Fig. 6: Effects of csa1 amplification on the immunostimulatory capacity of strains.

a Confluent lawns of HEK-hTLR2 cells were stimulated for 18 h with 0.5% culture filtrates of csa1 copy number variants grown to stationary phase. IL-8 protein levels within the supernatants were quantified by ELISA (R&D Systems). IL-8 amounts are displayed as bars referring to the left Y-axis, data derived from a single stimulation experiment are shown. For each lineage, two parallel bacterial cultures (labeled with A and B) were used for stimulation and the csa1 copy number of each independent culture determined by qPCR (displayed as filled circle referring to the right Y-axis). Source data are provided as a Source Data file. b IL-8 protein levels shown in (a) are expressed in function of the csa1 copy number of the stimulating strain. Sample sizes were the following: 0 - n = 2; 1–30 - n = 7; 91–121 - n = 10; 121–151 - n = 3; 181–210 - n = 2). Mean and SEM is shown, statistical analysis was performed using one-way ANOVA (F = 7,710; DF=23) followed by Bonferroni’s multiple comparison test. Source data are provided as a Source Data file. c 5 × 10⁵ HL60 cells were stimulated for 5 h with 1.5% culture filtrates of csa1 copy number variants (∆lpl1lpl2lpl3 background) grown to stationary phase. IL-8 protein levels within the supernatants were quantified using ELISA (R&D Systems). Data represent three independent supernatants of each copy number variant used in three independent stimulations (n = 9 in each group). Mean and SEM are shown. Statistical analysis was performed using one-way ANOVA (F = 3,632; DF = 38) followed by Bonferroni’s multiple comparison test. Source data are provided as a Source Data file. d Polymorphonuclear cells (PMNs) were isolated from fresh blood of healthy human volunteers. 5 × 10⁵ PMNs were stimulated for 5 h with 1.5% culture filtrates of csa1 copy number variants (∆lplΔlpp3∆lpp4 background) grown to stationary phase. IL-8 protein levels within the supernatants were quantified using by ELISA (R&D Systems). Data represent three independent supernatants of each copy number variant used on PMNs of a single donor. Mean and SEM are shown. Statistical analysis was performed using one-way ANOVA (F = 15,74; DF = 11) followed by Bonferroni’s multiple comparison test. The experiment was repeated thrice with PMNs from different donors with similar results. Source data are provided as a Source Data file.