Proteomic Investigation Uncovers Potential Targets and Target Sites of Pneumococcal Serine-Threonine Kinase StkP and Phosphatase PhpP

Hirschfeld, Claudia; Gómez-Mejía, Alejandro; Bartel, Jürgen; Hentschker, Christian; Rohde, Manfred; Maaß, Sandra; Hammerschmidt, Sven; Becher, Dörte

doi:10.3389/fmicb.2019.03101

Cited by 32 publications

(46 citation statements)

References 98 publications

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“…(D) Total number of phosphosites and phosphoproteins identified from the three bacterial strains. (E) Comparison of these data to relevant related studies (Misra et al, 20 , 21 Prust et al, 12 Henry et al, 22 and Hirschfeld et al 23 ). (F) Histograms illustrating the distribution of the number of phosphosites per protein.…”

Section: Resultsmentioning

confidence: 95%

Automated Phosphopeptide Enrichment for Gram-Positive Bacteria

2021

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Protein phosphorylation in prokaryotes has gained more attention in recent years as several studies linked it to regulatory and signaling functions, indicating importance similar to protein phosphorylation in eukaryotes. Studies on bacterial phosphorylation have so far been conducted using manual or HPLC-supported phosphopeptide enrichment, whereas automation of phosphopeptide enrichment has been established in eukaryotes, allowing for high-throughput sampling. To facilitate the prospect of studying bacterial phosphorylation on a systems level, we here established an automated Ser/Thr/Tyr phosphopeptide enrichment workflow on the Agilent AssayMap platform. We present optimized buffer conditions for TiO 2 and Fe(III)-NTA-IMAC cartridge-based enrichment and the most advantageous, species-specific loading amounts for Streptococcus pyogenes , Listeria monocytogenes , and Bacillus subtilis . For higher sample amounts (≥250 μg), we observed superior performance of the Fe(III)-NTA cartridges, whereas for lower sample amounts (≤100 μg), TiO 2 -based enrichment is equally efficient. Both cartridges largely enriched the same set of phosphopeptides, suggesting no improvement of peptide yield by the complementary use of the two cartridges. Our data represent, to the best of our knowledge, the largest phosphoproteome identified in a single study for each of these bacteria.

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Section: Resultsmentioning

confidence: 95%

Automated Phosphopeptide Enrichment for Gram-Positive Bacteria

2021

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“…Many substrates of PASTA-eSTKs have been identified in the different Gram-positive bacteria. Among these kinase substrates are proteins from various cellular pathways, but some functional classes are overrepresented: (i) Proteins acting in carbon and cell wall metabolism such as the HPr and YvcK proteins of B. subtilis and L. monocytogenes (28, 42–44), (ii) regulatory proteins such as the WalR and GraR response regulators of B. subtilis and Staphylococcus aureus , respectively (45, 46), and (iii) proteins acting in cell division, among which GpsB is one of the proteins consistently found as a PASTA-eSTK substrate in B. subtilis, L. monocytogenes and Streptococcus pneumoniae (28, 47, 48). This diversity of substrates and their own – often pleiotropic - functions have impeded the identification of primary and the discrimination of less important kinase substrates.…”

Section: Discussionmentioning

confidence: 99%

MurA escape mutations uncouple peptidoglycan biosynthesis from PrkA signaling

Wamp

Rothe

Holland

et al. 2021

Preprint

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Gram-positive bacteria are protected by a thick mesh of peptidoglycan (PG) completely engulfing their cells. This PG network is the main component of the bacterial cell wall, it provides rigidity and acts as foundation for the attachment of other surface molecules. Biosynthesis of PG consumes a high amount of cellular resources and therefore requires careful adjustments to environmental conditions. An important switch in the control of PG biosynthesis of Listeria monocytogenes, a Gram-positive pathogen with a high infection fatality rate, is the serine/threonine protein kinase PrkA. A key substrate of this kinase is the small cytosolic protein ReoM. We have shown previously that ReoM phosphorylation regulates PG formation through control of MurA stability. MurA catalyzes the first step in PG biosynthesis and the current model suggests that phosphorylated ReoM prevents MurA degradation by the ClpCP protease. In contrast, conditions leading to ReoM dephosphorylation stimulate MurA degradation. How ReoM controls degradation of MurA and potential other substrates is not understood. Also, the individual contribution of the ~20 other known PrkA targets to PG biosynthesis regulation is unknown. We here present murA mutants which escape proteolytic degradation. The release of MurA from ClpCP-dependent proteolysis was able to constitutively activate PG biosynthesis and further enhances the intrinsic cephalosporin resistance of L. monocytogenes. This activation required the RodA3/PBP B3 transglycosylase/transpeptidase pair as additional effectors of the PrkA signaling route. One murA escape mutation not only fully rescued an otherwise non-viable prkA mutant during growth in batch culture and inside macrophages but also overcompensated cephalosporin hypersensitivity. Our data collectively indicate that the main purpose of PrkA-mediated signaling in L. monocytogenes is control of MurA stability during extra- and intracellular growth. These findings have important implications for the understanding of PG biosynthesis regulation and β-lactam resistance of L. monocytogenes and related Gram-positive bacteria.

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“…In S.pneumoniae , a mass-spectrometry based label-free quantitative (LFQ) approach was used to characterize and determine the impact of StkP on the protein expression profiles. Notable changes in the proteome of the kinase mutant ΔstkP in comparison to the WT strain have been observed especially in the cluster of amino acid metabolism, energy metabolism, regulatory fuction and transcription(Hirschfeld et al 2019 ). In the present study, comparative proteomics approaches revealed that SsSTK can regulate the expression of proteins involved in bacterial central metabolism, stress response and virulence.…”

Section: Discussionmentioning

confidence: 99%

Quantitative proteomic analysis reveals that serine/threonine kinase is involved in Streptococcus suis virulence and adaption to stress conditions

et al. 2021

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The eukaryotic-type serine/threonine kinase of Streptococcus suis serotype 2 (SS2) performs critical roles in bacterial pathogenesis. In this study, isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were used to analyze the protein profiles of wild type strain SS2-1 and its isogenic STK deletion mutant (Δstk). A total of 281 significant differential proteins, including 147 up-regulated and 134 down-regulated proteins, were found in Δstk. Moreover, 69 virulence factors (VFs) among these 281 proteins were predicted by the Virulence Factor Database (VFDB), including 38 downregulated and 31 up-regulated proteins in Δstk, among which 15 down regulated VFs were known VFs of SS2. Among the down-regulated proteins, high temperature requirement A (HtrA), glutamine synthase (GlnA), ferrichrome ABC transporter substrate-binding protein FepB, and Zinc-binding protein AdcA are known to be involved in bacterial survival and/or nutrient and energy acquisition under adverse host conditions. Overall, our results indicate that STK regulates the expression of proteins involved in virulence of SS2 and its adaption to stress environments.

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Proteomic Investigation Uncovers Potential Targets and Target Sites of Pneumococcal Serine-Threonine Kinase StkP and Phosphatase PhpP

Cited by 32 publications

References 98 publications

Automated Phosphopeptide Enrichment for Gram-Positive Bacteria

Automated Phosphopeptide Enrichment for Gram-Positive Bacteria

MurA escape mutations uncouple peptidoglycan biosynthesis from PrkA signaling

Quantitative proteomic analysis reveals that serine/threonine kinase is involved in Streptococcus suis virulence and adaption to stress conditions

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