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Proteomic analysis of the Ehrlichia chaffeensis phagosome in cultured DH82 cells.

Cheng Y, Liu Y, Wu B, Zhang JZ, Gu J, Liao YL, Wang FK, Mao XH, Yu XJ - PLoS ONE (2014)

Bottom Line: The ECV composition was compared with that of phagolysosomes containing latex beads.Confocal microscopy confirmed that E. chaffeensis phagosomes contained Rab7 and were acidified at approximately pH 5.2, suggesting that the E. chaffeensis vacuole was an acidified late endosomal compartment.Our results also demonstrated by mass spectrometry and immunofluorescence analysis that Ehrlichia morulae were not associated with the autophagic pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Microbiology and Immunology, College of Medical Laboratory, Third Military Medical University, Chongqing, China ; Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America ; Bethune International Peace Hospital, Shijiazhuang, China.

ABSTRACT
Ehrlichia chaffeensis is an obligately intracellular bacterium that resides and multiplies within cytoplasmic vacuoles of phagocytes. The Ehrlichia-containing vacuole (ECV) does not fuse with lysosomes, an essential condition for Ehrlichia to survive inside phagocytes, but the mechanism of inhibiting the fusion of the phagosome with lysosomes is not clear. Understanding the ECV molecular composition may decipher the mechanism by which Ehrlichia inhibits phagosome-lysosome fusion. In this study, we obtained highly purified ECVs from E. chaffeensis-infected DH82 cells by sucrose density gradient centrifugation and analyzed their composition by mass spectrometry-based proteomics. The ECV composition was compared with that of phagolysosomes containing latex beads. Lysosomal proteins such as cathepsin D, cathepsin S, and lysosomal acid phosphatase were not detected in E. chaffeensis phagosome preparations. Some small GTPases, involved in membrane dynamics and phagocytic trafficking, were detected in ECVs. A notable finding was that Rab7, a late endosomal marker, was consistently detected in E. chaffeensis phagosomes by mass spectrometry. Confocal microscopy confirmed that E. chaffeensis phagosomes contained Rab7 and were acidified at approximately pH 5.2, suggesting that the E. chaffeensis vacuole was an acidified late endosomal compartment. Our results also demonstrated by mass spectrometry and immunofluorescence analysis that Ehrlichia morulae were not associated with the autophagic pathway. Ehrlichia chaffeensis did not inhibit phagosomes containing latex beads from fusing with lysosomes in infected cells. We concluded that the E. chaffeensis vacuole was a late endosome and E. chaffeensis might inhibit phagosome-lysosome fusion by modifying its vacuolar membrane composition, rather than by regulating the expression of host genes involved in trafficking.

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LysoSensor Green DND-189 and DND-153 labeling of E. chaffeensis-infected DH82 cells.(A and D): The confocal images containing DAPI-stained nuclei and E. chaffeensis morulae (blue); (B): LysoSensor Green DND-153-labeled infected DH82 cells; (C): merged image of A and B; (E): LysoSensor Green DND-189-labeled infected DH82 cells (green); (F): merged image of D and E; (G): DAPI-stained uninfected DH82 cells; (H): LysoSensor probe-labeled uninfected DH82 cells; (I): merged image of G and H. Arrows indicate E. chaffeensis morulae. These results were confirmed in three independent labeling experiments.
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pone-0088461-g003: LysoSensor Green DND-189 and DND-153 labeling of E. chaffeensis-infected DH82 cells.(A and D): The confocal images containing DAPI-stained nuclei and E. chaffeensis morulae (blue); (B): LysoSensor Green DND-153-labeled infected DH82 cells; (C): merged image of A and B; (E): LysoSensor Green DND-189-labeled infected DH82 cells (green); (F): merged image of D and E; (G): DAPI-stained uninfected DH82 cells; (H): LysoSensor probe-labeled uninfected DH82 cells; (I): merged image of G and H. Arrows indicate E. chaffeensis morulae. These results were confirmed in three independent labeling experiments.

Mentions: Confocal microscopy demonstrated that E. chaffeensis vacuoles co-localized with LysoSensor Green DND-189 with a pKa value of 5.2, but did not co-localize with LysoSensor Green DND-153 with a pKa value of 7.5. This result suggested that E. chaffeensis vacuoles were acidified with an approximate pH of 5.2 (Fig. 3).


Proteomic analysis of the Ehrlichia chaffeensis phagosome in cultured DH82 cells.

Cheng Y, Liu Y, Wu B, Zhang JZ, Gu J, Liao YL, Wang FK, Mao XH, Yu XJ - PLoS ONE (2014)

LysoSensor Green DND-189 and DND-153 labeling of E. chaffeensis-infected DH82 cells.(A and D): The confocal images containing DAPI-stained nuclei and E. chaffeensis morulae (blue); (B): LysoSensor Green DND-153-labeled infected DH82 cells; (C): merged image of A and B; (E): LysoSensor Green DND-189-labeled infected DH82 cells (green); (F): merged image of D and E; (G): DAPI-stained uninfected DH82 cells; (H): LysoSensor probe-labeled uninfected DH82 cells; (I): merged image of G and H. Arrows indicate E. chaffeensis morulae. These results were confirmed in three independent labeling experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928192&req=5

pone-0088461-g003: LysoSensor Green DND-189 and DND-153 labeling of E. chaffeensis-infected DH82 cells.(A and D): The confocal images containing DAPI-stained nuclei and E. chaffeensis morulae (blue); (B): LysoSensor Green DND-153-labeled infected DH82 cells; (C): merged image of A and B; (E): LysoSensor Green DND-189-labeled infected DH82 cells (green); (F): merged image of D and E; (G): DAPI-stained uninfected DH82 cells; (H): LysoSensor probe-labeled uninfected DH82 cells; (I): merged image of G and H. Arrows indicate E. chaffeensis morulae. These results were confirmed in three independent labeling experiments.
Mentions: Confocal microscopy demonstrated that E. chaffeensis vacuoles co-localized with LysoSensor Green DND-189 with a pKa value of 5.2, but did not co-localize with LysoSensor Green DND-153 with a pKa value of 7.5. This result suggested that E. chaffeensis vacuoles were acidified with an approximate pH of 5.2 (Fig. 3).

Bottom Line: The ECV composition was compared with that of phagolysosomes containing latex beads.Confocal microscopy confirmed that E. chaffeensis phagosomes contained Rab7 and were acidified at approximately pH 5.2, suggesting that the E. chaffeensis vacuole was an acidified late endosomal compartment.Our results also demonstrated by mass spectrometry and immunofluorescence analysis that Ehrlichia morulae were not associated with the autophagic pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Microbiology and Immunology, College of Medical Laboratory, Third Military Medical University, Chongqing, China ; Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America ; Bethune International Peace Hospital, Shijiazhuang, China.

ABSTRACT
Ehrlichia chaffeensis is an obligately intracellular bacterium that resides and multiplies within cytoplasmic vacuoles of phagocytes. The Ehrlichia-containing vacuole (ECV) does not fuse with lysosomes, an essential condition for Ehrlichia to survive inside phagocytes, but the mechanism of inhibiting the fusion of the phagosome with lysosomes is not clear. Understanding the ECV molecular composition may decipher the mechanism by which Ehrlichia inhibits phagosome-lysosome fusion. In this study, we obtained highly purified ECVs from E. chaffeensis-infected DH82 cells by sucrose density gradient centrifugation and analyzed their composition by mass spectrometry-based proteomics. The ECV composition was compared with that of phagolysosomes containing latex beads. Lysosomal proteins such as cathepsin D, cathepsin S, and lysosomal acid phosphatase were not detected in E. chaffeensis phagosome preparations. Some small GTPases, involved in membrane dynamics and phagocytic trafficking, were detected in ECVs. A notable finding was that Rab7, a late endosomal marker, was consistently detected in E. chaffeensis phagosomes by mass spectrometry. Confocal microscopy confirmed that E. chaffeensis phagosomes contained Rab7 and were acidified at approximately pH 5.2, suggesting that the E. chaffeensis vacuole was an acidified late endosomal compartment. Our results also demonstrated by mass spectrometry and immunofluorescence analysis that Ehrlichia morulae were not associated with the autophagic pathway. Ehrlichia chaffeensis did not inhibit phagosomes containing latex beads from fusing with lysosomes in infected cells. We concluded that the E. chaffeensis vacuole was a late endosome and E. chaffeensis might inhibit phagosome-lysosome fusion by modifying its vacuolar membrane composition, rather than by regulating the expression of host genes involved in trafficking.

Show MeSH
Related in: MedlinePlus