Fig 1. The growth of a ΔyopH mutant is restored in absence of ROS in vivo and YopH is necessary and sufficient to block extracellular ROS produced by BM neutrophils in vitro.

(A-B) Female C57BL/6J or C57BL/6J gp91^phox-/- mice were inoculated with a 1:1 mixture of IP2666 WT-Yptb and ΔyopH-Kan^R (A) intravenously with 1x10³ CFU or (B) intratracheally with 5x10³ CFU. Spleens and lungs were collected, weighed, homogenized, and plated for CFU two days post- infection. The competitive indices (C.I.) were determined by patching 100 colonies on L-Irgasan and L-Irgasan+Kanamycin plates. Each symbol represents an individual mouse; horizontal bars represent the geometric mean. (C-F) BM neutrophils were infected at a MOI of 20:1 with (C-D) WT-Yptb, ΔyopH, or Δ5 (a mutant lacking 5 effector Yops, H, E, O, M, J), (E-F) WT-Yptb+pBAD, Δ5+YopH (a strain expressing only YopH at native levels from an arabinose inducible plasmid),or Δ5+pBAD, and monitored for extracellular ROS production using isoluminol chemiluminescence assay. (D and F) Total chemiluminescence was determined by computing the area under the curve (AUC) using GraphPad Prism version 7. Mean ±SD of a representative experiment from 2–3 independent experiments done in 2–3 replicates is presented. Statistical significance was calculated using (A) the Mann-Whitney t-test, (B) one-way ANOVA followed by Tukey’s Post-test after log₁₀ transformation of the data, or (D, F) one-way ANOVA followed by Tukey’s Post-test for area under the curve (AUC).