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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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Blockage of Shigella invasion of mammalian cells by treatment with C3. (A) Invasion of CHO cells treated with various concentrations of C3 by S. flexneri or S. typhimurium. ○, S. flexneri YSH6000T; □,  S. typhimurium SB300. (B) Inhibitory effect of C3 on S. flexneri invasion  of various cell lines. Semiconfluent monolayers of mammalian cells were  incubated in MEM containing C3 at 1.25 μg/ml (CHO cells), 4 μg/ml  (MK2 cells and Caco-2 cells), 5 μg/ml (HeLa cells), and 1 μg/ml (Swiss  3T3 cells) for 24 h before the bacterial infection. The black and white  columns indicate the invasive capacity of S. flexneri YSH6000T and S. typhimurium SB300 relative to bacteria incubated in MEM in the absence of  C3. The invasive capacity was determined by the gentamicin-protection  assay. The data shown are the means of triplicate experiments.
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Figure 1: Blockage of Shigella invasion of mammalian cells by treatment with C3. (A) Invasion of CHO cells treated with various concentrations of C3 by S. flexneri or S. typhimurium. ○, S. flexneri YSH6000T; □, S. typhimurium SB300. (B) Inhibitory effect of C3 on S. flexneri invasion of various cell lines. Semiconfluent monolayers of mammalian cells were incubated in MEM containing C3 at 1.25 μg/ml (CHO cells), 4 μg/ml (MK2 cells and Caco-2 cells), 5 μg/ml (HeLa cells), and 1 μg/ml (Swiss 3T3 cells) for 24 h before the bacterial infection. The black and white columns indicate the invasive capacity of S. flexneri YSH6000T and S. typhimurium SB300 relative to bacteria incubated in MEM in the absence of C3. The invasive capacity was determined by the gentamicin-protection assay. The data shown are the means of triplicate experiments.

Mentions: To examine whether or not the invasion of mammalian cells by Shigella is dependent on the activity of rho, a ras-related GTP-binding protein (21, 22), semiconfluent (∼80%) CHO cell monolayers treated with various concentrations of C3 (0–2.0 μg/ml) for 1 d were infected with YSH6000T (wild-type S. flexneri) for 20 min, and the internalized bacterial numbers were measured by gentamicin-protection assay. As shown in Fig. 1 A, the invasive capacity of the bacteria decreased upon pretreatment of the CHO cells with C3, with the invasive capacity for the CHO cells pretreated with 2.0 μg/ml of C3 being only 10% of the initial invasiveness (100%). Under the same conditions, we observed that the invasion of the C3-treated CHO cells by a S. typhimurium strain, SB300 (31), with its characteristic apical entry and induction of membrane ruffling (17), was not decreased (Fig. 1 A). The inhibitory effect of C3 treatment on the invasiveness of YSH6000T was also revealed in other tissue cultured cell lines such as LLC-MK2, Caco-2, HeLa, and Swiss3T3 cells (Fig. 1 B); YSH6000T invasiveness for LLC-MK2, Caco-2, HeLa, and Swiss3T3 cells pretreated with C3 was decreased to 13, 9, 11, and 13% of the initial invasive capacity (100%), respectively. Unlike YSH6000T, SB300 invasiveness for these cell lines treated with C3 were not decreased at all, suggesting that the inactivation of rho function specifically prevents Shigella invasion of mammalian 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)

Blockage of Shigella invasion of mammalian cells by treatment with C3. (A) Invasion of CHO cells treated with various concentrations of C3 by S. flexneri or S. typhimurium. ○, S. flexneri YSH6000T; □,  S. typhimurium SB300. (B) Inhibitory effect of C3 on S. flexneri invasion  of various cell lines. Semiconfluent monolayers of mammalian cells were  incubated in MEM containing C3 at 1.25 μg/ml (CHO cells), 4 μg/ml  (MK2 cells and Caco-2 cells), 5 μg/ml (HeLa cells), and 1 μg/ml (Swiss  3T3 cells) for 24 h before the bacterial infection. The black and white  columns indicate the invasive capacity of S. flexneri YSH6000T and S. typhimurium SB300 relative to bacteria incubated in MEM in the absence of  C3. The invasive capacity was determined by the gentamicin-protection  assay. The data shown are the means of triplicate experiments.
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: Blockage of Shigella invasion of mammalian cells by treatment with C3. (A) Invasion of CHO cells treated with various concentrations of C3 by S. flexneri or S. typhimurium. ○, S. flexneri YSH6000T; □, S. typhimurium SB300. (B) Inhibitory effect of C3 on S. flexneri invasion of various cell lines. Semiconfluent monolayers of mammalian cells were incubated in MEM containing C3 at 1.25 μg/ml (CHO cells), 4 μg/ml (MK2 cells and Caco-2 cells), 5 μg/ml (HeLa cells), and 1 μg/ml (Swiss 3T3 cells) for 24 h before the bacterial infection. The black and white columns indicate the invasive capacity of S. flexneri YSH6000T and S. typhimurium SB300 relative to bacteria incubated in MEM in the absence of C3. The invasive capacity was determined by the gentamicin-protection assay. The data shown are the means of triplicate experiments.
Mentions: To examine whether or not the invasion of mammalian cells by Shigella is dependent on the activity of rho, a ras-related GTP-binding protein (21, 22), semiconfluent (∼80%) CHO cell monolayers treated with various concentrations of C3 (0–2.0 μg/ml) for 1 d were infected with YSH6000T (wild-type S. flexneri) for 20 min, and the internalized bacterial numbers were measured by gentamicin-protection assay. As shown in Fig. 1 A, the invasive capacity of the bacteria decreased upon pretreatment of the CHO cells with C3, with the invasive capacity for the CHO cells pretreated with 2.0 μg/ml of C3 being only 10% of the initial invasiveness (100%). Under the same conditions, we observed that the invasion of the C3-treated CHO cells by a S. typhimurium strain, SB300 (31), with its characteristic apical entry and induction of membrane ruffling (17), was not decreased (Fig. 1 A). The inhibitory effect of C3 treatment on the invasiveness of YSH6000T was also revealed in other tissue cultured cell lines such as LLC-MK2, Caco-2, HeLa, and Swiss3T3 cells (Fig. 1 B); YSH6000T invasiveness for LLC-MK2, Caco-2, HeLa, and Swiss3T3 cells pretreated with C3 was decreased to 13, 9, 11, and 13% of the initial invasive capacity (100%), respectively. Unlike YSH6000T, SB300 invasiveness for these cell lines treated with C3 were not decreased at all, suggesting that the inactivation of rho function specifically prevents Shigella invasion of mammalian 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