Figure 3.

EspK, EspJ, and PPE68 are Esx-1 substrates. (A) nLC–MRM analysis of the levels of EspK in cell lysate (CL, blue bars) and culture filtrate (CF, red bars) relative to the levels of EspK in the WT M strain. The ΔRD1 and espK::Tn strains served as a negative control for EspK detection and demonstrate specificity of the approach. (B) nLC–MRM analysis of the levels of the EspJ substrate in the CL and CF. The ΔRD1 strain and espJ::Tn strain serve as a negative control for EspJ detection. We report a weak false positive signal indicating extremely low levels of EspJ peptides in the espJ::Tn strain. One EspJ peptide was observed at <0.5% of the levels in the espB::Tn strain and <0.2% of the EspJ levels observed in the WT M strain. This low false-positive signal was due to the large EspJ signal observed in the espB::Tn strain, which was the previous nLC injection on the mass spectrometer. For example, the EspJ tryptic peptide TSSMSTAADIYAK was present at ∼1e⁴cps2 in the M strain and was not detected in ΔRD1 or the espJ::Tn strains. AEPLAVDPAR is a high-intensity proteotypic peptide for EspJ that was measured at >2e⁶cps2 in the WT strain and 1.3e⁶cps2 in the espB::Tn strain, which was injected immediately prior to espJ::Tn analysis. Further evidence of this is the gradual reduction in carryover signal with each successive analysis of the espJ::Tn strain. (C) nLC–MRM analysis of the levels of PPE68 in the CL and CF. The ΔRD1 strain served as a negative control for PPE68 detection. Error bars represent the average propagated standard error and were calculated as described in the Experimental Procedures. The differences between the levels of each protein in the WT and each Esx-1-deficient strain were considered statistically significant if p values were less than 0.05 based on a two-tailed Student’s t test. The levels of the indicated protein in each Esx-1-deficient strain were significantly different from the wild-type levels with the following exceptions: The levels of EspK in the cell lysates of the espG strain were not different from the levels of EspK in the wild-type lysate. The levels of PPE68 in the cell lysate from the espJ strain were not significantly different from the wild-type strain. The levels of PPE68 in the culture filtrate generated from the espK strain were not significantly different from the levels of PPE68 in the culture filtrate from the wild-type strain. The actual p values are listed in Supplemental Table S2. Log₂ transformed versions of the CF data are available in Supplemental Figure S4.