Acinetobacter type VI secretion system comprises a non-canonical membrane complex
Fig 2
A. baumannii T6SS essential for the MC biogenesis.
(A) Fluorescent cluster of the TssMsfGFP protein in A. baumannii tssM-sfGFP strain. On the left panel, the images represent the average over a time lapse acquisition of 61 frames (10s / frame). scale = 2 μm. The right panel represents the histograms of cluster distribution of the main axis of the cell with a preferential accumulation at the poles of the cell. (n > 1000 cells, two biological replicates). Comparison of the cellular distribution of TssM-sfGFP foci between the WT and tsmK mutant (right panels). (B) Statistical analysis of the amount of fluorescence foci in the different mutants. On the left, a graphical representation of the percentage of cells with zero (n = 0), one (n = 1), two (n = 2), or more than three (n = 3) foci in the different A. baumannii mutants. On the right, a graphical representation of the average number of foci per cell in the different mutants. The experiment was performed in triplicate on three different groups of cells. (C-E) Dynamic study of the fluorescence foci of the TssM-sfGFP in WT and tsmK mutant. Fluorescence microscopy in TIRF mode captured an average of 61 images every 10 seconds, revealing multiple fluorescent foci within the cells. Cell masks were generated using the Cellpose cyto2 model, and FIJI’s MicrobeJ plugin, set in "rod-shape" mode. The lower panel shows the average of images processed with a bandpass FFT filter and background subtraction that enhances the foci contrast. (D) Kymographs derived from intensity profiles measured along the cell contour, representing time (61 images every 10 seconds) on the vertical axis and cell contour length in microns on the horizontal axis. Traces of foci movement along the cell contour are evident, with fixed and stable foci yielding vertical traces, some corresponding to the whole acquisition duration (the height of the kymograph = 610 seconds). (E). Violin plots for the quantitative analysis of foci traces between the WT and tsmK mutant (3 biological replications). The difference in means was tested by t.test (R software) and yields a p value < 0.0001. (F) Biogenesis of the A. baumannii-T6SS MC and the original degradation of the tail after contraction. Time-lapse fluorescence microscopy recordings showing localization and dynamics of the mCherryClpV and TssMsfGFP fusions proteins. Individual images were taken every 40 sec. The positions of the foci are indicated by an asterisk. The scale bars are 1 μm. The lines (from top to bottom) represent the phase contrast, the mCherry channel, the sfGFP channel and the superposition of the two channels. Below, a schematic representation of the sequential biogenesis of the T6SS membrane complex.