GRA7-III-induced activation of PLD1 and phagosomal maturation were required for antimicrobial activity in MTB-infected macrophages.

<p>(<b>A</b> and <b>B</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 (<b>A</b>) or IP with αPLD1 and IB with αHis, αPLD1, and αActin (<b>B</b>). Quantitative analysis of the PLD1 interacts with His-rGRA7 in MTB-containing phagosomes band normalized to Actin is shown (lower). (<b>C</b> and <b>D</b>) 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. (<b>D</b>, right) IB with αPLD1, αΑSC, and αActin in BMDMs. The data are representative of five independent experiments with similar results (<b>A</b> and <b>B</b>). Data shown are the mean ± SD of five experiments (<b>C</b> and <b>D</b>). Significant differences (*<i>P</i> < 0.05; **<i>P</i> < 0.01; ***<i>P</i> < 0.001) compared with PKCα+/+ and PKCα-/- (<b>B</b>) or rVector (<b>C</b> and <b>D</b>). CFU, colony-forming units. ns, not significant.</p>