The four cysteines conserved in PAAR-like proteins are required for IglG secretion and function.

(A) Levels of 2HA-tagged IglG variants were assessed in the concentrated supernatant fractions or in bacterial pellets of the indicated strains grown in the presence of 5% KCl. Since all of the IglG constructs were expressed in the U112 background, IglC was similarly secreted in these strains. The non-secreted inner membrane protein PdpB was included as a lysis control. (B) J774 macrophages were infected at an MOI of one with the indicated strains. Intracellular burden was assessed by determination of viable counts at 2 and 24 h. (C) Phagosomal rupture in BMDMs infected with the indicated strains at an MOI of 100 was determined at 2 h by flow cytometry using the β-lactamase/CCF4 assay. (D) Type I IFN secretion in the supernatant of BMDMs infected with the indicated strains at an MOI of 1 was determined by the ISRE-luciferase bioassay and normalized to the value of the bioassay in uninfected macrophages. (E) Cell death of BMDMs infected with the indicated strains at an MOI of 1 was monitored in real time in the presence of propidium iodide by fluorescence readings every 15 min. (F) Survival of mice following intradermal challenge with U112 strains. Mice were challenged with the dose indicated in Materials and Methods and monitored for 21 days for signs of illness. (A-E) Mean and (A-D) SD of triplicates from one experiment representative of 2 to 3 independent experiments are shown. Unpaired t-tests were performed; two-tailed P-values are shown. (F) Mantel-Cox test was performed, P-values for the comparison with WT survival curves are shown (ns: not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).