Deep phosphoproteomics of Klebsiella pneumoniae reveals HipA-mediated tolerance to ciprofloxacin

by Payal Nashier, Isabell Samp, Marvin Adler, Fiona Ebner, Lisa Thai Lê, Marc Göppel, Carsten Jers, Ivan Mijakovic, Sandra Schwarz, Boris Macek

Klebsiella pneumoniae belongs to the group of bacterial pathogens causing the majority of antibiotic-resistant nosocomial infections worldwide; however, the molecular mechanisms underlying post-translational regulation of its physiology are poorly understood. Here we perform a comprehensive analysis of Klebsiella phosphoproteome, focusing on HipA, a Ser/Thr kinase involved in antibiotic tolerance in Escherichia coli. We show that overproduced K. pneumoniae HipA (HipA_kp) is toxic to both E. coli and K. pneumoniae and its toxicity can be rescued by overproduction of the antitoxin HipB_kp. Importantly, HipA_kp overproduction leads to increased tolerance against ciprofloxacin, a commonly used antibiotic in the treatment of K. pneumoniae infections. Proteome and phosphoproteome analyses in the absence and presence of ciprofloxacin confirm that HipA_kp has Ser/Thr kinase activity, auto-phosphorylates at S150, and shares multiple substrates with HipA_ec, thereby providing a valuable resource to clarify the molecular basis of tolerance and the role of Ser/Thr phosphorylation in this human pathogen.