M. marinum induces an early autophagic response in D. discoideum.
A. Two representative maximum projections showing GFP-Atg8-expressing cells infected (bottom) or mock-infected (top) with mCherry-expressing wt M. marinum. Images were recorded live 1.5 h after infection. White arrowheads point to GFP-Atg8 structures. Scale bars, 10 μm; B. The median and interquartile ranges of the number of GFP-Atg8 structures per cell were calculated. For each condition, 300 cells from independent triplicates were counted. Mann-Whitney test (****p ≤ 0.0001); C. Single sections of live GFP-Atg8-expressing amoebae 1.5 h after infection with mCherry-expressing M. marinum wt. White, magenta and yellow arrowheads point to GFP-Atg8 dots, patches and GFP-Atg8-positive (GFP-Atg8+) MCV, respectively. Scale bars, 5 μm; D. At 1.5 hpi, 256 cells (100%) with GFP-Atg8+ M. marinum were classified as in C. Means and standard deviations from three independent infections are represented; E. EM of the different types of autophagosomes in the vicinity of the MCV at 7 hpi. White asterisks label bacteria, white arrowheads point to round phagophores and autophagosomes, magenta arrowheads point to extended autophagosomes, and the yellow arrowhead indicates a double membrane compartment containing mycobacteria. Nuclei . Scale bars, 1 μm; F. Sections (top) and maximum projections (bottom) showing co-localization (white arrowheads) of autophagy markers with bacteria at 1–1.5 hpi. Around 50% and 30% of the Atg8-positive bacteria were also positive for Ub and GFP-p62, respectively. Scale bars, 5 μm; G. qPCR results of relative abundance of atg8a, atg8b, atg1 and p62 mRNAs at 3 hpi. The mRNA level in non-infected cells is indicated as 1. The mRNA level of the housekeeping gene gapdh was used for normalization. Means and standard deviations from three independent experiments performed in triplicates are represented.