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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

Visualisation and quantification of intracellular bacteria in fixed macrophages.Macrophages infected with PAO1 or ΔmgtC mutant expressing mCherry were fixed after phagocytosis and 20 min (t0) or 2 hours (t2h) treatment with amikacin. (A) Visualization of intracellular bacteria. The images shown were obtained by making a maximum projection of 12 central planes from a Z-stack. The merged image shows Differential Interference Contrast (DIC), nucleus staining (blue) and bacteria expressing mCherry (red). (B) Count of the number of bacteria in infected macrophages from images obtained with the maximum projection. The numbers of bacteria per cell (b/c) were classified in three groups and percentage of each class is shown. Count is done from at least 20 cells and results are expressed as means from three independent experiments.
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ppat.1004969.g004: Visualisation and quantification of intracellular bacteria in fixed macrophages.Macrophages infected with PAO1 or ΔmgtC mutant expressing mCherry were fixed after phagocytosis and 20 min (t0) or 2 hours (t2h) treatment with amikacin. (A) Visualization of intracellular bacteria. The images shown were obtained by making a maximum projection of 12 central planes from a Z-stack. The merged image shows Differential Interference Contrast (DIC), nucleus staining (blue) and bacteria expressing mCherry (red). (B) Count of the number of bacteria in infected macrophages from images obtained with the maximum projection. The numbers of bacteria per cell (b/c) were classified in three groups and percentage of each class is shown. Count is done from at least 20 cells and results are expressed as means from three independent experiments.

Mentions: To visualize intracellular bacteria upon infection of J774 cells, we imaged slides of fixed macrophages infected with fluorescent bacteria (Fig 4A). Moreover, bacterial counts on images were made at time 0 and 2 hrs after phagocytosis (Fig 4B). Our results indicated that the number of bacteria per cell after phagocytosis is slightly higher for the mutant strain than for the wild-type strain. We further quantified the phagocytosis rate with CFUs counts and showed that the mgtC mutant is phagocytosed at a slightly but significantly higher rate than the wild-type strain (S4 Fig). The evolution of the patterns between time 0 and time 2 hrs (Fig 4B) indicated that the bacterial number per cell rather increased with the wild-type but not with the mutant. These experiments, which do not discriminate between live and dead/dying bacteria, suggested a survival of wild-type bacteria with limited bacterial multiplication.


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)

Visualisation and quantification of intracellular bacteria in fixed macrophages.Macrophages infected with PAO1 or ΔmgtC mutant expressing mCherry were fixed after phagocytosis and 20 min (t0) or 2 hours (t2h) treatment with amikacin. (A) Visualization of intracellular bacteria. The images shown were obtained by making a maximum projection of 12 central planes from a Z-stack. The merged image shows Differential Interference Contrast (DIC), nucleus staining (blue) and bacteria expressing mCherry (red). (B) Count of the number of bacteria in infected macrophages from images obtained with the maximum projection. The numbers of bacteria per cell (b/c) were classified in three groups and percentage of each class is shown. Count is done from at least 20 cells and results are expressed as means from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004969.g004: Visualisation and quantification of intracellular bacteria in fixed macrophages.Macrophages infected with PAO1 or ΔmgtC mutant expressing mCherry were fixed after phagocytosis and 20 min (t0) or 2 hours (t2h) treatment with amikacin. (A) Visualization of intracellular bacteria. The images shown were obtained by making a maximum projection of 12 central planes from a Z-stack. The merged image shows Differential Interference Contrast (DIC), nucleus staining (blue) and bacteria expressing mCherry (red). (B) Count of the number of bacteria in infected macrophages from images obtained with the maximum projection. The numbers of bacteria per cell (b/c) were classified in three groups and percentage of each class is shown. Count is done from at least 20 cells and results are expressed as means from three independent experiments.
Mentions: To visualize intracellular bacteria upon infection of J774 cells, we imaged slides of fixed macrophages infected with fluorescent bacteria (Fig 4A). Moreover, bacterial counts on images were made at time 0 and 2 hrs after phagocytosis (Fig 4B). Our results indicated that the number of bacteria per cell after phagocytosis is slightly higher for the mutant strain than for the wild-type strain. We further quantified the phagocytosis rate with CFUs counts and showed that the mgtC mutant is phagocytosed at a slightly but significantly higher rate than the wild-type strain (S4 Fig). The evolution of the patterns between time 0 and time 2 hrs (Fig 4B) indicated that the bacterial number per cell rather increased with the wild-type but not with the mutant. These experiments, which do not discriminate between live and dead/dying bacteria, suggested a survival of wild-type bacteria with limited bacterial multiplication.

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