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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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PrPC-influenced B. abortus infection. (A–F) Wild-type (solid bars) or virB4 mutant (open bars) were deposited onto macro-phages from normal (A–C) or Ngsk PrPC-deficient mice (D–F), and then incubated for the periods of time indicated. Uptake (A, C, D, and F) and macropinosome formation (B and E) were quantified as described in Materials and Methods. (A, B, D, and E) 100 macrophages were examined per coverslip. (C and F) Uptake efficiency by macrophages was determined by protection of internalized bacteria from gentamicin killing. Data are the average of triplicate samples from three identical experiments, and the error bars represent the standard deviation. (G and H) Intracellular replication of B. abortus. Macrophages from BALB/c, C57BL/6, Ngsk, or Zrch PrPC-deficient mice, or PrPC transgenic Ngsk PrPC-deficient mice were infected with wild-type B. abortus (G), or virB4 mutant (virB4−) and complemented strain (virB4+). (H). Data points and error bars represent the mean CFUs of triplicate samples from a typical experiment (performed at least four times) and their standard deviation, respectively. (I) Proliferation in mice. The CFUs of each strain were enumerated in the spleens of five mice from each group at 10 d after infection. For each mouse, the results are indicated by one open circle (log CFU of the wild-type) and one solid circle (log CFU of the virB4 mutant). The means of the data are indicated by horizontal lines. The competitive index was calculated by dividing the mean ratio of mutant CFUs to the wild-type CFUs recovered from spleens by the ratio of the mutant CFUs to the wild-type CFUs in the inoculum.
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fig7: PrPC-influenced B. abortus infection. (A–F) Wild-type (solid bars) or virB4 mutant (open bars) were deposited onto macro-phages from normal (A–C) or Ngsk PrPC-deficient mice (D–F), and then incubated for the periods of time indicated. Uptake (A, C, D, and F) and macropinosome formation (B and E) were quantified as described in Materials and Methods. (A, B, D, and E) 100 macrophages were examined per coverslip. (C and F) Uptake efficiency by macrophages was determined by protection of internalized bacteria from gentamicin killing. Data are the average of triplicate samples from three identical experiments, and the error bars represent the standard deviation. (G and H) Intracellular replication of B. abortus. Macrophages from BALB/c, C57BL/6, Ngsk, or Zrch PrPC-deficient mice, or PrPC transgenic Ngsk PrPC-deficient mice were infected with wild-type B. abortus (G), or virB4 mutant (virB4−) and complemented strain (virB4+). (H). Data points and error bars represent the mean CFUs of triplicate samples from a typical experiment (performed at least four times) and their standard deviation, respectively. (I) Proliferation in mice. The CFUs of each strain were enumerated in the spleens of five mice from each group at 10 d after infection. For each mouse, the results are indicated by one open circle (log CFU of the wild-type) and one solid circle (log CFU of the virB4 mutant). The means of the data are indicated by horizontal lines. The competitive index was calculated by dividing the mean ratio of mutant CFUs to the wild-type CFUs recovered from spleens by the ratio of the mutant CFUs to the wild-type CFUs in the inoculum.

Mentions: The differences in rate of phagocytosis and macropinosome formation for parent or Ngsk PrPC-deficient mice were quantitated microscopically at various times of incubation. The kinetics of bacterial internalization and macropinosome formation in macrophages from parent C57BL/6 mice were almost identical to those observed for macrophages from BALB/c mice (Figs. 2, B and C, and 7, A–C). Internalization of wild-type B. abortus into macrophages from Ngsk PrPC-deficient mice, in contrast, was much quicker and macropinosome formation was hardly detectable (Fig. 7, D–F). The internalized wild-type strain did not replicate in the macrophages from Ngsk PrPC-deficient mice (Fig. 7 G). Macrophages from parent and Ngsk PrPC-deficient mice showed no significant difference in the internalization, macropinosome formation, and intracellular replication of virB4 mutant (Fig. 7, A–F and H). In macrophages from Ngsk PrPC-deficient mice, wild-type strain failed to block phagosome maturation as shown by colocalization of phagosomes containing the bacteria and the late endocytic marker, LAMP-1, at 35 min after infection (Fig. 8, A and C). In contrast, wild-type strain prevented phagosome-lysosome fusion, and therefore phagosomes containing wild-type strain do not have LAMP-1 in macrophages from parent mice (Fig. 8, A and B).


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)

PrPC-influenced B. abortus infection. (A–F) Wild-type (solid bars) or virB4 mutant (open bars) were deposited onto macro-phages from normal (A–C) or Ngsk PrPC-deficient mice (D–F), and then incubated for the periods of time indicated. Uptake (A, C, D, and F) and macropinosome formation (B and E) were quantified as described in Materials and Methods. (A, B, D, and E) 100 macrophages were examined per coverslip. (C and F) Uptake efficiency by macrophages was determined by protection of internalized bacteria from gentamicin killing. Data are the average of triplicate samples from three identical experiments, and the error bars represent the standard deviation. (G and H) Intracellular replication of B. abortus. Macrophages from BALB/c, C57BL/6, Ngsk, or Zrch PrPC-deficient mice, or PrPC transgenic Ngsk PrPC-deficient mice were infected with wild-type B. abortus (G), or virB4 mutant (virB4−) and complemented strain (virB4+). (H). Data points and error bars represent the mean CFUs of triplicate samples from a typical experiment (performed at least four times) and their standard deviation, respectively. (I) Proliferation in mice. The CFUs of each strain were enumerated in the spleens of five mice from each group at 10 d after infection. For each mouse, the results are indicated by one open circle (log CFU of the wild-type) and one solid circle (log CFU of the virB4 mutant). The means of the data are indicated by horizontal lines. The competitive index was calculated by dividing the mean ratio of mutant CFUs to the wild-type CFUs recovered from spleens by the ratio of the mutant CFUs to the wild-type CFUs in the inoculum.
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fig7: PrPC-influenced B. abortus infection. (A–F) Wild-type (solid bars) or virB4 mutant (open bars) were deposited onto macro-phages from normal (A–C) or Ngsk PrPC-deficient mice (D–F), and then incubated for the periods of time indicated. Uptake (A, C, D, and F) and macropinosome formation (B and E) were quantified as described in Materials and Methods. (A, B, D, and E) 100 macrophages were examined per coverslip. (C and F) Uptake efficiency by macrophages was determined by protection of internalized bacteria from gentamicin killing. Data are the average of triplicate samples from three identical experiments, and the error bars represent the standard deviation. (G and H) Intracellular replication of B. abortus. Macrophages from BALB/c, C57BL/6, Ngsk, or Zrch PrPC-deficient mice, or PrPC transgenic Ngsk PrPC-deficient mice were infected with wild-type B. abortus (G), or virB4 mutant (virB4−) and complemented strain (virB4+). (H). Data points and error bars represent the mean CFUs of triplicate samples from a typical experiment (performed at least four times) and their standard deviation, respectively. (I) Proliferation in mice. The CFUs of each strain were enumerated in the spleens of five mice from each group at 10 d after infection. For each mouse, the results are indicated by one open circle (log CFU of the wild-type) and one solid circle (log CFU of the virB4 mutant). The means of the data are indicated by horizontal lines. The competitive index was calculated by dividing the mean ratio of mutant CFUs to the wild-type CFUs recovered from spleens by the ratio of the mutant CFUs to the wild-type CFUs in the inoculum.
Mentions: The differences in rate of phagocytosis and macropinosome formation for parent or Ngsk PrPC-deficient mice were quantitated microscopically at various times of incubation. The kinetics of bacterial internalization and macropinosome formation in macrophages from parent C57BL/6 mice were almost identical to those observed for macrophages from BALB/c mice (Figs. 2, B and C, and 7, A–C). Internalization of wild-type B. abortus into macrophages from Ngsk PrPC-deficient mice, in contrast, was much quicker and macropinosome formation was hardly detectable (Fig. 7, D–F). The internalized wild-type strain did not replicate in the macrophages from Ngsk PrPC-deficient mice (Fig. 7 G). Macrophages from parent and Ngsk PrPC-deficient mice showed no significant difference in the internalization, macropinosome formation, and intracellular replication of virB4 mutant (Fig. 7, A–F and H). In macrophages from Ngsk PrPC-deficient mice, wild-type strain failed to block phagosome maturation as shown by colocalization of phagosomes containing the bacteria and the late endocytic marker, LAMP-1, at 35 min after infection (Fig. 8, A and C). In contrast, wild-type strain prevented phagosome-lysosome fusion, and therefore phagosomes containing wild-type strain do not have LAMP-1 in macrophages from parent mice (Fig. 8, A and B).

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