Journal: Infection and Immunity

Article Title: Ehrlichia chaffeensis TRP120 Effector Targets and Recruits Host Polycomb Group Proteins for Degradation To Promote Intracellular Infection

doi: 10.1128/IAI.00845-17

Figure Lengend Snippet: PCGF isoforms colocalize with cytoplasmic E. chaffeensis vacuoles during the late stage (48 h) of infection. (A) Immunofluorescence analysis of E. chaffeensis-infected THP-1 cells. Immunofluorescence micrographs demonstrate distribution of PCGF4 isoforms in uninfected and E. chaffeensis-infected cells at 12, 24, and 48 hpi. PCGF4 was mainly localized in the nucleus of an uninfected cell or during early infection (12 and 24 hpi) but was distributed to the ehrlichial vacuole (rectangle) at a late stage (48 hpi). Scale bar, 10 μm. (B) Bar graph depicting percentages of PCGF-TRP colocalized cells for individual PCGF isoforms (n = 3; Student's two-tailed t test; *, P ≤ 0.05; **, P ≤ 0.005). (C) Immunofluorescence micrographs demonstrating colocalization (infected, rectangles) of E. chaffeensis expressing TRP120 (green) with PCGF2, -3, -4, -5, and -6 isoforms (red) at the ehrlichial vacuole (arrow) at 48 hpi compared to uninfected cells. The signal intensities of red, green, and blue fluorescence channels are shown on each image using a table (a.u., arbitrary units) to cross-reference index numbers to output values. The color map is used to look up the actual colors corresponding to each index number. The Pearson's correlation coefficients shown next to the regions of interest (rectangles) indicate a strong/very strong colocalization for PCGF2, -3, -4, -5, and -6 isoforms with TRP120 in the ehrlichial vacuole. Scale bar, 10 μm. (D) Coimmunoprecipitation using PCGF2, -3, -4, -5, and -6 antibodies and chemiluminescence detection of TRP120 using TRP120-specific antibody from E. chaffeensis-infected and uninfected THP-1 cell lysate. (E) Western blot analysis of E. chaffeensis-infected and uninfected input lysates using TRP120, PCGF2, PCGF3, PCGF4, PCGF5, and PCGF6 antibodies; GAPDH was used as a loading control. (F) Western blot analysis of immunoprecipitation eluates using PCGF2, -3, -4, -5, and -6 isoform-specific antibodies.

Article Snippet: E. chaffeensis (Arkansas strain) was cultivated in THP-1 cells as previously described ( 54 ). siRNAs and antibodies. siRNAs used in this study were PCGF2 (sc-38191), PCGF3 (sc-89157), PCGF4 (sc-29814), PCGF6 (sc-90663), and nontargeting siRNA (sc-37007) (Santa Cruz Biotechnology, Santa Cruz, CA). siRNA for PCGF5 (M-007089-01) was obtained from GE Dharmacon (Lafayette, CO).

Techniques: Infection, Immunofluorescence, Two Tailed Test, Expressing, Fluorescence, Western Blot, Control, Immunoprecipitation

Journal: Infection and Immunity

Article Title: Ehrlichia chaffeensis TRP120 Effector Targets and Recruits Host Polycomb Group Proteins for Degradation To Promote Intracellular Infection

doi: 10.1128/IAI.00845-17

Figure Lengend Snippet: Decrease in PCGF isoforms occurs via proteosomal degradation in E. chaffeensis-infected cells. (A) Graph representing fold changes in PRC1 core component gene expression in E. chaffeensis-infected cells at 4, 10, 24, and 48 hpi; n = 3. Biologically significant (approximately ≤2-fold increase or decrease) and statistically significant (Student's two-tailed t test; *, P ≤ 0.05) changes in gene transcription were observed for CBX7, PCGF2, PCGF5, and RING1 genes. (B) Heat map showing relative expression levels of PRC1 core component genes at 4, 10, 24, and 48 hpi. Each position in the heat map represents an individual gene (listed next to the heat map). The colors indicate differential expression (red indicates induction, green indicates repression, and white represents no significant change) from the average gene expression level in uninfected cells. The intensity of the color represents the amplitude of induction/repression. (C) Western blot analysis of bortezomib (BORTZ)- or DMSO-treated THP-1 (E. chaffeensis-infected and uninfected) whole-cell lysates using isoform-specific anti-PCGF antibodies. E. chaffeensis-infected or uninfected THP-1 cells were treated with 100 nM bortezomib 38 h postinfection for 10 h. The whole-cell lysates were then subjected to SDS-PAGE separation, and the relative abundance of PCGF isoforms was determined using chemiluminescence. (D) Graph representing GAPDH-normalized relative abundance of PCGF isoforms in bortezomib- or DMSO-treated E. chaffeensis-infected or uninfected THP-1 cell lysates. Error bars indicate standard deviations between experiments (n = 3; Student's two-tailed t test; *, P ≤ 0.05).

Article Snippet: E. chaffeensis (Arkansas strain) was cultivated in THP-1 cells as previously described ( 54 ). siRNAs and antibodies. siRNAs used in this study were PCGF2 (sc-38191), PCGF3 (sc-89157), PCGF4 (sc-29814), PCGF6 (sc-90663), and nontargeting siRNA (sc-37007) (Santa Cruz Biotechnology, Santa Cruz, CA). siRNA for PCGF5 (M-007089-01) was obtained from GE Dharmacon (Lafayette, CO).

Techniques: Infection, Gene Expression, Two Tailed Test, Expressing, Quantitative Proteomics, Western Blot, SDS Page

Journal: Infection and Immunity

Article Title: Ehrlichia chaffeensis TRP120 Effector Targets and Recruits Host Polycomb Group Proteins for Degradation To Promote Intracellular Infection

doi: 10.1128/IAI.00845-17

Figure Lengend Snippet: siRNA-mediated silencing of PCGF isoforms increases E. chaffeensis infection. THP-1 cells were transfected with isoform-specific siRNA and then infected with E. chaffeensis at 24 h posttransfection. (A) Alexa Fluor 488-conjugated siRNA-transfected cell, showing high efficiency of RNA transfection using Lipofectamine 3000. (B) Western blot analysis of the total cell lysate from control and siRNA-transfected THP-1 cells confirmed the decrease in PCGF2, PCGF3, PCGF4, and PCGF5 48 h posttransfection. GAPDH was used as the loading control. The relative abundance of PCGF isoforms in siRNA-transfected cells was determined after normalization to the loading control and then represented as the percentage remaining after the knockdown. (C) Table representing the percentage increase in ehrlichial morulae and the average number of morulae/cell for each PCGF isoform-specific knockdown. The average morula counts were determined by counting the number of morula present in each field of view and then dividing that by the number of cells counted. The experiment was repeated three times in duplicate, and the values shown are means ± standard deviations (Stdev). (D) The fold change in ehrlichial infection was determined by comparing the ehrlichial dsb to the host cell gapdh in individual PCGF knockdown using real-time qPCR at 48 hpi (n = 3; *, P ≤ 0.05).

Article Snippet: E. chaffeensis (Arkansas strain) was cultivated in THP-1 cells as previously described ( 54 ). siRNAs and antibodies. siRNAs used in this study were PCGF2 (sc-38191), PCGF3 (sc-89157), PCGF4 (sc-29814), PCGF6 (sc-90663), and nontargeting siRNA (sc-37007) (Santa Cruz Biotechnology, Santa Cruz, CA). siRNA for PCGF5 (M-007089-01) was obtained from GE Dharmacon (Lafayette, CO).

Techniques: Infection, Transfection, Western Blot, Control, Knockdown