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Role of JAK-STAT signaling in maturation of phagosomes containing Staphylococcus aureus.

Zhu F, Zhou Y, Jiang C, Zhang X - Sci Rep (2015)

Bottom Line: In this study, the S. aureus was engulfed by macrophages (RAW264.7 cells) but not digested by the cells, suggesting that the phagosomes did not maturate in macrophages.Further investigation revealed that peptidoglycan (PG) induced the phagosome maturation of macrophages, resulting in the eradication of S. aureus.Therefore, our study contributed evidence that revealed a novel aspect of PG-triggered JAK-STAT pathway in the phagosome maturation of macrophages.

View Article: PubMed Central - PubMed

Affiliation: Collaborative Innovation Center of Deep Sea Biology, Key Laboratory of Animal Virology of Ministry of Agriculture and College of Life Sciences, Zhejiang University, Hangzhou 310058, China.

ABSTRACT
Phagocytosis is a required mechanism for the defense against pathogens. Staphylococcus aureus, an important bacterial pathogen, can promptly escape from phagosomes and proliferate within the cytoplasm of host. However, the mechanism of phagocytosis against S. aureus has not been intensively investigated. In this study, the S. aureus was engulfed by macrophages (RAW264.7 cells) but not digested by the cells, suggesting that the phagosomes did not maturate in macrophages. Further investigation revealed that peptidoglycan (PG) induced the phagosome maturation of macrophages, resulting in the eradication of S. aureus. Genome-wide analysis and quantitative real-time PCR indicated that the JAK-STAT pathway was activated by PG during the phagosome maturation of macrophages against S. aureus. This finding presented that the PG-activated JAK-STAT pathway was required for phagosome maturation. Therefore, our study contributed evidence that revealed a novel aspect of PG-triggered JAK-STAT pathway in the phagosome maturation of macrophages.

No MeSH data available.


Related in: MedlinePlus

Peptidoglycan induces phagocytosis after inoculation with inactivated S. aureus.(A) RAW264.7 cells were induced with LPS or PG inoculated with inactivated S. aureus and inactivated S. aureus. One day later, the cells were examined with transmission electron microscope. RAW264.7 cells only and PG alone were used as controls. Lane headings indicated the treatments. The box indicated the enlarged image. Scale bar, 5 μm (up) or 1 μm (down). (B) RAW264.7 cells were treated with LPS or PG and then with FITC-labeled inactivated S. aureus. After incubation for 1 day, the cells were subjected to phagocytosis assays. The filamentous actin was stained with rhodamine phalloidin and the cellular DNA with DAPI. The green spots indicated the internalized S. aureus. Non-treated cells and PG alone were used as controls. Scale bar, 10 μm (up) or 5 μm (down). (C) RAW264.7 cells were incubated with LPS or PG and pHrodo-labeled heat-inactivated S. aureus. The treatment chloroquine + heat-inactivated S. aureus +PG was included in the incubations. At 1 h after incubation, the cells were examined with confocal microscopy. The red spots showed the formation of phagolysosome. RAW264.7 cells and PG alone were used as controls. Scale bar, 10 μm (up) or 5 μm (down). (D) The detection of the total S. aureus using a pH-insensitive PCR. The RAW 264.7 cells were treated with PG/LPS and S. aureus. At 2 h after treatments, the cells were subjected to PCR with the S. aureus nuc gene-specific primers. M indicates the DNA marker. (E) The percentage of phagocytosed pHrodo-labeled inactivated S. aureus in RAW264.7 cells. The RAW264.7 cells were incubated with LPS or PG and pHrodo-labeled inactivated S. aureus. At 1 h after incubation, the cells were evaluated using flow cytometry. Non-treated cells and PG alone were used as controls. Statistically significant differences between treatments were indicated with asterisks (**P < 0.01).
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f1: Peptidoglycan induces phagocytosis after inoculation with inactivated S. aureus.(A) RAW264.7 cells were induced with LPS or PG inoculated with inactivated S. aureus and inactivated S. aureus. One day later, the cells were examined with transmission electron microscope. RAW264.7 cells only and PG alone were used as controls. Lane headings indicated the treatments. The box indicated the enlarged image. Scale bar, 5 μm (up) or 1 μm (down). (B) RAW264.7 cells were treated with LPS or PG and then with FITC-labeled inactivated S. aureus. After incubation for 1 day, the cells were subjected to phagocytosis assays. The filamentous actin was stained with rhodamine phalloidin and the cellular DNA with DAPI. The green spots indicated the internalized S. aureus. Non-treated cells and PG alone were used as controls. Scale bar, 10 μm (up) or 5 μm (down). (C) RAW264.7 cells were incubated with LPS or PG and pHrodo-labeled heat-inactivated S. aureus. The treatment chloroquine + heat-inactivated S. aureus +PG was included in the incubations. At 1 h after incubation, the cells were examined with confocal microscopy. The red spots showed the formation of phagolysosome. RAW264.7 cells and PG alone were used as controls. Scale bar, 10 μm (up) or 5 μm (down). (D) The detection of the total S. aureus using a pH-insensitive PCR. The RAW 264.7 cells were treated with PG/LPS and S. aureus. At 2 h after treatments, the cells were subjected to PCR with the S. aureus nuc gene-specific primers. M indicates the DNA marker. (E) The percentage of phagocytosed pHrodo-labeled inactivated S. aureus in RAW264.7 cells. The RAW264.7 cells were incubated with LPS or PG and pHrodo-labeled inactivated S. aureus. At 1 h after incubation, the cells were evaluated using flow cytometry. Non-treated cells and PG alone were used as controls. Statistically significant differences between treatments were indicated with asterisks (**P < 0.01).

Mentions: To evaluate the phagocytic activity of RAW264.7 cells against Staphylococcus aureus, the bacteria were inactivated and then subjected to phagocytosis assay in RAW264.7 cells. The use of inactivated bacteria in the evaluations of phagocytic activity of macrophages could exclude the effects of bacterium infection on phagocytosis. The heat-inactivated S. aureus was engulfed, but not digested by RAW264.7 cells (Fig. 1A,B). We hypothesized that the receptors on the surface of macrophages were not activated to recognize and subsequently induce the digestion of S. aureus.


Role of JAK-STAT signaling in maturation of phagosomes containing Staphylococcus aureus.

Zhu F, Zhou Y, Jiang C, Zhang X - Sci Rep (2015)

Peptidoglycan induces phagocytosis after inoculation with inactivated S. aureus.(A) RAW264.7 cells were induced with LPS or PG inoculated with inactivated S. aureus and inactivated S. aureus. One day later, the cells were examined with transmission electron microscope. RAW264.7 cells only and PG alone were used as controls. Lane headings indicated the treatments. The box indicated the enlarged image. Scale bar, 5 μm (up) or 1 μm (down). (B) RAW264.7 cells were treated with LPS or PG and then with FITC-labeled inactivated S. aureus. After incubation for 1 day, the cells were subjected to phagocytosis assays. The filamentous actin was stained with rhodamine phalloidin and the cellular DNA with DAPI. The green spots indicated the internalized S. aureus. Non-treated cells and PG alone were used as controls. Scale bar, 10 μm (up) or 5 μm (down). (C) RAW264.7 cells were incubated with LPS or PG and pHrodo-labeled heat-inactivated S. aureus. The treatment chloroquine + heat-inactivated S. aureus +PG was included in the incubations. At 1 h after incubation, the cells were examined with confocal microscopy. The red spots showed the formation of phagolysosome. RAW264.7 cells and PG alone were used as controls. Scale bar, 10 μm (up) or 5 μm (down). (D) The detection of the total S. aureus using a pH-insensitive PCR. The RAW 264.7 cells were treated with PG/LPS and S. aureus. At 2 h after treatments, the cells were subjected to PCR with the S. aureus nuc gene-specific primers. M indicates the DNA marker. (E) The percentage of phagocytosed pHrodo-labeled inactivated S. aureus in RAW264.7 cells. The RAW264.7 cells were incubated with LPS or PG and pHrodo-labeled inactivated S. aureus. At 1 h after incubation, the cells were evaluated using flow cytometry. Non-treated cells and PG alone were used as controls. Statistically significant differences between treatments were indicated with asterisks (**P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1: Peptidoglycan induces phagocytosis after inoculation with inactivated S. aureus.(A) RAW264.7 cells were induced with LPS or PG inoculated with inactivated S. aureus and inactivated S. aureus. One day later, the cells were examined with transmission electron microscope. RAW264.7 cells only and PG alone were used as controls. Lane headings indicated the treatments. The box indicated the enlarged image. Scale bar, 5 μm (up) or 1 μm (down). (B) RAW264.7 cells were treated with LPS or PG and then with FITC-labeled inactivated S. aureus. After incubation for 1 day, the cells were subjected to phagocytosis assays. The filamentous actin was stained with rhodamine phalloidin and the cellular DNA with DAPI. The green spots indicated the internalized S. aureus. Non-treated cells and PG alone were used as controls. Scale bar, 10 μm (up) or 5 μm (down). (C) RAW264.7 cells were incubated with LPS or PG and pHrodo-labeled heat-inactivated S. aureus. The treatment chloroquine + heat-inactivated S. aureus +PG was included in the incubations. At 1 h after incubation, the cells were examined with confocal microscopy. The red spots showed the formation of phagolysosome. RAW264.7 cells and PG alone were used as controls. Scale bar, 10 μm (up) or 5 μm (down). (D) The detection of the total S. aureus using a pH-insensitive PCR. The RAW 264.7 cells were treated with PG/LPS and S. aureus. At 2 h after treatments, the cells were subjected to PCR with the S. aureus nuc gene-specific primers. M indicates the DNA marker. (E) The percentage of phagocytosed pHrodo-labeled inactivated S. aureus in RAW264.7 cells. The RAW264.7 cells were incubated with LPS or PG and pHrodo-labeled inactivated S. aureus. At 1 h after incubation, the cells were evaluated using flow cytometry. Non-treated cells and PG alone were used as controls. Statistically significant differences between treatments were indicated with asterisks (**P < 0.01).
Mentions: To evaluate the phagocytic activity of RAW264.7 cells against Staphylococcus aureus, the bacteria were inactivated and then subjected to phagocytosis assay in RAW264.7 cells. The use of inactivated bacteria in the evaluations of phagocytic activity of macrophages could exclude the effects of bacterium infection on phagocytosis. The heat-inactivated S. aureus was engulfed, but not digested by RAW264.7 cells (Fig. 1A,B). We hypothesized that the receptors on the surface of macrophages were not activated to recognize and subsequently induce the digestion of S. aureus.

Bottom Line: In this study, the S. aureus was engulfed by macrophages (RAW264.7 cells) but not digested by the cells, suggesting that the phagosomes did not maturate in macrophages.Further investigation revealed that peptidoglycan (PG) induced the phagosome maturation of macrophages, resulting in the eradication of S. aureus.Therefore, our study contributed evidence that revealed a novel aspect of PG-triggered JAK-STAT pathway in the phagosome maturation of macrophages.

View Article: PubMed Central - PubMed

Affiliation: Collaborative Innovation Center of Deep Sea Biology, Key Laboratory of Animal Virology of Ministry of Agriculture and College of Life Sciences, Zhejiang University, Hangzhou 310058, China.

ABSTRACT
Phagocytosis is a required mechanism for the defense against pathogens. Staphylococcus aureus, an important bacterial pathogen, can promptly escape from phagosomes and proliferate within the cytoplasm of host. However, the mechanism of phagocytosis against S. aureus has not been intensively investigated. In this study, the S. aureus was engulfed by macrophages (RAW264.7 cells) but not digested by the cells, suggesting that the phagosomes did not maturate in macrophages. Further investigation revealed that peptidoglycan (PG) induced the phagosome maturation of macrophages, resulting in the eradication of S. aureus. Genome-wide analysis and quantitative real-time PCR indicated that the JAK-STAT pathway was activated by PG during the phagosome maturation of macrophages against S. aureus. This finding presented that the PG-activated JAK-STAT pathway was required for phagosome maturation. Therefore, our study contributed evidence that revealed a novel aspect of PG-triggered JAK-STAT pathway in the phagosome maturation of macrophages.

No MeSH data available.


Related in: MedlinePlus