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Cellular prion protein promotes Brucella infection into macrophages.

Watarai M, Kim S, Erdenebaatar J, Makino S, Horiuchi M, Shirahata T, Sakaguchi S, Katamine S - J. Exp. Med. (2003)

Bottom Line: Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex-associated process.Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC.These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro-shi, Hokkaido 080-8555, Japan. watarai@obihiro.ac.jp

ABSTRACT
The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles. The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified. We report here that Hsp60, a member of the GroEL family of chaperonins, of B. abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells. Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B. abortus. Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex-associated process. Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC. Hsp60 of B. abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages. The PrPC deficiency prevented swimming internalization and intracellular replication of B. abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network. These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection.

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Binding of Hsp60 to PrPC. (A) Demonstration of affinity of Hsp60 for PrPC by pull-down assay with Hsp60-coated beads. Control was assessed with beads only, the addition of anti-Hsp60 antibody, or purified Hsp60. Precipitates were analyzed by immunoblotting with anti-PrPC antibody. (B) Cell lysate or immunoprecipitates with anti-PrPC antibody and affinity of PrPC for Hsp60 by pull-down assay with PrPC-coated beads was analyzed by immunoblotting with anti-Hsp60 antibody. Control was assessed with beads only, or the addition of purified PrPC. (C) Silver staining of precipitates by pull-down assay. Samples were purified with Hsp60 (control) and precipitates of pull-down with Hsp60-coated beads (PD). (D) Labeling of internalized bacteria in macrophages with antibody specific for Hsp60. Macrophages were incubated with B. abortus for 5 or 15 min, and Hsp60 were localized by immunofluorescence as described in Materials and Methods. Fluorescence microscopy of stained GFP-expressed wild-type strain with anti-Hsp60 and phase contrast microscopy of the corresponding microscopic fields are shown. Arrows point to bacteria.
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fig4: Binding of Hsp60 to PrPC. (A) Demonstration of affinity of Hsp60 for PrPC by pull-down assay with Hsp60-coated beads. Control was assessed with beads only, the addition of anti-Hsp60 antibody, or purified Hsp60. Precipitates were analyzed by immunoblotting with anti-PrPC antibody. (B) Cell lysate or immunoprecipitates with anti-PrPC antibody and affinity of PrPC for Hsp60 by pull-down assay with PrPC-coated beads was analyzed by immunoblotting with anti-Hsp60 antibody. Control was assessed with beads only, or the addition of purified PrPC. (C) Silver staining of precipitates by pull-down assay. Samples were purified with Hsp60 (control) and precipitates of pull-down with Hsp60-coated beads (PD). (D) Labeling of internalized bacteria in macrophages with antibody specific for Hsp60. Macrophages were incubated with B. abortus for 5 or 15 min, and Hsp60 were localized by immunofluorescence as described in Materials and Methods. Fluorescence microscopy of stained GFP-expressed wild-type strain with anti-Hsp60 and phase contrast microscopy of the corresponding microscopic fields are shown. Arrows point to bacteria.

Mentions: Because Hsp60 expressed on the bacterial surface by the type IV secretion system was most likely interacting with the target cell, we tested Hsp60 for its ability to bind to PrPC on macrophages by pull-down assay with Hsp60 or PrPC beads. Analysis of the precipitated proteins by immunoblotting with anti-PrPC or Hsp60 antibody showed that a 29-kD PrPC was associated with Hsp60, but not beads alone (Fig. 4, A and B). To confirm this association, Hsp60 was added to macrophage lysate and the proteins in the mixture were then immunoprecipitated with anti-PrPC antibody. The precipitated proteins were analyzed by immunoblotting with anti-Hsp60 antibody. The precipitates contained Hsp60 (Fig. 4 B). Because the anti-Hsp60 antibody did not recognize macrophages Hsp60, the antibody showed specific for bacterial Hsp60 (Fig. 4 B). This Hsp60 and PrPC association was inhibited by the addition of anti-Hsp60 polyclonal antibody, purified Hsp60, or PrPC (Fig. 4, A and B). These results indicated that the interaction between Hsp60 and PrPC would be specific. The precipitated proteins were also analyzed by silver staining. The precipitates contained two major bands (60 and 29 kD) and two weak minor bands (74 and 27 kD; Fig. 4 C). These results suggested that Hsp60 bound to PrPC mostly, but there is possibility that Hsp60 might interact indirectly with PrPC mediated by other cellular components.


Cellular prion protein promotes Brucella infection into macrophages.

Watarai M, Kim S, Erdenebaatar J, Makino S, Horiuchi M, Shirahata T, Sakaguchi S, Katamine S - J. Exp. Med. (2003)

Binding of Hsp60 to PrPC. (A) Demonstration of affinity of Hsp60 for PrPC by pull-down assay with Hsp60-coated beads. Control was assessed with beads only, the addition of anti-Hsp60 antibody, or purified Hsp60. Precipitates were analyzed by immunoblotting with anti-PrPC antibody. (B) Cell lysate or immunoprecipitates with anti-PrPC antibody and affinity of PrPC for Hsp60 by pull-down assay with PrPC-coated beads was analyzed by immunoblotting with anti-Hsp60 antibody. Control was assessed with beads only, or the addition of purified PrPC. (C) Silver staining of precipitates by pull-down assay. Samples were purified with Hsp60 (control) and precipitates of pull-down with Hsp60-coated beads (PD). (D) Labeling of internalized bacteria in macrophages with antibody specific for Hsp60. Macrophages were incubated with B. abortus for 5 or 15 min, and Hsp60 were localized by immunofluorescence as described in Materials and Methods. Fluorescence microscopy of stained GFP-expressed wild-type strain with anti-Hsp60 and phase contrast microscopy of the corresponding microscopic fields are shown. Arrows point to bacteria.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196088&req=5

fig4: Binding of Hsp60 to PrPC. (A) Demonstration of affinity of Hsp60 for PrPC by pull-down assay with Hsp60-coated beads. Control was assessed with beads only, the addition of anti-Hsp60 antibody, or purified Hsp60. Precipitates were analyzed by immunoblotting with anti-PrPC antibody. (B) Cell lysate or immunoprecipitates with anti-PrPC antibody and affinity of PrPC for Hsp60 by pull-down assay with PrPC-coated beads was analyzed by immunoblotting with anti-Hsp60 antibody. Control was assessed with beads only, or the addition of purified PrPC. (C) Silver staining of precipitates by pull-down assay. Samples were purified with Hsp60 (control) and precipitates of pull-down with Hsp60-coated beads (PD). (D) Labeling of internalized bacteria in macrophages with antibody specific for Hsp60. Macrophages were incubated with B. abortus for 5 or 15 min, and Hsp60 were localized by immunofluorescence as described in Materials and Methods. Fluorescence microscopy of stained GFP-expressed wild-type strain with anti-Hsp60 and phase contrast microscopy of the corresponding microscopic fields are shown. Arrows point to bacteria.
Mentions: Because Hsp60 expressed on the bacterial surface by the type IV secretion system was most likely interacting with the target cell, we tested Hsp60 for its ability to bind to PrPC on macrophages by pull-down assay with Hsp60 or PrPC beads. Analysis of the precipitated proteins by immunoblotting with anti-PrPC or Hsp60 antibody showed that a 29-kD PrPC was associated with Hsp60, but not beads alone (Fig. 4, A and B). To confirm this association, Hsp60 was added to macrophage lysate and the proteins in the mixture were then immunoprecipitated with anti-PrPC antibody. The precipitated proteins were analyzed by immunoblotting with anti-Hsp60 antibody. The precipitates contained Hsp60 (Fig. 4 B). Because the anti-Hsp60 antibody did not recognize macrophages Hsp60, the antibody showed specific for bacterial Hsp60 (Fig. 4 B). This Hsp60 and PrPC association was inhibited by the addition of anti-Hsp60 polyclonal antibody, purified Hsp60, or PrPC (Fig. 4, A and B). These results indicated that the interaction between Hsp60 and PrPC would be specific. The precipitated proteins were also analyzed by silver staining. The precipitates contained two major bands (60 and 29 kD) and two weak minor bands (74 and 27 kD; Fig. 4 C). These results suggested that Hsp60 bound to PrPC mostly, but there is possibility that Hsp60 might interact indirectly with PrPC mediated by other cellular components.

Bottom Line: Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex-associated process.Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC.These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro-shi, Hokkaido 080-8555, Japan. watarai@obihiro.ac.jp

ABSTRACT
The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles. The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified. We report here that Hsp60, a member of the GroEL family of chaperonins, of B. abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells. Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B. abortus. Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex-associated process. Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC. Hsp60 of B. abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages. The PrPC deficiency prevented swimming internalization and intracellular replication of B. abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network. These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection.

Show MeSH
Related in: MedlinePlus