GRA7-III-induced activation of PLD1 and phagosomal maturation were required for antimicrobial activity in MTB-infected macrophages.
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(A and B) BMDMs were infected with MTB (MOI = 1) for 4 h and then stimulated with rGRA7 (5 μg/ml) and its mutants for 18 h. Mycobacteria-containing phagosome fractions were subsequently purified by sucrose-step-gradient-ultra-centrifugations, followed by IB to detect αRab5, αRab7, αLAMP1, αLAMP2, αPLD1, αPKCα, and αActin (A) or IP with αPLD1 and IB with αHis, αPLD1, and αActin (B). Quantitative analysis of the PLD1 interacts with His-rGRA7 in MTB-containing phagosomes band normalized to Actin is shown (lower). (C and D) Intracellular survival of MTB was assessed by CFU assay. BMDMs were infected with MTB for 4 h, followed by treatment with rGRA7, and then lysed to determine intracellular bacterial loads. (D, right) IB with αPLD1, αΑSC, and αActin in BMDMs. The data are representative of five independent experiments with similar results (A and B). Data shown are the mean ± SD of five experiments (C and D). Significant differences (*P < 0.05; **P < 0.01; ***P < 0.001) compared with PKCα+/+ and PKCα-/- (B) or rVector (C and D). CFU, colony-forming units. ns, not significant.