Fig. 4: ClyA-dependent cytolysis of various target cells.

The impact of ClyA on various host cells was analyzed by hemolysis tests and LDH-release assays. a Supernatants of different S. Paratyphi A strains (wild-type, wild-type with Prha:slyA, wild-type with Prha:clyA, ΔclyA, ΔclyA with Prha:clyA and wild-type with empty vector control) were collected after 24 h under plasmid- inducing conditions (LB + 0.1% rhamnose) filtered and then incubated with 0.1% human erythrocytes. Optical densities (OD₆₂₀) were measured every 2 min for 4 h in a plate reader (Clario Star). b–f Supernatants of S. Paratyphi A [Prha:clyA], ΔclyA mutant, complemented ΔclyA mutant [Prha:clyA], and empty vector controls grown for 24 h under plasmid-inducing conditions (LB + 0.1% rhamnose) were collected and filtered (0.45 µm). Cell-free supernatants were incubated with (b) THP-1 cells, (c) U937 cells, (d) HeLa cells, (e) non-polarized Caco-2 cells, and (f) HT-29 MTX cells for up to 4 h. Samples were taken every hour and LDH-release was measured in a luminescence plate reader (Clario Star). All data were acquired from three independent biological replicates. Significance was determined using 2way ANOVA Šídák’s multiple comparisons tests. b Not significant (ns): p > 0.05, **: p = 0.0015. c Not significant (ns): p > 0.05, *p = 0.0365, ***p = 0.0001. d–f Not significant (ns): p > 0.05. g HeLa cells were infected with S. Paratyphi wild-type, ΔclyA mutant strain, complemented ΔclyA mutant [Prha:clyA], and empty vector control (MOI 30). After 24 h post-infection (24 hpi), supernatants of infected HeLa cells were collected and added to THP-1 macrophages. Samples were taken every 2 h and LDH-release was measured in a luminescence plate reader (Clario Star). All data were acquired from three independent biological replicates Significance was determined using 2way ANOVA Šídák’s multiple comparisons tests. *p ≤ 0.0379, **p ≤ 0.0056, ***p ≤ 0.0006. Data are presented as mean values ± SD. Source data are provided as a Source Data file Graphs.