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A Macrophage Subversion Factor Is Shared by Intracellular and Extracellular Pathogens.

Belon C, Soscia C, Bernut A, Laubier A, Bleves S, Blanc-Potard AB - PLoS Pathog. (2015)

Bottom Line: MgtC is also found in pathogenic Pseudomonas species.Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role.In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus highlighting MgtC as a promising new target for anti-virulence strategies.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université de Montpellier, CNRS-UMR5235, Montpellier, France.

ABSTRACT
Pathogenic bacteria have developed strategies to adapt to host environment and resist host immune response. Several intracellular bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis, share the horizontally-acquired MgtC virulence factor that is important for multiplication inside macrophages. MgtC is also found in pathogenic Pseudomonas species. Here we investigate for the first time the role of MgtC in the virulence of an extracellular pathogen, Pseudomonas aeruginosa. A P. aeruginosa mgtC mutant is attenuated in the systemic infection model of zebrafish embryos, and strikingly, the attenuated phenotype is dependent on the presence of macrophages. In ex vivo experiments, the P. aeruginosa mgtC mutant is more sensitive to macrophage killing than the wild-type strain. However, wild-type and mutant strains behave similarly toward macrophage killing when macrophages are treated with an inhibitor of the vacuolar proton ATPase. Importantly, P. aeruginosa mgtC gene expression is strongly induced within macrophages and phagosome acidification contributes to an optimal expression of the gene. Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC shares a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to host immune response in relation to the different bacterial lifestyles. In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus highlighting MgtC as a promising new target for anti-virulence strategies.

No MeSH data available.


Related in: MedlinePlus

Infection of zebrafish embryos with the P. aeruginosa mgtC mutant.(A) Diagram of 30 hours post-fertilization (hpf) zebrafish embryo showing the injection site used in this study (arrow). All injections were done in the caudal vein (CV) just behind to the urogenital opening (UGO). Y: Yolk, CHT: Caudal Hematopoietic Tissue. Scale bar, 100 μm. (B) Survival curves of embryos infected with PAO1 wild-type stain or PAO1 ΔmgtC mutant and PBS-injected (control). Approximately 1200–1400 CFU P. aeruginosa were microinjected into the caudal vein (n = 20 per group). Results are expressed as the percentage of surviving on each hour post-infection. Representative results of three biologically independent replicates are shown. Embryos are significantly more susceptible to infection with PAO1 wild-type than ΔmgtC mutant (P<0.001). (C) 30 hpf embryons are intravenously infected with PAO1 or ΔmgtC mutant expressing mCherry. Representative fluorescence microscopy images of 18 hpi embryos infected with PAO1 (top panel) or PAO1 ΔmgtC mutant (bottow panel) are shown (1200–1400 CFU). Scale bar, 200 μm. (D) 30 hpf zebrafish embryos are intravenously infected with mCherry-expressing PAO1 (left panel) or PAO1 ΔmgtC mutant (right panel) and imaged by confocal microscopy. Arrows indicate maximum intensity projection of macrophages that phagocytose bacteria close to the site of injection at 1 hpi. Scale bar, 10 μm. (E) Survival curves of pu.1 morphant (pu.1 mo) embryos (n = 20 each) infected with 900 CFU of PAO1 wild type stain or PAO1 ΔmgtC mutant compared to the PBS- injected control. Representative results of three biologically independent replicates are shown. No statistically significant difference is obtained between wild type-infected embryos and ΔmgtC mutant-infected embryos (ns: non significant).
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ppat.1004969.g002: Infection of zebrafish embryos with the P. aeruginosa mgtC mutant.(A) Diagram of 30 hours post-fertilization (hpf) zebrafish embryo showing the injection site used in this study (arrow). All injections were done in the caudal vein (CV) just behind to the urogenital opening (UGO). Y: Yolk, CHT: Caudal Hematopoietic Tissue. Scale bar, 100 μm. (B) Survival curves of embryos infected with PAO1 wild-type stain or PAO1 ΔmgtC mutant and PBS-injected (control). Approximately 1200–1400 CFU P. aeruginosa were microinjected into the caudal vein (n = 20 per group). Results are expressed as the percentage of surviving on each hour post-infection. Representative results of three biologically independent replicates are shown. Embryos are significantly more susceptible to infection with PAO1 wild-type than ΔmgtC mutant (P<0.001). (C) 30 hpf embryons are intravenously infected with PAO1 or ΔmgtC mutant expressing mCherry. Representative fluorescence microscopy images of 18 hpi embryos infected with PAO1 (top panel) or PAO1 ΔmgtC mutant (bottow panel) are shown (1200–1400 CFU). Scale bar, 200 μm. (D) 30 hpf zebrafish embryos are intravenously infected with mCherry-expressing PAO1 (left panel) or PAO1 ΔmgtC mutant (right panel) and imaged by confocal microscopy. Arrows indicate maximum intensity projection of macrophages that phagocytose bacteria close to the site of injection at 1 hpi. Scale bar, 10 μm. (E) Survival curves of pu.1 morphant (pu.1 mo) embryos (n = 20 each) infected with 900 CFU of PAO1 wild type stain or PAO1 ΔmgtC mutant compared to the PBS- injected control. Representative results of three biologically independent replicates are shown. No statistically significant difference is obtained between wild type-infected embryos and ΔmgtC mutant-infected embryos (ns: non significant).

Mentions: To evaluate the role of MgtC in P. aeruginosa virulence, we used the zebrafish (Danio rerio) embryo model. This model, which has been used for various intracellular and extracellular bacterial pathogens, is a model of choice to investigate the contribution of cells from the innate immune system during infection [25]. The Danio has been successfully used to monitor the role of P. aeruginosa determinants in virulence based on survival curves of larvae infected with mutants deficient in type III secretion system (T3SS) or Quorum Sensing [20,26]. Bacteria producing a fluorescent protein were injected intravenously in the caudal vein of embryos (Fig 2A) at 30 hours post-fertilization, a time where macrophages, but not neutrophils, are fully functional and capable of engulfing invading bacteria [27]. The survival curves of infected embryos indicated that MgtC is a critical virulence determinant in this model since the mgtC mutant is significantly attenuated as compared to the wild-type PAO1 strain (Fig 2B). In agreement, fluorescence microscopy of 18 hpi infected embryos showed a lower bacterial burden with PAO1 strain than mgtC mutant (Fig 2C).


A Macrophage Subversion Factor Is Shared by Intracellular and Extracellular Pathogens.

Belon C, Soscia C, Bernut A, Laubier A, Bleves S, Blanc-Potard AB - PLoS Pathog. (2015)

Infection of zebrafish embryos with the P. aeruginosa mgtC mutant.(A) Diagram of 30 hours post-fertilization (hpf) zebrafish embryo showing the injection site used in this study (arrow). All injections were done in the caudal vein (CV) just behind to the urogenital opening (UGO). Y: Yolk, CHT: Caudal Hematopoietic Tissue. Scale bar, 100 μm. (B) Survival curves of embryos infected with PAO1 wild-type stain or PAO1 ΔmgtC mutant and PBS-injected (control). Approximately 1200–1400 CFU P. aeruginosa were microinjected into the caudal vein (n = 20 per group). Results are expressed as the percentage of surviving on each hour post-infection. Representative results of three biologically independent replicates are shown. Embryos are significantly more susceptible to infection with PAO1 wild-type than ΔmgtC mutant (P<0.001). (C) 30 hpf embryons are intravenously infected with PAO1 or ΔmgtC mutant expressing mCherry. Representative fluorescence microscopy images of 18 hpi embryos infected with PAO1 (top panel) or PAO1 ΔmgtC mutant (bottow panel) are shown (1200–1400 CFU). Scale bar, 200 μm. (D) 30 hpf zebrafish embryos are intravenously infected with mCherry-expressing PAO1 (left panel) or PAO1 ΔmgtC mutant (right panel) and imaged by confocal microscopy. Arrows indicate maximum intensity projection of macrophages that phagocytose bacteria close to the site of injection at 1 hpi. Scale bar, 10 μm. (E) Survival curves of pu.1 morphant (pu.1 mo) embryos (n = 20 each) infected with 900 CFU of PAO1 wild type stain or PAO1 ΔmgtC mutant compared to the PBS- injected control. Representative results of three biologically independent replicates are shown. No statistically significant difference is obtained between wild type-infected embryos and ΔmgtC mutant-infected embryos (ns: non significant).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4469704&req=5

ppat.1004969.g002: Infection of zebrafish embryos with the P. aeruginosa mgtC mutant.(A) Diagram of 30 hours post-fertilization (hpf) zebrafish embryo showing the injection site used in this study (arrow). All injections were done in the caudal vein (CV) just behind to the urogenital opening (UGO). Y: Yolk, CHT: Caudal Hematopoietic Tissue. Scale bar, 100 μm. (B) Survival curves of embryos infected with PAO1 wild-type stain or PAO1 ΔmgtC mutant and PBS-injected (control). Approximately 1200–1400 CFU P. aeruginosa were microinjected into the caudal vein (n = 20 per group). Results are expressed as the percentage of surviving on each hour post-infection. Representative results of three biologically independent replicates are shown. Embryos are significantly more susceptible to infection with PAO1 wild-type than ΔmgtC mutant (P<0.001). (C) 30 hpf embryons are intravenously infected with PAO1 or ΔmgtC mutant expressing mCherry. Representative fluorescence microscopy images of 18 hpi embryos infected with PAO1 (top panel) or PAO1 ΔmgtC mutant (bottow panel) are shown (1200–1400 CFU). Scale bar, 200 μm. (D) 30 hpf zebrafish embryos are intravenously infected with mCherry-expressing PAO1 (left panel) or PAO1 ΔmgtC mutant (right panel) and imaged by confocal microscopy. Arrows indicate maximum intensity projection of macrophages that phagocytose bacteria close to the site of injection at 1 hpi. Scale bar, 10 μm. (E) Survival curves of pu.1 morphant (pu.1 mo) embryos (n = 20 each) infected with 900 CFU of PAO1 wild type stain or PAO1 ΔmgtC mutant compared to the PBS- injected control. Representative results of three biologically independent replicates are shown. No statistically significant difference is obtained between wild type-infected embryos and ΔmgtC mutant-infected embryos (ns: non significant).
Mentions: To evaluate the role of MgtC in P. aeruginosa virulence, we used the zebrafish (Danio rerio) embryo model. This model, which has been used for various intracellular and extracellular bacterial pathogens, is a model of choice to investigate the contribution of cells from the innate immune system during infection [25]. The Danio has been successfully used to monitor the role of P. aeruginosa determinants in virulence based on survival curves of larvae infected with mutants deficient in type III secretion system (T3SS) or Quorum Sensing [20,26]. Bacteria producing a fluorescent protein were injected intravenously in the caudal vein of embryos (Fig 2A) at 30 hours post-fertilization, a time where macrophages, but not neutrophils, are fully functional and capable of engulfing invading bacteria [27]. The survival curves of infected embryos indicated that MgtC is a critical virulence determinant in this model since the mgtC mutant is significantly attenuated as compared to the wild-type PAO1 strain (Fig 2B). In agreement, fluorescence microscopy of 18 hpi infected embryos showed a lower bacterial burden with PAO1 strain than mgtC mutant (Fig 2C).

Bottom Line: MgtC is also found in pathogenic Pseudomonas species.Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role.In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus highlighting MgtC as a promising new target for anti-virulence strategies.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université de Montpellier, CNRS-UMR5235, Montpellier, France.

ABSTRACT
Pathogenic bacteria have developed strategies to adapt to host environment and resist host immune response. Several intracellular bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis, share the horizontally-acquired MgtC virulence factor that is important for multiplication inside macrophages. MgtC is also found in pathogenic Pseudomonas species. Here we investigate for the first time the role of MgtC in the virulence of an extracellular pathogen, Pseudomonas aeruginosa. A P. aeruginosa mgtC mutant is attenuated in the systemic infection model of zebrafish embryos, and strikingly, the attenuated phenotype is dependent on the presence of macrophages. In ex vivo experiments, the P. aeruginosa mgtC mutant is more sensitive to macrophage killing than the wild-type strain. However, wild-type and mutant strains behave similarly toward macrophage killing when macrophages are treated with an inhibitor of the vacuolar proton ATPase. Importantly, P. aeruginosa mgtC gene expression is strongly induced within macrophages and phagosome acidification contributes to an optimal expression of the gene. Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC shares a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to host immune response in relation to the different bacterial lifestyles. In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus highlighting MgtC as a promising new target for anti-virulence strategies.

No MeSH data available.


Related in: MedlinePlus