Proteomic Analysis of the Ehrlichia chaffeensis Phagosome in Cultured DH82 Cells

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.

Ehrlichia Activation of Wnt-PI3K-mTOR Signaling Inhibits Autolysosome Generation and Autophagic Destruction by the Mononuclear Phagocyte

“…To further understand the autophagic process as it relates to E. chaffeensis infection, localization of lysosomes in the infected cells was determined. Consistent with previous reports (21,47), colocalization of E. chaffeensis vacuoles with the lysosomal marker LAMP-2 was not observed, suggesting that autolysosome generation is inhibited. Similarly to reports from studies performed with A. phagocytophilum, E. chaffeensis appears to induce autophagosome formation through noncanonical mechanisms involving effector protein Etf-1 with the Beclin-1 complex as a mechanism of nutrient acquisition (21).…”

“…Two lysosomal membraneassociated proteins, LAMP-1 and LAMP-2, are essential for autolysosomal fusion during the autophagic process (35,46). Similarly to a previous study (47), colocalization of the ehrlichial vacuole with the lysosomal marker LAMP-2 was not detected ( Fig. 2A).…”

Purification and proteomics of pathogen-modified vacuoles and membranes

“…As a pathogen is residing in a specific compartment in the host, the first step for most proteome studies in this field comprises selective enrichment of pathogen-containing vacuoles (PCVs) or internalized bacteria. This may be achieved by subcellular/organellar fractionation based on physicochemical properties (Howe and Heinzen, 2008 ; He et al, 2012 ; Cheng et al, 2014 ), by immuno-affinity purification (Urwyler et al, 2010 ; Hoffmann et al, 2013 ; Vorwerk et al, 2015 ), or through single cell FACS enrichment by sorting internalized bacteria and lysed host cells/organelles (Becker et al, 2006 ; Pförtner et al, 2013 ; Surmann et al, 2014 ). These approaches greatly reduce sample complexity in proteomics approaches and increase the specificity and the quality of conclusions that can be drawn from the data.…”

Anaplasmataceae Subversion of the Lysosomal Activity

“…2,6,14 Despite the above mentioned studies demonstrated that Ehrlichiacontaining vacuole (ECV) does not fuse with lysosomes, an essential condition for Ehrlichia to survive inside phagocytes, the mechanism of inhibiting the fusion of the phagosome with lysosomes is not clear. Thus, Cheng et al,…”