EsxG_Mt EsxH_Mt arrests phagosome maturation.

(A) H37Rv transformed with empty vector, EsxGMt EsxHMt-FLAG, EsxGMs EsxHMs-FLAG or EsxGMt EsxHMt- H76A-E77A-FLAG were analyzed for the presence of EsxH in the pellet and culture filtrate (CF). DlaT (Rv2215), a cytosolic protein, was used as a loading control and to indicate the degree of bacterial lysis. (B) MFI of phagosomal LAMP1 24 hpi in RAW cells treated with siRNAs and infected with Mtb containing EsxGMt-EsxHMt plasmid (red) or vector control (black); data points are the mean fluorescence intensity (MFI) around at least 70 phagosomes for each condition; p<0.0001 between the two Mtb strains for all conditions, unpaired Student's t-test. (C) Composite images of cells infected with Mtb with autofluorescence of Mtb (blue) and LAMP1 (red). Regions indicated by yellow circles are shown in higher magnification in adjacent panels. (D) MFI of phagosomal TfR 24 hpi in RAW cells treated with siRNAs and infected with Mtb containing EsxGMt-EsxHMt plasmid (red) or vector control (black); data points are the mean fluorescence intensity (MFI) around at least 50 phagosomes for each condition; p<0.0001 between the two Mtb strains for all conditions, except Rab7 (p = 0.0005), unpaired Student's t-test. (E) Composite images of cells infected with Mtb with autofluorescence of Mtb (blue) and TfR (red). Regions indicated by yellow circles are shown in higher magnification. (F) MFI of phagosomal LAMP1 in RAW cells infected with Mtb containing the indicated plasmids 24 hpi. Bars show mean +/− SEM. ****p<0.0001, unpaired Student's t-test.