Figure 5.

Host preincorporated amino acids are taken up by E. chaffeensis (Ech) in a host class III PtdIns3K-dependent manner. (A) THP-1 cells were prelabeled with [³H]glutamine for 1 d and then infected with E. chaffeensis for 2 d in the absence of [³H]glutamine. Infected cells were treated with 2 mM 3-MA or solvent control for an additional 6 h. E. chaffeensis was purified from infected cells, and the [³H]glutamine incorporated in E. chaffeensis was determined by liquid scintillation counter and normalized to the total protein amount. Data are presented as the mean ± standard deviation of triplicate samples. *, Significantly different (P< 0.05) by the Student t test. (B and C) E. chaffeensis growth inhibition by 3-MA is partially rescued upon supplementation with essential amino acids. E. chaffeensis-infected cells were treated at 1 d p.i. with 3-MA or 3-MA and amino acids (3-MA + AA) for 2 d. The percentage of infected cells (B) and number of bacteria (C) were scored in each group. *, Significantly different (P < 0.05) by the Student t test. (D) DsRed-GAPDH (white arrows) was detected within E. chaffeensis-containing inclusions more with 0.1 μM DFP treatment for 2 h than without treatment before fixation by deconvolution microscopy. DAPI was pseudocolored in green. N, nucleus. Bar: 10 μm. (E) Percentage of inclusions that contain DsRed-GAPDH or DsRed with or without DFP treatment was determined by counting 10 to 20 inclusions per cell in 10 to 20 cells per experiment from 3 independent experiments. *, Significantly different (P < 0.01) by the Student t test.