FIG 4 .

E. chaffeensis can import proline and glutamine, but not glutamate, and requires proline and glutamine uptake for infection. (A) E. chaffeensis was incubated at 65°C for 15 min (Heat) or pretreated with 50 µM protamine, 10 mM AC or DHP, or 2 mM histidine (His) on ice for 30 min and incubated with ³H-labeled proline, glutamate, or glutamine at 37°C for 2 h to determine amino acid uptake. Relative radioactivity per milligram E. chaffeensis protein was measured. Data indicate the means ± standard deviations from three independent experiments. CTL, nontreated control. *, significantly different (P < 0.05; analysis of variance) among all groups. (B) Dose-dependent inhibitory effect of protamine, AC, or DHP on E. chaffeensis growth in THP-1 cells. DNA samples for qPCR were prepared at 72 h p.i. from THP-1 cells infected with host cell-free E. chaffeensis pretreated with protamine (2, 10, or 50 µM), AC (2, 6, or 10 mM), or DHP (2, 6, or 10 mM). The values reflect bacterial 16S rRNA gene normalized against human GAPDH DNA, relative to the amount determined for the sham-treated control (CTL). Data indicate the means ± standard deviations from three independent experiments performed in triplicate. (C) Dose-dependent inhibitory effect of histidine on E. chaffeensis growth in THP-1 cells. DNA samples for qPCR were prepared at 72 h p.i. from THP-1 cells infected with host cell-free E. chaffeensis pretreated with histidine (0.5, 1, or 2 mM). The values reflect bacterial 16S rRNA gene normalized against human GAPDH DNA, relative to the amount determined for the sham-treated control (CTL). Data indicate the means ± standard deviations from three independent experiments performed in triplicate. (D) Proline and glutamine, but not glutamate, enhance E. chaffeensis infection. DNA samples for qPCR were prepared at 48 h p.i. from THP-1 cells infected with host cell-free E. chaffeensis pretreated with 20 mM proline, glutamate, or glutamine. CTL, control pretreatment; Pro, proline pretreatment; Glu, glutamate pretreatment; Gln, glutamine pretreatment. The values reflect bacterial 16S rRNA gene normalized against human GAPDH DNA, relative to the amount determined for the sham-treated control (CTL). Data indicate the means ± standard deviations from three independent experiments performed in triplicate. *, significantly different (P < 0.05; analysis of variance) compared with the value of the control.