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Pseudomonas aeruginosa is a difficult-to-treat Gram-negative bacterial
pathogen causing life-threatening infections. Adaptive resistance (AR)
to cationic peptide antibiotics such as polymyxin B impairs the
therapeutic success. This self-protection is mediated by two component
systems (TCS) consisting of a membrane-bound histidine kinase and an
intracellular response regulator (RR). As phosphorylation of the key RR
aspartate residue is transient during signaling and hydrolytically
unstable, the study of these systems is challenging. Therefore, we
applied a tailored reverse polarity chemical proteomic strategy to
capture this transient modification and read-out RR phosphorylation in
complex proteomes using a nucleophilic probe. An ideal trapping
methodology was developed with a recombinant RR demonstrating the
importance of fine-tuned acidic pH values to facilitate the attack on
the aspartate carbonyl C-atom and prevent unproductive hydrolysis.
Analysis of Bacillus subtilis and P. aeruginosa proteomes revealed the
detection of multiple phosphoaspartate sites, which closely resembled
the conserved RR sequence motif. With this validated strategy we
dissected the signaling of dynorphin A, a human peptide stress hormone,
which is sensed by P. aeruginosa to mediate AR. Intriguingly, our
methodology identified CprR as an unprecedented RR in dynorphin A
This project was funded by the European Research Council (ERC) and the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 725085, CHEMMINE, ERC consolidator grant). SMH acknowledges funding by the Fonds der Chemischen Industrie (Liebig Fellowship) and the TUM Junior Fellow Fund. The authors gratefully acknowledge M. Wolff, K. Bäuml and K. Gliesche for technical assistance.