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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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Rearrangements of  F-actin and tyrosine phosphorylated  proteins in CHO cells upon incubation in MEM containing secreted  Ipa invasins. (A) Development of  actin stress fibers and tyrosine  phosphorylated proteins in CHO  cells in response to addition of secreted Ipa invasins. Serum-starved  CHO cells were incubated in MEM  containing secreted Ipa invasins  from S. flexneri YSH6000T for 0  min (a and e), 30 min (b and f), 60  min (c and g), or in MEM without secreted Ipa invasins for 60 min  (d and h) at 37°C. Actin filaments  were visualized with rhodaminephalloidin (a–d), and in the same  cells phosphorylated protein tyrosine  was localized by FITC-conjugated  anti-phosphotyrosine (e–h). Bar,  (h) 100 μm. (B) High magnification of A. Serum-starved CHO  cells (a and b) and Swiss 3T3 cells  (c and d) were incubated in MEM  containing secreted Ipa invasins  for 60 min at 37°C. Actin filaments  were visualized with rhodaminephalloidin (a and c), and in the  same cells phosphorylated protein  tyrosine was localized by FITCconjugated anti-phosphotyrosine  (b and d). Bar, (d) 10 μm. (C)  Quantification of development of  F-actin and assembly of focal adhesion proteins. Serum-starved CHO  cells were incubated with secreted  Ipa invasins for 0 min (lane 1), 30  min (lane 2), 60 min (lane 3),  without Ipa invasins (lane 4) or  were pretreated with C3 (1.25 μg/ ml) for 24 h (lane 5). Quantification of the pixel count was calculated using LaserSharp version 2.0  (Bio-Rad Labs.). Figures represent  relative numbers compared with 0  min (lane 1). The data shown are  the means of triplicate experiments. (D) Tyrosine phosphorylation of FAK and paxillin in serumstarved CHO cells upon incubation  with MEM containing secreted Ipa  invasins. Samples are from untreated cells (UC), and from cells  incubated with Ipa invasins for up  to 60 min. Cell lysate proteins were  immunoprecipitated with the antiFAK mAb 2A7 (a) or anti-paxillin  mAb 347 (b), separated by 10% SDSPAGE, and transferred to nitrocellulose, before probing with antiphosphotyrosine 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 7: Rearrangements of F-actin and tyrosine phosphorylated proteins in CHO cells upon incubation in MEM containing secreted Ipa invasins. (A) Development of actin stress fibers and tyrosine phosphorylated proteins in CHO cells in response to addition of secreted Ipa invasins. Serum-starved CHO cells were incubated in MEM containing secreted Ipa invasins from S. flexneri YSH6000T for 0 min (a and e), 30 min (b and f), 60 min (c and g), or in MEM without secreted Ipa invasins for 60 min (d and h) at 37°C. Actin filaments were visualized with rhodaminephalloidin (a–d), and in the same cells phosphorylated protein tyrosine was localized by FITC-conjugated anti-phosphotyrosine (e–h). Bar, (h) 100 μm. (B) High magnification of A. Serum-starved CHO cells (a and b) and Swiss 3T3 cells (c and d) were incubated in MEM containing secreted Ipa invasins for 60 min at 37°C. Actin filaments were visualized with rhodaminephalloidin (a and c), and in the same cells phosphorylated protein tyrosine was localized by FITCconjugated anti-phosphotyrosine (b and d). Bar, (d) 10 μm. (C) Quantification of development of F-actin and assembly of focal adhesion proteins. Serum-starved CHO cells were incubated with secreted Ipa invasins for 0 min (lane 1), 30 min (lane 2), 60 min (lane 3), without Ipa invasins (lane 4) or were pretreated with C3 (1.25 μg/ ml) for 24 h (lane 5). Quantification of the pixel count was calculated using LaserSharp version 2.0 (Bio-Rad Labs.). Figures represent relative numbers compared with 0 min (lane 1). The data shown are the means of triplicate experiments. (D) Tyrosine phosphorylation of FAK and paxillin in serumstarved CHO cells upon incubation with MEM containing secreted Ipa invasins. Samples are from untreated cells (UC), and from cells incubated with Ipa invasins for up to 60 min. Cell lysate proteins were immunoprecipitated with the antiFAK mAb 2A7 (a) or anti-paxillin mAb 347 (b), separated by 10% SDSPAGE, and transferred to nitrocellulose, before probing with antiphosphotyrosine mAb PT-66 (a and b, top), anti-FAK mAb 2A7 (a, bottom), or anti-paxillin mAb 347 (b, bottom).

Mentions: The secretion of Ipa invasins from the surface of S. flexneri into the external medium is essential for allowing the bacteria to invade epithelial cells (14, 35). We therefore wished to clarify that the CHO cell response to YSH6000T invasion observed in this study was the consequence of the activity displayed by the Ipa invasins secreted into the medium. To test this, semiconfluent CHO cell monolayers serum starved for 3 d were incubated in tissue culture medium (MEM) containing secreted IpaB, IpaC, and IpaD and examined for the appearance of actin polymerization, foci of protein tyrosine phosphorylation, and accumulation of vinculin and talin. As shown in Fig. 7 A, a dramatic change in the development of actin polymerization in CHO cells incubated in Ipa-containing MEM was accompanied by the appearance of foci of protein tyrosine phosphorylation around the peripheral edges. Similar responses were also observed in Swiss3T3 fibroblasts (Fig. 7 B). As shown in Fig. 7 C, the semiquantitative measurement of amounts of actin polymerization, and accumulation of vinculin, talin, paxillin, FAK, and tyrosine phosphorylated proteins in the CHO cells revealed that at 30 (lane 2) and 60 min (lane 3) after the incubation was increased to greatly higher levels as compared with those of the CHO cells incubated in the MEM at 0 min (lane 1). Such cellular responses were not evoked at 60 min incubation in the CHO cells incubated in the MEM whose Ipa invasins had been removed by immunoprecipitation with anti-IpaB, -IpaC, and -IpaD antibodies (lane 4). The cellular responses to the addition of Ipa invasins were almost completely shut off in CHO cells pretreated with C3 (lane 5). In fact, measurement of phosphorylated FAK and paxillin levels by immunoblottings with anti-phosphotyrosine revealed that they were elicited upon incubation of the CHO cells in Ipa-containing MEM 30 min after incubation (Fig. 7 D). These data thus indicate that the cellular responses to the addition of secreted Ipa invasins are 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)

Rearrangements of  F-actin and tyrosine phosphorylated  proteins in CHO cells upon incubation in MEM containing secreted  Ipa invasins. (A) Development of  actin stress fibers and tyrosine  phosphorylated proteins in CHO  cells in response to addition of secreted Ipa invasins. Serum-starved  CHO cells were incubated in MEM  containing secreted Ipa invasins  from S. flexneri YSH6000T for 0  min (a and e), 30 min (b and f), 60  min (c and g), or in MEM without secreted Ipa invasins for 60 min  (d and h) at 37°C. Actin filaments  were visualized with rhodaminephalloidin (a–d), and in the same  cells phosphorylated protein tyrosine  was localized by FITC-conjugated  anti-phosphotyrosine (e–h). Bar,  (h) 100 μm. (B) High magnification of A. Serum-starved CHO  cells (a and b) and Swiss 3T3 cells  (c and d) were incubated in MEM  containing secreted Ipa invasins  for 60 min at 37°C. Actin filaments  were visualized with rhodaminephalloidin (a and c), and in the  same cells phosphorylated protein  tyrosine was localized by FITCconjugated anti-phosphotyrosine  (b and d). Bar, (d) 10 μm. (C)  Quantification of development of  F-actin and assembly of focal adhesion proteins. Serum-starved CHO  cells were incubated with secreted  Ipa invasins for 0 min (lane 1), 30  min (lane 2), 60 min (lane 3),  without Ipa invasins (lane 4) or  were pretreated with C3 (1.25 μg/ ml) for 24 h (lane 5). Quantification of the pixel count was calculated using LaserSharp version 2.0  (Bio-Rad Labs.). Figures represent  relative numbers compared with 0  min (lane 1). The data shown are  the means of triplicate experiments. (D) Tyrosine phosphorylation of FAK and paxillin in serumstarved CHO cells upon incubation  with MEM containing secreted Ipa  invasins. Samples are from untreated cells (UC), and from cells  incubated with Ipa invasins for up  to 60 min. Cell lysate proteins were  immunoprecipitated with the antiFAK mAb 2A7 (a) or anti-paxillin  mAb 347 (b), separated by 10% SDSPAGE, and transferred to nitrocellulose, before probing with antiphosphotyrosine 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 7: Rearrangements of F-actin and tyrosine phosphorylated proteins in CHO cells upon incubation in MEM containing secreted Ipa invasins. (A) Development of actin stress fibers and tyrosine phosphorylated proteins in CHO cells in response to addition of secreted Ipa invasins. Serum-starved CHO cells were incubated in MEM containing secreted Ipa invasins from S. flexneri YSH6000T for 0 min (a and e), 30 min (b and f), 60 min (c and g), or in MEM without secreted Ipa invasins for 60 min (d and h) at 37°C. Actin filaments were visualized with rhodaminephalloidin (a–d), and in the same cells phosphorylated protein tyrosine was localized by FITC-conjugated anti-phosphotyrosine (e–h). Bar, (h) 100 μm. (B) High magnification of A. Serum-starved CHO cells (a and b) and Swiss 3T3 cells (c and d) were incubated in MEM containing secreted Ipa invasins for 60 min at 37°C. Actin filaments were visualized with rhodaminephalloidin (a and c), and in the same cells phosphorylated protein tyrosine was localized by FITCconjugated anti-phosphotyrosine (b and d). Bar, (d) 10 μm. (C) Quantification of development of F-actin and assembly of focal adhesion proteins. Serum-starved CHO cells were incubated with secreted Ipa invasins for 0 min (lane 1), 30 min (lane 2), 60 min (lane 3), without Ipa invasins (lane 4) or were pretreated with C3 (1.25 μg/ ml) for 24 h (lane 5). Quantification of the pixel count was calculated using LaserSharp version 2.0 (Bio-Rad Labs.). Figures represent relative numbers compared with 0 min (lane 1). The data shown are the means of triplicate experiments. (D) Tyrosine phosphorylation of FAK and paxillin in serumstarved CHO cells upon incubation with MEM containing secreted Ipa invasins. Samples are from untreated cells (UC), and from cells incubated with Ipa invasins for up to 60 min. Cell lysate proteins were immunoprecipitated with the antiFAK mAb 2A7 (a) or anti-paxillin mAb 347 (b), separated by 10% SDSPAGE, and transferred to nitrocellulose, before probing with antiphosphotyrosine mAb PT-66 (a and b, top), anti-FAK mAb 2A7 (a, bottom), or anti-paxillin mAb 347 (b, bottom).
Mentions: The secretion of Ipa invasins from the surface of S. flexneri into the external medium is essential for allowing the bacteria to invade epithelial cells (14, 35). We therefore wished to clarify that the CHO cell response to YSH6000T invasion observed in this study was the consequence of the activity displayed by the Ipa invasins secreted into the medium. To test this, semiconfluent CHO cell monolayers serum starved for 3 d were incubated in tissue culture medium (MEM) containing secreted IpaB, IpaC, and IpaD and examined for the appearance of actin polymerization, foci of protein tyrosine phosphorylation, and accumulation of vinculin and talin. As shown in Fig. 7 A, a dramatic change in the development of actin polymerization in CHO cells incubated in Ipa-containing MEM was accompanied by the appearance of foci of protein tyrosine phosphorylation around the peripheral edges. Similar responses were also observed in Swiss3T3 fibroblasts (Fig. 7 B). As shown in Fig. 7 C, the semiquantitative measurement of amounts of actin polymerization, and accumulation of vinculin, talin, paxillin, FAK, and tyrosine phosphorylated proteins in the CHO cells revealed that at 30 (lane 2) and 60 min (lane 3) after the incubation was increased to greatly higher levels as compared with those of the CHO cells incubated in the MEM at 0 min (lane 1). Such cellular responses were not evoked at 60 min incubation in the CHO cells incubated in the MEM whose Ipa invasins had been removed by immunoprecipitation with anti-IpaB, -IpaC, and -IpaD antibodies (lane 4). The cellular responses to the addition of Ipa invasins were almost completely shut off in CHO cells pretreated with C3 (lane 5). In fact, measurement of phosphorylated FAK and paxillin levels by immunoblottings with anti-phosphotyrosine revealed that they were elicited upon incubation of the CHO cells in Ipa-containing MEM 30 min after incubation (Fig. 7 D). These data thus indicate that the cellular responses to the addition of secreted Ipa invasins are 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