Abstract:
Post-translational modifications (PTMs) play an indispensable role in the rapid execution
of responses that regulate a number of biological functions. Protein Ser/Thr kinases
(STKs) are key regulators of vital cellular processes, including antibiotic tolerance,
metabolism, virulence, and stress response. Despite their importance, the molecular
mechanisms and targets of STKs are underinvestigated, especially in the context of
post-translational regulation in pathogenic bacteria. Antibiotic tolerance and persistence
are a major contributor to the relapse of many chronic infections and frequently result in
antibiotic overuse and the development of antibiotic resistance. One of the best-studied
drivers of persistence is a Ser/Thr kinase HipA, first characterized in Escherichia coli.
Multiple HipA-like kinases have been recently reported to be present in bacteria,
including pathogens such as Klebsiella pneumoniae, but their functions remain poorly
understood. In my thesis, I focused on elucidating the function of two such STKs, HipH
(YjjJ) in E. coli and HipA in K. pneumoniae to understand their potential role in regulating
cellular processes.
To explore this, I applied state-of-the-art mass spectrometry-based quantitative
phosphoproteomics to gain new insights into the functions and substrates of these
kinases and fill crucial gaps in knowledge of bacterial physiology and pathogenesis. I
first reviewed recent advances in quantitative phosphoproteomics to highlight the utility
of LC-MS/MS technologies combined with quantitative proteomics strategies to
investigate dynamic phosphorylation changes during various biological processes. I
then applied this technology to study HipA-family Ser/Thr kinase HipH (YjjJ) in E. coli.
Using quantitative phosphoproteomics based on stable isotope labeling by amino acids
in cell culture (SILAC) and in vitro kinase assay, I demonstrated that HipH
phosphorylates specific targets such as the ribosomal protein RpmE and the carbon
storage regulator CsrA. Therefore, HipH plays an important role in regulating ribosome
assembly, cell division, and central carbon metabolism, but it does not confer antibiotic
tolerance like its homolog HipA. I have also shown that HipH cross-talks with other
bacterial kinases, revealing a complex network of regulatory interactions. The final part
of my work focused on Klebsiella pneumoniae, a major cause of antibiotic-resistant
VIII
nosocomial infections worldwide. I demonstrated that overproduced K. pneumoniae
HipA (HipAkp) is toxic to both E. coli and K. pneumoniae, and this toxicity can be rescued
by overproduction of the antitoxin HipBkp. Importantly, I showed that HipAkp
overproduction leads to increased tolerance against ciprofloxacin, linking HipA activity
to antibiotic persistence in this organism. Through proteome and phosphoproteome
analyses, I confirmed that HipAkp has Ser/Thr kinase activity, auto-phosphorylates at
S150, and shares multiple substrates with its E. coli counterpart. I performed a
comprehensive analysis of the K. pneumoniae phosphoproteome with HipAkp
overproduction to generate the largest dataset of phosphorylated proteins for this
bacterium.
Overall, my work provides an in-depth analysis of the roles of the two HipA-like kinases
in antibiotic tolerance and metabolism, offering new insights into their functions and
regulatory networks. These findings also provide a foundation for future research on
post-translational regulation of bacterial physiology.
Phosphoproteome