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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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VirB complex–dependent surface expression of immunodominant Hsp60. Immunoblot analysis of whole cell lysates with serum from human brucellosis (A) and of purified Hsp60 with indicated serum (B). (C) Labeling of bacteria grown in vitro with antibody specific for Hsp60. Fluorescence microscopy of stained wild-type, virB2, or virB4 mutant, and complemented strain for each mutant, with anti-Hsp60 (top) or anti–B. abortus (middle) and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown. (D) Localization of G6PDH on permeabilized B. abortus by immunofluorescence microscopy. Bacteria were probed with anti-G6PDH (top) and stained for DNA with DAPI (middle) in either the absence (−lysozyme) or the presence (+lysozyme) of lysozyme, and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown.
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fig3: VirB complex–dependent surface expression of immunodominant Hsp60. Immunoblot analysis of whole cell lysates with serum from human brucellosis (A) and of purified Hsp60 with indicated serum (B). (C) Labeling of bacteria grown in vitro with antibody specific for Hsp60. Fluorescence microscopy of stained wild-type, virB2, or virB4 mutant, and complemented strain for each mutant, with anti-Hsp60 (top) or anti–B. abortus (middle) and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown. (D) Localization of G6PDH on permeabilized B. abortus by immunofluorescence microscopy. Bacteria were probed with anti-G6PDH (top) and stained for DNA with DAPI (middle) in either the absence (−lysozyme) or the presence (+lysozyme) of lysozyme, and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown.

Mentions: To investigate bacterial factors associated with PrPC tail formation, immunodominant proteins were examined by immunoblotting with human brucellosis sera, which recognized a major protein (60 kD) and two minor proteins (30∼25 kD; Fig. 3 A). In a previous report (22), immunodominant Hsp60 reacted with sera from mice experimentally infected with B. abortus. Therefore, the 60-kD protein was expected to be Hsp60. To confirm this, purified Hsp60 of B. abortus was analyzed by immunoblotting with sera from human and animal brucellosis. As expected, Hsp60 reacted with serum from human, cattle, and sheep with naturally acquired brucellosis (Fig. 3 B). Mutant strains (ΔvirB2 and ΔvirB4) also had immunoreactive Hsp60 (Fig. 3 A). To examine if Hsp60 was secreted into the external medium, culture supernatant of B. abortus was analyzed by immunoblotting. Immunoreactive proteins were not detected in culture supernatant (unpublished data). However, surface-exposed Hsp60 on wild-type strain, but not virB2 and virB4 mutants, was detected by immunofluorescence staining with anti-Hsp60 antibody (Fig. 3 C). Because introduction of complementing plasmid into each mutant restored surface expression of Hsp60, the expression of Hsp60 on the bacterial surface associates with the type IV secretion system (Fig. 3 C).


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)

VirB complex–dependent surface expression of immunodominant Hsp60. Immunoblot analysis of whole cell lysates with serum from human brucellosis (A) and of purified Hsp60 with indicated serum (B). (C) Labeling of bacteria grown in vitro with antibody specific for Hsp60. Fluorescence microscopy of stained wild-type, virB2, or virB4 mutant, and complemented strain for each mutant, with anti-Hsp60 (top) or anti–B. abortus (middle) and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown. (D) Localization of G6PDH on permeabilized B. abortus by immunofluorescence microscopy. Bacteria were probed with anti-G6PDH (top) and stained for DNA with DAPI (middle) in either the absence (−lysozyme) or the presence (+lysozyme) of lysozyme, and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: VirB complex–dependent surface expression of immunodominant Hsp60. Immunoblot analysis of whole cell lysates with serum from human brucellosis (A) and of purified Hsp60 with indicated serum (B). (C) Labeling of bacteria grown in vitro with antibody specific for Hsp60. Fluorescence microscopy of stained wild-type, virB2, or virB4 mutant, and complemented strain for each mutant, with anti-Hsp60 (top) or anti–B. abortus (middle) and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown. (D) Localization of G6PDH on permeabilized B. abortus by immunofluorescence microscopy. Bacteria were probed with anti-G6PDH (top) and stained for DNA with DAPI (middle) in either the absence (−lysozyme) or the presence (+lysozyme) of lysozyme, and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown.
Mentions: To investigate bacterial factors associated with PrPC tail formation, immunodominant proteins were examined by immunoblotting with human brucellosis sera, which recognized a major protein (60 kD) and two minor proteins (30∼25 kD; Fig. 3 A). In a previous report (22), immunodominant Hsp60 reacted with sera from mice experimentally infected with B. abortus. Therefore, the 60-kD protein was expected to be Hsp60. To confirm this, purified Hsp60 of B. abortus was analyzed by immunoblotting with sera from human and animal brucellosis. As expected, Hsp60 reacted with serum from human, cattle, and sheep with naturally acquired brucellosis (Fig. 3 B). Mutant strains (ΔvirB2 and ΔvirB4) also had immunoreactive Hsp60 (Fig. 3 A). To examine if Hsp60 was secreted into the external medium, culture supernatant of B. abortus was analyzed by immunoblotting. Immunoreactive proteins were not detected in culture supernatant (unpublished data). However, surface-exposed Hsp60 on wild-type strain, but not virB2 and virB4 mutants, was detected by immunofluorescence staining with anti-Hsp60 antibody (Fig. 3 C). Because introduction of complementing plasmid into each mutant restored surface expression of Hsp60, the expression of Hsp60 on the bacterial surface associates with the type IV secretion system (Fig. 3 C).

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