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EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells.

Watanabe K, Tachibana M, Kim S, Watarai M - J. Biomed. Sci. (2009)

Bottom Line: The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice.Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells.Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Public Health, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. kentaw@yamaguchi-u.ac.jp

ABSTRACT

Background: The uptake of abortion-inducing pathogens by trophoblast giant (TG) cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70) contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this.

Methods: Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR) domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice.

Results: The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70.

Conclusions: Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.

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Control of infectious abortion by protein interaction via the EEVD motif of Hsc70. Infectious abortion was prevented by blocking the EEVD motif of Hsc70. Pregnant mice were inoculated with R2-25, rat IgG (control), TPR-Ba and immunized with EEVD or sEEVD (CAPISEDGSGETV) peptides. Control received no treatment. Statistically significant differences between the untreated control and antibody treated mice are indicated by asterisks (*, P < 0.01).
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Figure 5: Control of infectious abortion by protein interaction via the EEVD motif of Hsc70. Infectious abortion was prevented by blocking the EEVD motif of Hsc70. Pregnant mice were inoculated with R2-25, rat IgG (control), TPR-Ba and immunized with EEVD or sEEVD (CAPISEDGSGETV) peptides. Control received no treatment. Statistically significant differences between the untreated control and antibody treated mice are indicated by asterisks (*, P < 0.01).

Mentions: We finally investigated the effect on pregnancy by blocking the interaction between Hsc70 and bacteria in pregnant mice. Pregnant mice were inoculated with R2-25 or TPR-Ba, evaluated as the most effective TPR protein according to the results above, 24 h before infection with the wild-type B. abortus strain on day 4.5 of gestation. While abortions were observed in the non-inoculated and Rat IgG inoculated mice, there was a significant increase in the number of live fetuses in the R2-25 or TPR-Ba inoculated mice. Abortion was also inhibited by immunization using a peptide containing the EEVD motif before infection (Fig. 5).


EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells.

Watanabe K, Tachibana M, Kim S, Watarai M - J. Biomed. Sci. (2009)

Control of infectious abortion by protein interaction via the EEVD motif of Hsc70. Infectious abortion was prevented by blocking the EEVD motif of Hsc70. Pregnant mice were inoculated with R2-25, rat IgG (control), TPR-Ba and immunized with EEVD or sEEVD (CAPISEDGSGETV) peptides. Control received no treatment. Statistically significant differences between the untreated control and antibody treated mice are indicated by asterisks (*, P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2800845&req=5

Figure 5: Control of infectious abortion by protein interaction via the EEVD motif of Hsc70. Infectious abortion was prevented by blocking the EEVD motif of Hsc70. Pregnant mice were inoculated with R2-25, rat IgG (control), TPR-Ba and immunized with EEVD or sEEVD (CAPISEDGSGETV) peptides. Control received no treatment. Statistically significant differences between the untreated control and antibody treated mice are indicated by asterisks (*, P < 0.01).
Mentions: We finally investigated the effect on pregnancy by blocking the interaction between Hsc70 and bacteria in pregnant mice. Pregnant mice were inoculated with R2-25 or TPR-Ba, evaluated as the most effective TPR protein according to the results above, 24 h before infection with the wild-type B. abortus strain on day 4.5 of gestation. While abortions were observed in the non-inoculated and Rat IgG inoculated mice, there was a significant increase in the number of live fetuses in the R2-25 or TPR-Ba inoculated mice. Abortion was also inhibited by immunization using a peptide containing the EEVD motif before infection (Fig. 5).

Bottom Line: The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice.Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells.Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Public Health, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. kentaw@yamaguchi-u.ac.jp

ABSTRACT

Background: The uptake of abortion-inducing pathogens by trophoblast giant (TG) cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70) contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this.

Methods: Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR) domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice.

Results: The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70.

Conclusions: Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.

Show MeSH
Related in: MedlinePlus