Recent progress in quantitative phosphoproteomics.

INTRODUCTION: Protein phosphorylation is a critical post-translational modification involved in the regulation of numerous cellular processes from signal transduction to modulation of enzyme activities. Knowledge of dynamic changes of phosphorylation levels during biological processes, under various treatments or between healthy and disease models is fundamental for understanding the role of each phosphorylation event. Thereby, LC-MS/MS based technologies in combination with quantitative proteomics strategies evolved as a powerful strategy to investigate the function of individual protein phosphorylation events. AREAS COVERED: State-of-the-art labeling techniques including stable isotope and isobaric labeling provide precise and accurate quantification of phosphorylation events. Here, we review the strengths and limitations of recent quantification methods and provide examples based on current studies, how quantitative phosphoproteomics can be further optimized for enhanced analytic depth, dynamic range, site localization, and data integrity. Specifically, reducing the input material demands is key to a broader implementation of quantitative phosphoproteomics, not least for clinical samples. EXPERT OPINION: Despite quantitative phosphoproteomics is one of the most thriving fields in the proteomics world, many challenges still have to be overcome to facilitate even deeper and more comprehensive analyses as required in the current research, especially at single cell levels and in clinical diagnostics.

Bacillus anthracis chain length, a virulence determinant, is regulated by membrane localized serine/threonine protein kinase PrkC.

Anthrax is a zoonotic disease caused by Bacillus anthracis, a spore-forming pathogen that displays a chaining phenotype. It has been reported that the chaining phenotype acts as a virulence factor in B. anthracis In this study, we identify a serine/threonine protein kinase of B. anthracis, PrkC, the only kinase localized at the bacteria-host interface, as a determinant of B. anthracis chain length. In vitro, prkC disruption strain (BAS ΔprkC) grew as shorter chains throughout the bacterial growth cycle. A comparative analysis between the parent strain and BAS ΔprkC indicated that the levels of proteins, BslO and Sap, associated with the regulation of the bacterial chain length, were upregulated in BAS ΔprkC BslO is a septal murein hydrolase that catalyzes daughter cell separation and Sap is an S-layer structural protein required for the septal localization of BslO. PrkC disruption also has a significant effect on bacterial growth, cell wall thickness, and septa formation. Upregulation of ftsZ in BAS ΔprkC was also observed. Altogether, our results indicate that PrkC is required for maintaining optimum growth, cell wall homeostasis and most importantly - for the maintenance of the chaining phenotype.IMPORTANCEChaining phenotype acts as a virulence factor in Bacillus anthracis This is the first study that identifies a 'signal transduction protein' with an ability to regulate the chaining phenotype in Bacillus anthracis We show that the disruption of the lone surface-localized serine/threonine protein kinase, PrkC, leads to the shortening of the bacterial chains. We report upregulation of the de-chaining proteins in the PrkC disruption strain. Apart from this, we also report for the first time that PrkC disruption results in an attenuated cell growth, a decrease in the cell wall thickness and aberrant cell septa formation during the logarithmic phase of growth - a growth phase where PrkC is expressed maximally.

E. coli Toxin YjjJ (HipH) Is a Ser/Thr Protein Kinase That Impacts Cell Division, Carbon Metabolism, and Ribosome Assembly.

Protein Ser/Thr kinases are posttranslational regulators of key molecular processes in bacteria, such as cell division and antibiotic tolerance. Here, we characterize the E. coli toxin YjjJ (HipH), a putative protein kinase annotated as a member of the family of HipA-like Ser/Thr kinases, which are involved in antibiotic tolerance. Using SILAC-based phosphoproteomics we provide experimental evidence that YjjJ is a Ser/Thr protein kinase and its primary protein substrates are the ribosomal protein RpmE (L31) and the carbon storage regulator CsrA. YjjJ activity impacts ribosome assembly, cell division, and central carbon metabolism but it does not increase antibiotic tolerance as does its homologue HipA. Intriguingly, overproduction of YjjJ and its kinase-deficient variant can activate HipA and other kinases, pointing to a cross talk between Ser/Thr kinases in E. coli. IMPORTANCE Adaptation to growth condition is the key for bacterial survival, and protein phosphorylation is one of the strategies adopted to transduce extracellular signal in physiological response. In a previous work, we identified YjjJ, a putative kinase, as target of the persistence-related HipA kinase. Here, we performed the characterization of this putative kinase, complementing phenotypical analysis with SILAC-based phosphoproteomics and proteomics. We provide the first experimental evidence that YjjJ is a Ser/Thr protein kinase, having as primary protein substrates the ribosomal protein RpmE (L31) and the carbon storage regulator CsrA. We show that overproduction of YjjJ has a major influence on bacterial physiology, impacting DNA segregation, cell division, glycogen production, and ribosome assembly.

Proteome Dynamics during Antibiotic Persistence and Resuscitation.

During antibiotic persistence, bacterial cells become transiently tolerant to antibiotics by restraining their growth and metabolic activity. Detailed molecular characterization of antibiotic persistence is hindered by low count of persisting cells and the need for their isolation. Here, we used sustained addition of stable isotope-labeled lysine to selectively label the proteome during hipA-induced persistence and hipB-induced resuscitation of Escherichia coli cells in minimal medium after antibiotic treatment. Time-resolved, 24-h measurement of label incorporation allowed detection of over 500 newly synthesized proteins in viable cells, demonstrating low but widespread protein synthesis during persistence. Many essential proteins were newly synthesized, and several ribosome-associated proteins such as RaiA and Sra showed high synthesis levels, pointing to their roles in maintenance of persistence. At the onset of resuscitation, cells synthesized the ribosome-splitting GTPase HflX and various ABC transporters, restored translation machinery, and resumed metabolism by inducing glycolysis and biosynthesis of amino acids. IMPORTANCE While bactericidal antibiotics typically require actively growing cells to exploit their function, persister cells are slowly replicating which makes them tolerant to the lethal action of antimicrobials. Here, we used an established in vitro model of bacterial persistence based on overexpression of the paradigm toxin-antitoxin (TA) system hipA/hipB to devise a generic method for temporal analysis of protein synthesis during toxin-induced persistence and antitoxin-mediated resuscitation. Our time-resolved, 24-h measurement of label incorporation demonstrated low but widespread protein synthesis during persistence. At the onset of resuscitation, cells restored translation machinery and resumed metabolism by inducing glycolysis and biosynthesis of amino acids. Our study provides the first global analysis of protein synthesis in persisting and resuscitating bacterial cells, and as such, presents an unprecedented resource to study the processes governing antibiotic persistence.