Figure 3. PGRPs induce membrane depolarization, •OH formation, and HtrA expression through the CssR-CssS two-component system, and •OH is responsible for PGRP-induced killing.

(a) Membrane depolarization or (b) •OH production in WT B. subtilis (closed symbols) or ΔcssS or ΔcssR mutants (open symbols) incubated with the indicated PGRPs, or CCCP, or gentamicin (positive controls), or BSA (negative control) measured by flow cytometry with fluorescent probes DiBAC₄(3) (a) or HPF (b) and expressed as ratios of mean fluorescence intensity of PGRP-, CCCP-, or gentamicin-treated to BSA-treated bacteria are shown. (c) •OH-dependent killing of WT B. subtilis incubated with the indicated PGRPs, 20 μM CCCP, or BSA as a control without or with 300 μM dipyridyl measured by colony counts is shown. (d) HtrA mRNA expression in WT B. subtilis or ΔcssS or ΔcssR mutants incubated with PGLYRP-1, PGLYRP-4, or BSA (100 μg ml⁻¹), or gentamicin (5 μg ml⁻¹) for 30 min measured by qRT-PCR is shown. The results are means of 3 to 6 experiments ± SEM; *, P≤ 0.05; **, P≤ 0.001 (t-test) versus WT (a, b, d) or versus no dipyridyl (c).