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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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Effect of C3 on S. flexneri invasion-induced tyrosine phosphorylation of FAK and paxillin in CHO cells. The CHO cells were  treated with C3 (1.25 μg/ml) (lanes 5–8) or left untreated (lanes 1–4) for  24 h and then infected by YSH6000T. Samples are from uninfected cells  (lanes 1 and 5), from cells infected for 10 min (lanes 2 and 6), 20 min  (lanes 3 and 7), and 30 min (lanes 4 and 8). Numbers to the left indicate  the positions of molecular weight standards. Cell lysate proteins were immunoprecipitated with anti-FAK mAb 2A7 (A) or anti-paxillin mAb 347  (B), separated on a 10% SDS-PAGE, transferred to nitrocellulose, and  probed with anti-phosphotyrosine mAb PT-66 (A and B, top), anti-FAK  mAb 2A7 (A, bottom) or anti-paxillin mAb 347 (B, bottom).
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Figure 3: Effect of C3 on S. flexneri invasion-induced tyrosine phosphorylation of FAK and paxillin in CHO cells. The CHO cells were treated with C3 (1.25 μg/ml) (lanes 5–8) or left untreated (lanes 1–4) for 24 h and then infected by YSH6000T. Samples are from uninfected cells (lanes 1 and 5), from cells infected for 10 min (lanes 2 and 6), 20 min (lanes 3 and 7), and 30 min (lanes 4 and 8). Numbers to the left indicate the positions of molecular weight standards. Cell lysate proteins were immunoprecipitated with anti-FAK mAb 2A7 (A) or anti-paxillin mAb 347 (B), separated on a 10% SDS-PAGE, transferred to nitrocellulose, and probed with anti-phosphotyrosine mAb PT-66 (A and B, top), anti-FAK mAb 2A7 (A, bottom) or anti-paxillin mAb 347 (B, bottom).

Mentions: We recently showed that localized accumulation of F-actin, vinculin, or talin was elicited by the attachment of S. flexneri to CHO cells, and that this was also accompanied by tyrosine phosphorylation of FAK and paxillin (19). Hence, we examined whether such cellular responses to Shigella invasion were regulated by rho. Semiconfluent CHO cell monolayers treated with or without C3 (at 1.25 μg/ml) for 1 d were infected with YSH6000T, and, at 10, 20, and 30 min after the infection, whole cell protein extracts were immunoprecipitated with the antiFAK or anti-paxillin antibody. The proteins in the precipitates were separated by SDS-PAGE and immunoblotted with each of the anti-FAK, anti-paxillin, and anti-phosphotyrosine antibodies. As shown in Fig. 3, both the CHO cells with and without C3 treatment expressed some amounts of FAK and paxillin (nonphosphorylated), but the tyrosine phosphorylated FAK and paxillin were evoked in nontreated CHO cells after 20–30 min infection with YSH6000T. Under the same conditions, neither of these proteins was tyrosine phosphorylated in the C3-treated CHO cells.


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)

Effect of C3 on S. flexneri invasion-induced tyrosine phosphorylation of FAK and paxillin in CHO cells. The CHO cells were  treated with C3 (1.25 μg/ml) (lanes 5–8) or left untreated (lanes 1–4) for  24 h and then infected by YSH6000T. Samples are from uninfected cells  (lanes 1 and 5), from cells infected for 10 min (lanes 2 and 6), 20 min  (lanes 3 and 7), and 30 min (lanes 4 and 8). Numbers to the left indicate  the positions of molecular weight standards. Cell lysate proteins were immunoprecipitated with anti-FAK mAb 2A7 (A) or anti-paxillin mAb 347  (B), separated on a 10% SDS-PAGE, transferred to nitrocellulose, and  probed with anti-phosphotyrosine mAb PT-66 (A and B, top), anti-FAK  mAb 2A7 (A, bottom) or anti-paxillin mAb 347 (B, bottom).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196126&req=5

Figure 3: Effect of C3 on S. flexneri invasion-induced tyrosine phosphorylation of FAK and paxillin in CHO cells. The CHO cells were treated with C3 (1.25 μg/ml) (lanes 5–8) or left untreated (lanes 1–4) for 24 h and then infected by YSH6000T. Samples are from uninfected cells (lanes 1 and 5), from cells infected for 10 min (lanes 2 and 6), 20 min (lanes 3 and 7), and 30 min (lanes 4 and 8). Numbers to the left indicate the positions of molecular weight standards. Cell lysate proteins were immunoprecipitated with anti-FAK mAb 2A7 (A) or anti-paxillin mAb 347 (B), separated on a 10% SDS-PAGE, transferred to nitrocellulose, and probed with anti-phosphotyrosine mAb PT-66 (A and B, top), anti-FAK mAb 2A7 (A, bottom) or anti-paxillin mAb 347 (B, bottom).
Mentions: We recently showed that localized accumulation of F-actin, vinculin, or talin was elicited by the attachment of S. flexneri to CHO cells, and that this was also accompanied by tyrosine phosphorylation of FAK and paxillin (19). Hence, we examined whether such cellular responses to Shigella invasion were regulated by rho. Semiconfluent CHO cell monolayers treated with or without C3 (at 1.25 μg/ml) for 1 d were infected with YSH6000T, and, at 10, 20, and 30 min after the infection, whole cell protein extracts were immunoprecipitated with the antiFAK or anti-paxillin antibody. The proteins in the precipitates were separated by SDS-PAGE and immunoblotted with each of the anti-FAK, anti-paxillin, and anti-phosphotyrosine antibodies. As shown in Fig. 3, both the CHO cells with and without C3 treatment expressed some amounts of FAK and paxillin (nonphosphorylated), but the tyrosine phosphorylated FAK and paxillin were evoked in nontreated CHO cells after 20–30 min infection with YSH6000T. Under the same conditions, neither of these proteins was tyrosine phosphorylated in the C3-treated CHO cells.

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