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

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. Antibiotic tolerance plays a significant role in shaping the evolutionary dynamics of bacterial populations subjected to repeated antibiotic treatments. A subset of the population developing tolerance to high concentrations of antibiotics and characterized by slow growth are called persisters. Exposure to various bactericidal antibiotics commonly employed in the treatment of K. pneumoniae infections has revealed the presence of multidrug-tolerant persister cells. Persister cells have also been identified in clinical isolates from individuals experiencing recurring bloodstream infections, demonstrating genomic alterations in relapsed isolates that evolved within the host. However, the mechanism of formation of these multidrug-tolerant persister cells in K. pneumoniae is understudied. One of the best-studied drivers of persistence is the eukaryotic-type Ser/Thr kinase, HipA in E. coli and a recent study has identified homologs of the hipA in many bacterial species.

We used a comprehensive analysis of Klebsiella phosphoproteome, focusing on HipA, a Ser/Thr kinase involved in antibiotic tolerance in Escherichia coli. We showed that overproduced K. pneumoniae HipA (HipAkp) is toxic to both E. coli and K. pneumoniae and its toxicity can be rescued by overproduction of the antitoxin HipBkp. Importantly, HipAkp 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 confirmed that HipAkp has Ser/Thr kinase activity, auto-phosphorylates at S150, and shares multiple substrates with HipAec including GltX (glutamate tRNA synthetase) at S239. In E. coli, this phosphorylation leads to the accumulation of uncharged glutamate tRNA and activates the stringent response in cells, ultimately leading to antibiotic tolerance and persistence. Our study provides a valuable resource to clarify the molecular basis of tolerance and the role of Ser/Thr phosphorylation in this human pathogen.