Fig. 3: Secretion of ClyA by the TtsA peptidoglycan hydrolase-dependent Type 10 secretion system.

a Immunofluorescence microscopy images of HeLa cells infected with S. Paratyphi wild-type or ΔttsA mutant strain with epitope tagged ClyA:3xFLAG (MOI 30). After 24 hpi the cells were harvested and stained for S. Paratyphi A using an α-LPS antibody and for ClyA:3xFLAG using an α-FLAG antibody. As secondary antibodies Alexa Fluor^TM 594 goat anti-rabbit and Alexa Fluor^TM 488 rabbit anti mouse were used. For DNA staining, DAPI was used. If indicated, lysozyme (100 μg/ml, Sigma Aldrich) was added. b The percentage of ClyA-secreting and, consequently, ClyA:3xFLAG-positive bacteria (green fluorescence) was quantified relative to the total bacterial population (LPS staining, red fluorescence), within infected HeLa cells (MOI 30). A series of 10 regions of interest (ROIs) were selected for this analysis. Each ROI encompassed approximately 20 host cells (n = 200) and was examined quantitatively at the specified time points. Data are presented as mean values ± SD. Source data are provided as a Source Data file Graphs (c) Subcellular ClyA localization in intracellular S. Paratyphi A. HeLa cells were infected with S. Paratyphi A wild-type or ΔttsA mutant expressing chromosomally epitope tagged ClyA:3xFLAG. The subcellular fractions were collected as described in the methods and analyzed for ClyA detection via Western blot analyses. Periplasmic maltose-binding protein (MBP:3xFLAG) and cytoplasmic RecA (RecA:3xFLAG) were used as cross-contamination controls. Ultracentrifugation to detect membrane-associated ClyA in (d) infected THP-1 cells or during growth in (e) low Mg²⁺ medium. Infected THP-1 cells (MOI 100), incubated for 24 h, were gently lysed, and the samples were centrifuged at a standard speed of 10,000 g for 10 min to pellet bacteria and cellular components. The resulting supernatant was filtered (0.22 µm) and subjected to ultracentrifugation. For growth in low Mg²⁺ medium, bacteria were pelleted by standard centrifugation, and the filtered cell-free supernatant was used for ultracentrifugation. Standard Ponceau staining of the same blots was used to visualize protein contents. c–e Western blot analyses were repeated with three independent biological replicates. Source data are provided as a Source Data file Blots.