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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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Aggregation of PrPC by Hsp60 expressed on the surface of L. lactis. Macrophages were incubated with surface Hsp60+ (top) or Hsp60− (bottom) L. lactis for 5 min, and PrPC was localized by immunofluorescence as described in Materials and Methods. Phase contrast microscopy of the corresponding microscopic fields are shown. Bacteria (shown by arrows) were stained with DAPI. (B) Labeling of L. lactis grown in vitro, with antibody specific for Hsp60. Fluorescence microscopy of stained surface Hsp60+ or Hsp60− L. lactis with anti-Hsp60 (top) or DAPI (middle) and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown. (C) PrPC-binding activity. Measurement of PrPC-binding activity was performed by ELISA (refer to Materials and Methods). (D and E) Macrophages were incubated with surface Hsp60+ (solid bars) or Hsp60− (open bars) L. lactis for the indicated time, and association of PrPC was determined by immunofluorescence microscopy. Hsp60 of B. abortus (D) or E. coli (E) is expressing on L. lactis surface. % PrPC positive refers to percentage of bacteria that showed costaining with PrPC. 100 bacteria were examined per coverslip. Data are the average of triplicate samples from three identical experiments, and the error bars represent the standard deviation.
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fig5: Aggregation of PrPC by Hsp60 expressed on the surface of L. lactis. Macrophages were incubated with surface Hsp60+ (top) or Hsp60− (bottom) L. lactis for 5 min, and PrPC was localized by immunofluorescence as described in Materials and Methods. Phase contrast microscopy of the corresponding microscopic fields are shown. Bacteria (shown by arrows) were stained with DAPI. (B) Labeling of L. lactis grown in vitro, with antibody specific for Hsp60. Fluorescence microscopy of stained surface Hsp60+ or Hsp60− L. lactis with anti-Hsp60 (top) or DAPI (middle) and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown. (C) PrPC-binding activity. Measurement of PrPC-binding activity was performed by ELISA (refer to Materials and Methods). (D and E) Macrophages were incubated with surface Hsp60+ (solid bars) or Hsp60− (open bars) L. lactis for the indicated time, and association of PrPC was determined by immunofluorescence microscopy. Hsp60 of B. abortus (D) or E. coli (E) is expressing on L. lactis surface. % PrPC positive refers to percentage of bacteria that showed costaining with PrPC. 100 bacteria were examined per coverslip. Data are the average of triplicate samples from three identical experiments, and the error bars represent the standard deviation.

Mentions: To investigate if Hsp60 exposed on bacterial surface could aggregate PrPC on macrophages, macrophages were infected with L. lactis expressing Hsp60 of B. abortus on its surface (Fig. 5 B), and then PrPC was detected by immunofluorescence microscopy. After 5 min incubation, PrPC accumulated around internalized Hsp60+ L. lactis but not Hsp60− L. lactis (Fig. 5 A). Quantitative data showed that >70% of L. lactis expressing Hsp60 colocalized with PrPC at all time points (Fig. 5 D). PrPC tail formation was not observed with either Hsp60+ or Hsp60− L. lactis. L. lactis was seeded on the wells of a microtiter plate, macrophage lysate was added, and then binding activity was measured by ELISA with anti-PrPC antibody. The binding of PrPC to Hsp60 on the L. lactis surface was detected but not with Hsp60− L. lactis (Fig. 5 C). L. lactis expressing Hsp60 of E. coli also colocalized with PrPC at all time points, but the percentage of colocalization was lower than Hsp60 of B. abortus (Fig. 5, C–E). These results suggested that Hsp60 expressed on the bacterial surface promoted accumulation of PrPC, but is not sufficient for PrPC tail formation.


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)

Aggregation of PrPC by Hsp60 expressed on the surface of L. lactis. Macrophages were incubated with surface Hsp60+ (top) or Hsp60− (bottom) L. lactis for 5 min, and PrPC was localized by immunofluorescence as described in Materials and Methods. Phase contrast microscopy of the corresponding microscopic fields are shown. Bacteria (shown by arrows) were stained with DAPI. (B) Labeling of L. lactis grown in vitro, with antibody specific for Hsp60. Fluorescence microscopy of stained surface Hsp60+ or Hsp60− L. lactis with anti-Hsp60 (top) or DAPI (middle) and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown. (C) PrPC-binding activity. Measurement of PrPC-binding activity was performed by ELISA (refer to Materials and Methods). (D and E) Macrophages were incubated with surface Hsp60+ (solid bars) or Hsp60− (open bars) L. lactis for the indicated time, and association of PrPC was determined by immunofluorescence microscopy. Hsp60 of B. abortus (D) or E. coli (E) is expressing on L. lactis surface. % PrPC positive refers to percentage of bacteria that showed costaining with PrPC. 100 bacteria were examined per coverslip. Data are the average of triplicate samples from three identical experiments, and the error bars represent the standard deviation.
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Related In: Results  -  Collection

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fig5: Aggregation of PrPC by Hsp60 expressed on the surface of L. lactis. Macrophages were incubated with surface Hsp60+ (top) or Hsp60− (bottom) L. lactis for 5 min, and PrPC was localized by immunofluorescence as described in Materials and Methods. Phase contrast microscopy of the corresponding microscopic fields are shown. Bacteria (shown by arrows) were stained with DAPI. (B) Labeling of L. lactis grown in vitro, with antibody specific for Hsp60. Fluorescence microscopy of stained surface Hsp60+ or Hsp60− L. lactis with anti-Hsp60 (top) or DAPI (middle) and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown. (C) PrPC-binding activity. Measurement of PrPC-binding activity was performed by ELISA (refer to Materials and Methods). (D and E) Macrophages were incubated with surface Hsp60+ (solid bars) or Hsp60− (open bars) L. lactis for the indicated time, and association of PrPC was determined by immunofluorescence microscopy. Hsp60 of B. abortus (D) or E. coli (E) is expressing on L. lactis surface. % PrPC positive refers to percentage of bacteria that showed costaining with PrPC. 100 bacteria were examined per coverslip. Data are the average of triplicate samples from three identical experiments, and the error bars represent the standard deviation.
Mentions: To investigate if Hsp60 exposed on bacterial surface could aggregate PrPC on macrophages, macrophages were infected with L. lactis expressing Hsp60 of B. abortus on its surface (Fig. 5 B), and then PrPC was detected by immunofluorescence microscopy. After 5 min incubation, PrPC accumulated around internalized Hsp60+ L. lactis but not Hsp60− L. lactis (Fig. 5 A). Quantitative data showed that >70% of L. lactis expressing Hsp60 colocalized with PrPC at all time points (Fig. 5 D). PrPC tail formation was not observed with either Hsp60+ or Hsp60− L. lactis. L. lactis was seeded on the wells of a microtiter plate, macrophage lysate was added, and then binding activity was measured by ELISA with anti-PrPC antibody. The binding of PrPC to Hsp60 on the L. lactis surface was detected but not with Hsp60− L. lactis (Fig. 5 C). L. lactis expressing Hsp60 of E. coli also colocalized with PrPC at all time points, but the percentage of colocalization was lower than Hsp60 of B. abortus (Fig. 5, C–E). These results suggested that Hsp60 expressed on the bacterial surface promoted accumulation of PrPC, but is not sufficient for PrPC tail formation.

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