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. 2020 Feb 4;11:684. doi: 10.1038/s41467-020-14508-5

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a–c Comparative analysis of the percentage of phagocytosis (a) and growth (b, c) of TbD1-intact and ΔTbD1 WT and mutant strains in human THP-1 cells. d–f Comparative analysis of the percentage of uptake (d) and growth (e–f) of TbD1-intact and ΔTbD1 WT and mutant strains in human A549 cells. For determination of the percentage of uptake, THP-1 cells were infected at m.o.i 20:1 and 1:1 (cells: bacteria), while A549 cells were infected at m.o.i 10:1 and 1:1 (cells: bacteria). The comparison of growth kinetics of different TbD1-intact and ΔTbD1 WT and complemented strains was undertaken at m.o.i 20:1 (cells:bacteria) for THP-1, and 10:1 (cells:bacteria) for A549. These m.o.i ensured the integrity of the infected cell monolayer over a 6-day period. In both these ex vivo models, the numbers of intracellular bacteria (CFU) were determined immediately after phagocytosis and at different time points (as indicated in panels b, e). CFU ratio values (CFU at different time points/CFU at day 0) are reported in c, f. The statistical significance of differences in CFU and CFU ratio values among the strains were determined by one-way Anova with Bonferroni post hoc test. Only the statistical significance of differences between the 79112∆TbD1 mutant and its corresponding control strains (79112 and 79112∆TbD1-C) or H37Rv and its derivative H37Rv::TbD1 and related control Tb36, at days 4 and 6 post infection are depicted in the figure (**P < 0.01; ***P < 0.001). The figure shows the single data points, mean and standard deviations of uptake percentage, CFU numbers and CFU ratio values obtained in a representative experiment performed in quadruplicate (or in triplicate for determination of uptake percentage at m.o.i 1:1).