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A Tailored Phosphoaspartate Probe Unravels CprR as a Response Regulator in Pseudomonas Aeruginosa Interkingdom Signaling

submitted on 11.11.2020, 22:35 and posted on 16.11.2020, 04:36 by Patrick Allihn, Mathias W. Hackl, Christina Ludwig, Stephan M. Hacker, Stephan A. Sieber
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 interkingdom signaling.


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.


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Technical University of Munich



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Declaration of Conflict of Interest

There are no conflicts to declare.