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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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Enhancement of S. flexneri invasion of CHO cells by  Val14RhoA injection. A, C, and E represent serum-starved CHO cells  (without microinjection) infected with YSH6000T. B, D, and F represent  serum-starved CHO cells (microinjection with Val14rhoA at 300 μg/ml)  followed by infection with YSH6000T. The asterisks in panels B, D, and  F indicate the microinjected CHO cells. A and B show rhodaminelabeled phalloidin. C and D show mouse FITC-labeled anti-phosphotyrosine. E and F show Cy5-labeled anti–S. flexneri 2a LPS. The arrowhead  marks the bacterial attachment sites. Bar, (F) 10 μm.
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Figure 2: Enhancement of S. flexneri invasion of CHO cells by Val14RhoA injection. A, C, and E represent serum-starved CHO cells (without microinjection) infected with YSH6000T. B, D, and F represent serum-starved CHO cells (microinjection with Val14rhoA at 300 μg/ml) followed by infection with YSH6000T. The asterisks in panels B, D, and F indicate the microinjected CHO cells. A and B show rhodaminelabeled phalloidin. C and D show mouse FITC-labeled anti-phosphotyrosine. E and F show Cy5-labeled anti–S. flexneri 2a LPS. The arrowhead marks the bacterial attachment sites. Bar, (F) 10 μm.

Mentions: To visualize CHO cell responses such as development of actin polymerization and protein tyrosine phosphorylation, the microinjection of Val14RhoA, or the effect on infecting bacteria, the microinjected CHO cells infected with YSH6000T were examined by triple fluorescence staining with Cy5-labeled anti–S. flexneri 2a LPS antibody, rhodamine-phalloidin, and FITC-labeled anti-phosphotyrosine antibody. As shown in Fig. 2, the Val14RhoA-injected CHO cells displayed dramatic actin polymerization that was accompanied by the appearance of tyrosine phosphorylated protein foci concentrated around the bacterial particles at the periphery of the CHO cells (Fig. 2, B, D, and F). Since bacterial particles associated with the CHO cells were visualized in immunostainings of cells permiabilized by 0.2%Triton X-100 but not in unpermiabilized cells, the bacterial particles appeared to be internalized into the cytoplasm. This data thus indicates that Shigella invasion of epithelial cells can be promoted upon activation of 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)

Enhancement of S. flexneri invasion of CHO cells by  Val14RhoA injection. A, C, and E represent serum-starved CHO cells  (without microinjection) infected with YSH6000T. B, D, and F represent  serum-starved CHO cells (microinjection with Val14rhoA at 300 μg/ml)  followed by infection with YSH6000T. The asterisks in panels B, D, and  F indicate the microinjected CHO cells. A and B show rhodaminelabeled phalloidin. C and D show mouse FITC-labeled anti-phosphotyrosine. E and F show Cy5-labeled anti–S. flexneri 2a LPS. The arrowhead  marks the bacterial attachment sites. Bar, (F) 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196126&req=5

Figure 2: Enhancement of S. flexneri invasion of CHO cells by Val14RhoA injection. A, C, and E represent serum-starved CHO cells (without microinjection) infected with YSH6000T. B, D, and F represent serum-starved CHO cells (microinjection with Val14rhoA at 300 μg/ml) followed by infection with YSH6000T. The asterisks in panels B, D, and F indicate the microinjected CHO cells. A and B show rhodaminelabeled phalloidin. C and D show mouse FITC-labeled anti-phosphotyrosine. E and F show Cy5-labeled anti–S. flexneri 2a LPS. The arrowhead marks the bacterial attachment sites. Bar, (F) 10 μm.
Mentions: To visualize CHO cell responses such as development of actin polymerization and protein tyrosine phosphorylation, the microinjection of Val14RhoA, or the effect on infecting bacteria, the microinjected CHO cells infected with YSH6000T were examined by triple fluorescence staining with Cy5-labeled anti–S. flexneri 2a LPS antibody, rhodamine-phalloidin, and FITC-labeled anti-phosphotyrosine antibody. As shown in Fig. 2, the Val14RhoA-injected CHO cells displayed dramatic actin polymerization that was accompanied by the appearance of tyrosine phosphorylated protein foci concentrated around the bacterial particles at the periphery of the CHO cells (Fig. 2, B, D, and F). Since bacterial particles associated with the CHO cells were visualized in immunostainings of cells permiabilized by 0.2%Triton X-100 but not in unpermiabilized cells, the bacterial particles appeared to be internalized into the cytoplasm. This data thus indicates that Shigella invasion of epithelial cells can be promoted upon activation of 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