Proteomic analysis of the Ehrlichia chaffeensis phagosome in cultured DH82 cells

Yan Cheng, Yan Liu, Bin Wu, Jian Zhi Zhang, Jiang Gu, Ya Ling Liao, Fu Kun Wang, Xu Hu Mao, Xue Jie Yu

Research output: Contribution to journalArticle

11 Scopus citations

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 phagosomelysosome 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.

Original languageEnglish (US)
Article numbere88461
JournalPloS one
Volume9
Issue number2
DOIs
StatePublished - Feb 18 2014

    Fingerprint

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

Cite this

Cheng, Y., Liu, Y., Wu, B., Zhang, J. Z., Gu, J., Liao, Y. L., Wang, F. K., Mao, X. H., & Yu, X. J. (2014). Proteomic analysis of the Ehrlichia chaffeensis phagosome in cultured DH82 cells. PloS one, 9(2), [e88461]. https://doi.org/10.1371/journal.pone.0088461