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Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum.

Celli J, de Chastellier C, Franchini DM, Pizarro-Cerda J, Moreno E, Gorvel JP - J. Exp. Med. (2003)

Bottom Line: The acquisition of ER membranes by replicating Brucella is independent of ER-Golgi COPI-dependent vesicular transport.A mutant of the VirB type IV secretion system, which is necessary for intracellular survival, was unable to sustain interactions and fuse with the ER, and was killed via eventual fusion with lysosomes.Moreover, we assign an intracellular function to the VirB system, as being required for late maturation events necessary for the biogenesis of an ER-derived replicative organelle.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie INSERM-CNRS-Université Mediterranée de Marseille-Luminy, 13288 Marseille cedex 09, France.

ABSTRACT
The intracellular pathogen Brucella is the causative agent of brucellosis, a worldwide zoonosis that affects mammals, including humans. Essential to Brucella virulence is its ability to survive and replicate inside host macrophages, yet the underlying mechanisms and the nature of the replicative compartment remain unclear. Here we show in a model of Brucella abortus infection of murine bone marrow-derived macrophages that a fraction of the bacteria that survive an initial macrophage killing proceed to replicate in a compartment segregated from the endocytic pathway. The maturation of the Brucella-containing vacuole involves sustained interactions and fusion with the endoplasmic reticulum (ER), which creates a replicative compartment with ER-like properties. The acquisition of ER membranes by replicating Brucella is independent of ER-Golgi COPI-dependent vesicular transport. A mutant of the VirB type IV secretion system, which is necessary for intracellular survival, was unable to sustain interactions and fuse with the ER, and was killed via eventual fusion with lysosomes. Thus, we demonstrate that live intracellular Brucella evade macrophage killing through VirB-dependent sustained interactions with the ER. Moreover, we assign an intracellular function to the VirB system, as being required for late maturation events necessary for the biogenesis of an ER-derived replicative organelle.

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VirB mutant–containing vacuoles fail to sustain fusion-proficient interactions with the ER. BMDM were infected with either the wild-type Brucella strain GFP-2308 or the mutant strain GFP-virB10 for various times. Samples were processed for immunofluorescence (A–D) or EM staining for G6Pase (E–G). (A) Confocal images of infected BMDM showing acquisition of LAMP-1 on 2308 or virB10-containing vacuoles after 4 or 24 h of infection. (B) Quantitation of LAMP-1 acquisition by 2308 (○)- or virB10 (□)-containing vacuoles. Data are means ± SD of three independent experiments. (C) Confocal images of GFP Brucella–infected BMDM showing association with, or acquisition of, Sec61β+ structures on 2308 or virB10-containing vacuoles after 4 or 24 h of infection. (D) Quantitation of association of Sec61β1 structures with 2308 (○)- or virB10 (□)-containing vacuoles. Data are means ± SD of five independent experiments. (E) Staining for G6Pase in virB10-infected BMDM at 2 h after infection. The arrowhead shows a close association of BCVs with ER. (F) Staining for G6Pase in virB10-infected BMDM at 4 h after infection. The BCV is surrounded by ER (arrowheads). (G) Staining for G6Pase in virB10-infected BMDM at 8 h after infection Arrowheads show that the ER is no longer in the close vicinity of the BCV. G6Pase reaction product is not detectable inside the BCV. Bars, 5 μm (A and C), 1 μm (insets in A and C), and 0.5 μm (E–G).
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fig8: VirB mutant–containing vacuoles fail to sustain fusion-proficient interactions with the ER. BMDM were infected with either the wild-type Brucella strain GFP-2308 or the mutant strain GFP-virB10 for various times. Samples were processed for immunofluorescence (A–D) or EM staining for G6Pase (E–G). (A) Confocal images of infected BMDM showing acquisition of LAMP-1 on 2308 or virB10-containing vacuoles after 4 or 24 h of infection. (B) Quantitation of LAMP-1 acquisition by 2308 (○)- or virB10 (□)-containing vacuoles. Data are means ± SD of three independent experiments. (C) Confocal images of GFP Brucella–infected BMDM showing association with, or acquisition of, Sec61β+ structures on 2308 or virB10-containing vacuoles after 4 or 24 h of infection. (D) Quantitation of association of Sec61β1 structures with 2308 (○)- or virB10 (□)-containing vacuoles. Data are means ± SD of five independent experiments. (E) Staining for G6Pase in virB10-infected BMDM at 2 h after infection. The arrowhead shows a close association of BCVs with ER. (F) Staining for G6Pase in virB10-infected BMDM at 4 h after infection. The BCV is surrounded by ER (arrowheads). (G) Staining for G6Pase in virB10-infected BMDM at 8 h after infection Arrowheads show that the ER is no longer in the close vicinity of the BCV. G6Pase reaction product is not detectable inside the BCV. Bars, 5 μm (A and C), 1 μm (insets in A and C), and 0.5 μm (E–G).

Mentions: Our previous results (Fig. 1) suggested a role for VirB in BCV late maturation events because the intracellular fate of wild-type and virB10 Brucella diverged after 4 h after infection. To clearly determine a role for VirB in BCV maturation in BMDM, we examined the vacuole maturation of the surviving GFP-expressing virB10 Brucella and compared their interactions with the ER to those of the wild-type GFP-2308. Using LAMP-1 as a marker of BCV maturation (Fig. 2 C), we observed that the majority of vacuoles containing either strain were LAMP-1+ until 4 h after infection (Fig. 8, A and B) . However, although the percentage of LAMP-1+ wild-type BCVs decreased after 4 h after infection, vacuoles containing the virB10 mutant remained positive (Fig. 8, A and B), as previously described in epithelial cells (18). This indicates that virB mutants are deficient in controlling vacuole maturation in BMDM after 4 h after infection.


Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum.

Celli J, de Chastellier C, Franchini DM, Pizarro-Cerda J, Moreno E, Gorvel JP - J. Exp. Med. (2003)

VirB mutant–containing vacuoles fail to sustain fusion-proficient interactions with the ER. BMDM were infected with either the wild-type Brucella strain GFP-2308 or the mutant strain GFP-virB10 for various times. Samples were processed for immunofluorescence (A–D) or EM staining for G6Pase (E–G). (A) Confocal images of infected BMDM showing acquisition of LAMP-1 on 2308 or virB10-containing vacuoles after 4 or 24 h of infection. (B) Quantitation of LAMP-1 acquisition by 2308 (○)- or virB10 (□)-containing vacuoles. Data are means ± SD of three independent experiments. (C) Confocal images of GFP Brucella–infected BMDM showing association with, or acquisition of, Sec61β+ structures on 2308 or virB10-containing vacuoles after 4 or 24 h of infection. (D) Quantitation of association of Sec61β1 structures with 2308 (○)- or virB10 (□)-containing vacuoles. Data are means ± SD of five independent experiments. (E) Staining for G6Pase in virB10-infected BMDM at 2 h after infection. The arrowhead shows a close association of BCVs with ER. (F) Staining for G6Pase in virB10-infected BMDM at 4 h after infection. The BCV is surrounded by ER (arrowheads). (G) Staining for G6Pase in virB10-infected BMDM at 8 h after infection Arrowheads show that the ER is no longer in the close vicinity of the BCV. G6Pase reaction product is not detectable inside the BCV. Bars, 5 μm (A and C), 1 μm (insets in A and C), and 0.5 μm (E–G).
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fig8: VirB mutant–containing vacuoles fail to sustain fusion-proficient interactions with the ER. BMDM were infected with either the wild-type Brucella strain GFP-2308 or the mutant strain GFP-virB10 for various times. Samples were processed for immunofluorescence (A–D) or EM staining for G6Pase (E–G). (A) Confocal images of infected BMDM showing acquisition of LAMP-1 on 2308 or virB10-containing vacuoles after 4 or 24 h of infection. (B) Quantitation of LAMP-1 acquisition by 2308 (○)- or virB10 (□)-containing vacuoles. Data are means ± SD of three independent experiments. (C) Confocal images of GFP Brucella–infected BMDM showing association with, or acquisition of, Sec61β+ structures on 2308 or virB10-containing vacuoles after 4 or 24 h of infection. (D) Quantitation of association of Sec61β1 structures with 2308 (○)- or virB10 (□)-containing vacuoles. Data are means ± SD of five independent experiments. (E) Staining for G6Pase in virB10-infected BMDM at 2 h after infection. The arrowhead shows a close association of BCVs with ER. (F) Staining for G6Pase in virB10-infected BMDM at 4 h after infection. The BCV is surrounded by ER (arrowheads). (G) Staining for G6Pase in virB10-infected BMDM at 8 h after infection Arrowheads show that the ER is no longer in the close vicinity of the BCV. G6Pase reaction product is not detectable inside the BCV. Bars, 5 μm (A and C), 1 μm (insets in A and C), and 0.5 μm (E–G).
Mentions: Our previous results (Fig. 1) suggested a role for VirB in BCV late maturation events because the intracellular fate of wild-type and virB10 Brucella diverged after 4 h after infection. To clearly determine a role for VirB in BCV maturation in BMDM, we examined the vacuole maturation of the surviving GFP-expressing virB10 Brucella and compared their interactions with the ER to those of the wild-type GFP-2308. Using LAMP-1 as a marker of BCV maturation (Fig. 2 C), we observed that the majority of vacuoles containing either strain were LAMP-1+ until 4 h after infection (Fig. 8, A and B) . However, although the percentage of LAMP-1+ wild-type BCVs decreased after 4 h after infection, vacuoles containing the virB10 mutant remained positive (Fig. 8, A and B), as previously described in epithelial cells (18). This indicates that virB mutants are deficient in controlling vacuole maturation in BMDM after 4 h after infection.

Bottom Line: The acquisition of ER membranes by replicating Brucella is independent of ER-Golgi COPI-dependent vesicular transport.A mutant of the VirB type IV secretion system, which is necessary for intracellular survival, was unable to sustain interactions and fuse with the ER, and was killed via eventual fusion with lysosomes.Moreover, we assign an intracellular function to the VirB system, as being required for late maturation events necessary for the biogenesis of an ER-derived replicative organelle.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie INSERM-CNRS-Université Mediterranée de Marseille-Luminy, 13288 Marseille cedex 09, France.

ABSTRACT
The intracellular pathogen Brucella is the causative agent of brucellosis, a worldwide zoonosis that affects mammals, including humans. Essential to Brucella virulence is its ability to survive and replicate inside host macrophages, yet the underlying mechanisms and the nature of the replicative compartment remain unclear. Here we show in a model of Brucella abortus infection of murine bone marrow-derived macrophages that a fraction of the bacteria that survive an initial macrophage killing proceed to replicate in a compartment segregated from the endocytic pathway. The maturation of the Brucella-containing vacuole involves sustained interactions and fusion with the endoplasmic reticulum (ER), which creates a replicative compartment with ER-like properties. The acquisition of ER membranes by replicating Brucella is independent of ER-Golgi COPI-dependent vesicular transport. A mutant of the VirB type IV secretion system, which is necessary for intracellular survival, was unable to sustain interactions and fuse with the ER, and was killed via eventual fusion with lysosomes. Thus, we demonstrate that live intracellular Brucella evade macrophage killing through VirB-dependent sustained interactions with the ER. Moreover, we assign an intracellular function to the VirB system, as being required for late maturation events necessary for the biogenesis of an ER-derived replicative organelle.

Show MeSH
Related in: MedlinePlus