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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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Inhibition of bacterial internalization by the R2-25 antibody treatment. (A) The purified R2-25 antibody was added to the cell culture medium for the bacterial internalization assay at the indicated concentrations 2 h before infection. Data are the averages of triplicate samples from three identical experiments, and the error bars represent standard deviations. Statistically significant differences between bacterial internalization into TG cells treated with the R2-25 antibody and those treated with rat IgG are indicated by asterisks (*, P < 0.01). Immunofluorescence staining of B. abortus or L. monocytogenes (green) and Hsc70 (B) or Hsp90 (red) (C) in TG cells. Colocalized bacteria with Hsc70 are shown in yellow in the merged images. Arrows point to colocalized bacteria. Scale bar indicates 10 μm.
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Figure 1: Inhibition of bacterial internalization by the R2-25 antibody treatment. (A) The purified R2-25 antibody was added to the cell culture medium for the bacterial internalization assay at the indicated concentrations 2 h before infection. Data are the averages of triplicate samples from three identical experiments, and the error bars represent standard deviations. Statistically significant differences between bacterial internalization into TG cells treated with the R2-25 antibody and those treated with rat IgG are indicated by asterisks (*, P < 0.01). Immunofluorescence staining of B. abortus or L. monocytogenes (green) and Hsc70 (B) or Hsp90 (red) (C) in TG cells. Colocalized bacteria with Hsc70 are shown in yellow in the merged images. Arrows point to colocalized bacteria. Scale bar indicates 10 μm.

Mentions: In a previous study [7], we isolated a monoclonal antibody R2-25 that inhibits the uptake of Brucella abortus by trophoblast giant (TG) cells; this antibody reacts with heat shock cognate protein (Hsc70). Since Hsc70 on TG cells may be able to take up extracellular antigens such as bacteria, uptake of another abortion-inducing bacterium, Listeria monocytogenes, was examined. The purified R2-25 antibody significantly inhibited both B. abortus and L. monocytogenes uptake by TG cells concentration dependently, but there was no inhibition with rat IgG (negative control) (Fig. 1A). Hsc70 colocalized with B. abortus and L. monocytogenes at the site of bacterial uptake by TG cells (Fig. 1B), but the colocalization between Hsp90, other heat shock protein containing EEVD motif, and the bacteria was not detected (Fig. 1C).


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)

Inhibition of bacterial internalization by the R2-25 antibody treatment. (A) The purified R2-25 antibody was added to the cell culture medium for the bacterial internalization assay at the indicated concentrations 2 h before infection. Data are the averages of triplicate samples from three identical experiments, and the error bars represent standard deviations. Statistically significant differences between bacterial internalization into TG cells treated with the R2-25 antibody and those treated with rat IgG are indicated by asterisks (*, P < 0.01). Immunofluorescence staining of B. abortus or L. monocytogenes (green) and Hsc70 (B) or Hsp90 (red) (C) in TG cells. Colocalized bacteria with Hsc70 are shown in yellow in the merged images. Arrows point to colocalized bacteria. Scale bar indicates 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Inhibition of bacterial internalization by the R2-25 antibody treatment. (A) The purified R2-25 antibody was added to the cell culture medium for the bacterial internalization assay at the indicated concentrations 2 h before infection. Data are the averages of triplicate samples from three identical experiments, and the error bars represent standard deviations. Statistically significant differences between bacterial internalization into TG cells treated with the R2-25 antibody and those treated with rat IgG are indicated by asterisks (*, P < 0.01). Immunofluorescence staining of B. abortus or L. monocytogenes (green) and Hsc70 (B) or Hsp90 (red) (C) in TG cells. Colocalized bacteria with Hsc70 are shown in yellow in the merged images. Arrows point to colocalized bacteria. Scale bar indicates 10 μm.
Mentions: In a previous study [7], we isolated a monoclonal antibody R2-25 that inhibits the uptake of Brucella abortus by trophoblast giant (TG) cells; this antibody reacts with heat shock cognate protein (Hsc70). Since Hsc70 on TG cells may be able to take up extracellular antigens such as bacteria, uptake of another abortion-inducing bacterium, Listeria monocytogenes, was examined. The purified R2-25 antibody significantly inhibited both B. abortus and L. monocytogenes uptake by TG cells concentration dependently, but there was no inhibition with rat IgG (negative control) (Fig. 1A). Hsc70 colocalized with B. abortus and L. monocytogenes at the site of bacterial uptake by TG cells (Fig. 1B), but the colocalization between Hsp90, other heat shock protein containing EEVD motif, and the bacteria was not detected (Fig. 1C).

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