Phosphoproteomic analysis of bacillus Calmette–Guérin using gel-based and gel-free approaches

Zheng, Jianhua; Liu, Liguo; Liu, Bo; Jin, Qi

doi:10.1016/j.jprot.2015.06.003

Cited by 10 publications

(9 citation statements)

References 68 publications

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“…2A ; see also Table S3A in the supplemental material). Although these results partially overlap those from prior studies ( 21 , 26 – 28 ) ( Fig. 2B ), many new phosphoproteins were identified, substantially expanding the M. tuberculosis phosphoproteome.…”

Section: Resultssupporting

confidence: 74%

Multisystem Analysis of Mycobacterium tuberculosis Reveals Kinase-Dependent Remodeling of the Pathogen-Environment Interface

Carette

Platig

Young

et al. 2018

mBio

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Tuberculosis is the leading killer among infectious diseases worldwide. Increasing multidrug resistance has prompted new approaches for tuberculosis drug development, including targeted inhibition of virulence determinants and of signaling cascades that control many downstream pathways. We used a multisystem approach to determine the effects of a potent small-molecule inhibitor of the essential Mycobacterium tuberculosis Ser/Thr protein kinases PknA and PknB. We observed differential levels of phosphorylation of many proteins and extensive changes in levels of gene expression, protein abundance, cell wall lipids, and intracellular metabolites. The patterns of these changes indicate regulation by PknA and PknB of several pathways required for cell growth, including ATP synthesis, DNA synthesis, and translation. These data also highlight effects on pathways for remodeling of the mycobacterial cell envelope via control of peptidoglycan turnover, lipid content, a SigE-mediated envelope stress response, transmembrane transport systems, and protein secretion systems. Integrated analysis of phosphoproteins, transcripts, proteins, and lipids identified an unexpected pathway whereby threonine phosphorylation of the essential response regulator MtrA decreases its DNA binding activity. Inhibition of this phosphorylation is linked to decreased expression of genes for peptidoglycan turnover, and of genes for mycolyl transferases, with concomitant changes in mycolates and glycolipids in the cell envelope. These findings reveal novel roles for PknA and PknB in regulating multiple essential cell functions and confirm that these kinases are potentially valuable targets for new antituberculosis drugs. In addition, the data from these linked multisystems provide a valuable resource for future targeted investigations into the pathways regulated by these kinases in the M. tuberculosis cell.

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Section: Resultssupporting

confidence: 74%

Multisystem Analysis of Mycobacterium tuberculosis Reveals Kinase-Dependent Remodeling of the Pathogen-Environment Interface

Carette

Platig

Young

et al. 2018

mBio

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“…The potential adaptive advantage for the pathogen may result in a fitness cost of slower growth. In a follow-up phosphoproteomic study, Zheng et al ( 2015 ) found 659 phosphosites on 398 proteins in BCG harvested during stationary phase. The majority (40.1%) of identified phosphoproteins in this case were involved in regulation of metabolism.…”

Section: Comparative Phosphoproteomic Analysis Of Mycobacteriamentioning

confidence: 99%

“…Mycobacterial STPK phosphorylation has been long associated with pathogenicity (Sherman and Grundner, 2014 ), which has driven efforts to improve our understanding of the role of phosphorylation in M. tuberculosis (Prisic and Husson, 2014 ). Currently, there are six published manuscripts describing the phosphoproteome of a member of the Mycobacteria— M. tuberculosis H37Rv (Prisic et al, 2010 ; Kusebauch et al, 2014 ), M. smegmatis , and M. bovis BCG (Nakedi et al, 2015 ; Zheng et al, 2015 ), a clinical isolate of M. tuberculosis Beijing lineage (Fortuin et al, 2015 ) and a ΔpknE deletion mutant strain of M. tuberculosis (Parandhaman et al, 2014b ). While these studies are discussed in more detail below, a summary of the methods used and relevant results for each of them is presented in Table 1 .…”

Section: Introductionmentioning

confidence: 99%

Mass Spectrometry Offers Insight into the Role of Ser/Thr/Tyr Phosphorylation in the Mycobacteria

et al. 2016

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Phosphorylation is a post translational modification which can rapidly regulate biochemical pathways by altering protein function, and has been associated with pathogenicity in bacteria. Once engulfed by host macrophages, pathogenic bacteria are exposed to harsh conditions and must respond rapidly in order to survive. The causative agent of TB, Mycobacterium tuberculosis, is unusual amongst the bacteria because it can survive within the host macrophage for decades in a latent state, demonstrating a remarkable capacity to successfully evade the host immune response. This ability may be mediated in part by regulatory mechanisms such as ser/thr/tyr phosphorylation. Mass spectrometry-based proteomics has afforded us the capacity to identify hundreds of phosphorylation sites in the bacterial proteome, allowing for comparative phosphoproteomic studies in the mycobacteria. There remains an urgent need to validate the reported phosphosites, and to elucidate their biological function in the context of pathogenicity. However, given the sheer number of putative phosphorylation events in the mycobacterial proteome, and the technical difficulty of assigning biological function to a phosphorylation event, it will not be trivial to do so. There are currently six published phosphoproteomic investigations of a member of mycobacteria. Here, we combine the datasets from these studies in order to identify commonly detected phosphopeptides and phosphosites in order to present high confidence candidates for further validation. By applying modern mass spectrometry-based techniques to improve our understanding of phosphorylation and other PTMs in pathogenic bacteria, we may identify candidates for therapeutic intervention.

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“…Among the 301 proteins identified in a high throughput phosphoproteomic study from M. tuberculosis H37Rv, 69 proteins were linked to cell division and cell wall synthesis processes (54). In a recent study, 398 M. bovis BCG proteins were identified to be phosphorylated, and 19.5% of these phosphoproteins were involved in various cellular processes (57). Similarly, phosphoproteomic analysis of M. smegmatis mc 2 and M. bovis BCG has revealed the phosphorylation of a number of cell division proteins, such as FtsQ, FtsW, CwsA, FtsK, and FtsY (56).…”

Section: Discussionmentioning

confidence: 99%

Serine/Threonine Protein Phosphatase PstP of Mycobacterium tuberculosis Is Necessary for Accurate Cell Division and Survival of Pathogen

Sharma

Arora

Singh

et al. 2016

Journal of Biological Chemistry

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Protein phosphatases play vital roles in phosphorylation-mediated cellular signaling. Although there are 11 serine/threonine protein kinases in Mycobacterium tuberculosis, only one serine/threonine phosphatase, PstP, has been identified. Although PstP has been biochemically characterized and multiple in vitro substrates have been identified, its physiological role has not yet been elucidated. In this study, we have investigated the impact of PstP on cell growth and survival of the pathogen in the host. Overexpression of PstP led to elongated cells and partially compromised survival. We find that depletion of PstP is detrimental to cell survival, eventually leading to cell death. PstP depletion results in elongated multiseptate cells, suggesting a role for PstP in regulating cell division events. Complementation experiments performed with PstP deletion mutants revealed marginally compromised survival, suggesting that all of the domains, including the extracellular domain, are necessary for complete rescue. On the other hand, the catalytic activity of PstP is absolutely essential for the in vitro growth. Mice infection experiments establish a definitive role for PstP in pathogen survival within the host. Depletion of PstP from established infections causes pathogen clearance, indicating that the continued presence of PstP is necessary for pathogen survival. Taken together, our data suggest an important role for PstP in establishing and maintaining infection, possibly via the modulation of cell division events.

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Phosphoproteomic analysis of bacillus Calmette–Guérin using gel-based and gel-free approaches

Cited by 10 publications

References 68 publications

Multisystem Analysis of Mycobacterium tuberculosis Reveals Kinase-Dependent Remodeling of the Pathogen-Environment Interface

Multisystem Analysis of Mycobacterium tuberculosis Reveals Kinase-Dependent Remodeling of the Pathogen-Environment Interface

Mass Spectrometry Offers Insight into the Role of Ser/Thr/Tyr Phosphorylation in the Mycobacteria

Serine/Threonine Protein Phosphatase PstP of Mycobacterium tuberculosis Is Necessary for Accurate Cell Division and Survival of Pathogen

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