (I) 10 μm.
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Figure 5: Inhibitory effect of C3 on the rearrangement of F-actin and vinculin at the site of S. flexneri attachment to CHO cells. The CHO cells were treated with C3 (1.25 μg/ml) (C, F, and I) or left untreated (A, D, and G) for 24 h and then infected by YSH6000T for 20 min. B, E, and H are the enlarged inset of A, D, and G, respectively. A–C represent CHO cells infected with YSH6000T and stained by rhodamine-labeled phalloidin. D–F represent the same CHO cells but stained by mouse FITC-labeled anti-vinculin. G–I represent the same CHO cells but stained by Cy5-labeled anti–S. flexneri 2a LPS. Arrowheads indicate F-actin and vinculin accumulation induced upon attachment of YSH6000T to CHO cells (A, D, and G). Bars: (H) 5 μm; and (I) 10 μm.
Mentions: To visualize the protein tyrosine phosphorylation elicited by Shigella invasion of CHO cells and the effect of C3 treatment, the semiconfluent CHO cell monolayers pretreated with or without C3 were infected with YSH6000T for 20 min and examined by triple fluorescence staining with rhodamine-labeled phalloidin, FITC-labeled anti-phosphotyrosine antibody and Cy5-labeled anti–S. flexneri LPS. As shown in Fig. 4, actin polymerization and foci of protein tyrosine phosphorylation was concentrated around the bacteria accumulated at the periphery of the untreated CHO cells (Fig. 4, A, B, D, E, G, and H). On the other hand, the CHO cells treated with C3 (at 1.25 μl/ml) for 1 d had an altered cell shape and became surrounded with an F-actin sash (Fig. 4 C). As has been indicated above, only a few bacteria were associated with the CHO cells in which accumulation of foci of protein tyrosine phosphorylation had disappeared (Fig. 4, F and I). Indeed, the bacteria associated with the C3-treated CHO cells (as counted in 20 different fields of the immunofluorescence micrograpy) were less than one tenth of those associated with the nontreated CHO cells. Furthermore, examination of the infected CHO cells using triple fluorescence staining with rhodamine-labeled phalloidin, FITC-labeled anti-vinculin, and Cy5-labeled anti–S. flexneri LPS revealed that localized actin polymerization (Fig. 5, A and B) and accumulation of vinculin (Fig. 5, D and E) were also associated with the infecting bacteria (Fig. 5, G and H). None of those events were observed in C3-treated CHO cells (Fig. 5, C, F and I), suggesting that localized rearrangement of the host cytoskeleton elements such as actin, vinculin, and protein tyrosine phosphorylation elicited around invading Shigella is regulated by rho.

Bottom Line: Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with alpha5beta1 integrin.Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA.Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology, Institute of Medical Science, University of Tokyo, Minato-ku, Japan.

ABSTRACT
Shigella, the causative agents of bacillary dysentery, are capable of invading mammalian cells that are not normally phagocytic. Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with alpha5beta1 integrin. We report here that Shigella invasion of epithelial cells requires rho activity, a ras-related GTP-binding protein. The invasive capacity of Shigella flexneri for Chinese hamister ovary (CHO) cells and other epithelial cells were greatly reduced when treated with Clostridium botulinum exoenzyme C3 transferase. Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA. Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase. Our results indicate that cellular signal transduction regulated by rho is essential for Shigella invasion of epithelial cells.

Show MeSH
Related in: MedlinePlus