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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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Activation of PKC in S. flexneri invasion of CHO cells. A, B, and C show the effect of C3 upon YSH6000T invasion-induced PKC activation. The PKC activity present in the cytosolic (•) or the membrane (○) fraction of the CHO cells was assayed (33). The PKC activity in the CHO cells  treated with C3 (1.25 μg/ml) (B) or left untreated (A and C) were measured at the indicated times. The kinase activity is expressed as picomoles of  phosphate incorporated into a synthetic substrate per min. Data shown represent the mean of triplicate experiments. Bars, SEM. (A and B) YSH6000T,  (C) CS2585 (Δspa32). The effect of PKC inhibitors calphostin C (D), H7 (E), and HA1004 (F) on YSH6000T invasion of CHO cells was measured.  CHO cells pretreated with various concentrations of the inhibitors for 60 min were infected with S. flexneri YSH6000T (○) or S. typhimurium SB300  (•). The invasive capacity was determined by the gentamicin-protection assay. The data shown are the mean of triplicate experiments.
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Figure 6: Activation of PKC in S. flexneri invasion of CHO cells. A, B, and C show the effect of C3 upon YSH6000T invasion-induced PKC activation. The PKC activity present in the cytosolic (•) or the membrane (○) fraction of the CHO cells was assayed (33). The PKC activity in the CHO cells treated with C3 (1.25 μg/ml) (B) or left untreated (A and C) were measured at the indicated times. The kinase activity is expressed as picomoles of phosphate incorporated into a synthetic substrate per min. Data shown represent the mean of triplicate experiments. Bars, SEM. (A and B) YSH6000T, (C) CS2585 (Δspa32). The effect of PKC inhibitors calphostin C (D), H7 (E), and HA1004 (F) on YSH6000T invasion of CHO cells was measured. CHO cells pretreated with various concentrations of the inhibitors for 60 min were infected with S. flexneri YSH6000T (○) or S. typhimurium SB300 (•). The invasive capacity was determined by the gentamicin-protection assay. The data shown are the mean of triplicate experiments.

Mentions: Since intracellular signals regulated by rho accompany the activation of PKC (25), we thus looked to see if PKC was indeed activated in CHO cells in response to infection by S. flexneri. Since the active form of PKC exists in the plasma membrane while the nonactive form remains in the cytoplasm (37), the changes of PKC activity in the two subcellular fractions of CHO cells were monitored after infection by YSH6000T or CS2585 (noninvasive spa32 mutant) at 5 min intervals up to 30 min. As shown in Fig. 6 A, the PKC was activated in the CHO cells upon invasion by YSH6000T, but not by CS2585 (Fig. 6 C). However, the activation of PKC was not evoked in C3-treated CHO cells (Fig. 6 B). Since Salmonella invasion of CHO cells was not blocked by the treatment with C3 (Fig. 1), we tested whether or not S. typhimurium can activate PKC upon invasion of CHO cells. Under the same conditions, Salmonella invasion did not activate the PKC at all (data not shown). To further confirm this, PKC inhibitors calphostin C, H7, and HA1004 (38–40) were tested for their effects on the invasiveness of YSH6000T using the gentamicinprotection assay. As shown in Fig. 6, D–F, the PKC inhibitors greatly diminished the Shigella invasive capacity for the CHO cells as their concentration was increased. Under the same conditions, none of the PKC inhibitors blocked Salmonella invasiveness.


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)

Activation of PKC in S. flexneri invasion of CHO cells. A, B, and C show the effect of C3 upon YSH6000T invasion-induced PKC activation. The PKC activity present in the cytosolic (•) or the membrane (○) fraction of the CHO cells was assayed (33). The PKC activity in the CHO cells  treated with C3 (1.25 μg/ml) (B) or left untreated (A and C) were measured at the indicated times. The kinase activity is expressed as picomoles of  phosphate incorporated into a synthetic substrate per min. Data shown represent the mean of triplicate experiments. Bars, SEM. (A and B) YSH6000T,  (C) CS2585 (Δspa32). The effect of PKC inhibitors calphostin C (D), H7 (E), and HA1004 (F) on YSH6000T invasion of CHO cells was measured.  CHO cells pretreated with various concentrations of the inhibitors for 60 min were infected with S. flexneri YSH6000T (○) or S. typhimurium SB300  (•). The invasive capacity was determined by the gentamicin-protection assay. The data shown are the mean of triplicate experiments.
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Related In: Results  -  Collection

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Figure 6: Activation of PKC in S. flexneri invasion of CHO cells. A, B, and C show the effect of C3 upon YSH6000T invasion-induced PKC activation. The PKC activity present in the cytosolic (•) or the membrane (○) fraction of the CHO cells was assayed (33). The PKC activity in the CHO cells treated with C3 (1.25 μg/ml) (B) or left untreated (A and C) were measured at the indicated times. The kinase activity is expressed as picomoles of phosphate incorporated into a synthetic substrate per min. Data shown represent the mean of triplicate experiments. Bars, SEM. (A and B) YSH6000T, (C) CS2585 (Δspa32). The effect of PKC inhibitors calphostin C (D), H7 (E), and HA1004 (F) on YSH6000T invasion of CHO cells was measured. CHO cells pretreated with various concentrations of the inhibitors for 60 min were infected with S. flexneri YSH6000T (○) or S. typhimurium SB300 (•). The invasive capacity was determined by the gentamicin-protection assay. The data shown are the mean of triplicate experiments.
Mentions: Since intracellular signals regulated by rho accompany the activation of PKC (25), we thus looked to see if PKC was indeed activated in CHO cells in response to infection by S. flexneri. Since the active form of PKC exists in the plasma membrane while the nonactive form remains in the cytoplasm (37), the changes of PKC activity in the two subcellular fractions of CHO cells were monitored after infection by YSH6000T or CS2585 (noninvasive spa32 mutant) at 5 min intervals up to 30 min. As shown in Fig. 6 A, the PKC was activated in the CHO cells upon invasion by YSH6000T, but not by CS2585 (Fig. 6 C). However, the activation of PKC was not evoked in C3-treated CHO cells (Fig. 6 B). Since Salmonella invasion of CHO cells was not blocked by the treatment with C3 (Fig. 1), we tested whether or not S. typhimurium can activate PKC upon invasion of CHO cells. Under the same conditions, Salmonella invasion did not activate the PKC at all (data not shown). To further confirm this, PKC inhibitors calphostin C, H7, and HA1004 (38–40) were tested for their effects on the invasiveness of YSH6000T using the gentamicinprotection assay. As shown in Fig. 6, D–F, the PKC inhibitors greatly diminished the Shigella invasive capacity for the CHO cells as their concentration was increased. Under the same conditions, none of the PKC inhibitors blocked Salmonella invasiveness.

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