Dynamic post-translational modification profiling of Mycobacterium tuberculosis-infected primary macrophages

Budzik, Jonathan M.; Swaney, Danielle L.; Jimenez-Morales, David; Johnson, Jeffrey R.; Garelis, Nicholas E; Repasy, Teresa; Roberts, Allison; Popov, Lauren M.; Parry, Trevor J.; Pratt, Dexter; Ideker, Trey; Krogan, Nevan J.; Cox, Jeffery S.

doi:10.7554/elife.51461

Cited by 50 publications

(51 citation statements)

References 106 publications

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“…Glycosylation, phosphorylation, lipidation, formylation, pupylation, acetylation, and methylation have been identified in M. tuberculosis [88,89]. Several studies have elucidated extensive networks of reversible (S/T/Y) protein phosphorylation in mycobacteria [90][91][92][93], during mycobacterial infection [91,94], and the identification of irreversible protein N-terminal acetylation as an abundant PTM in mycobacteria associated with virulence and survival [17,95].…”

Section: Targeted and Comprehensive Proteomic Approaches Define The Mmentioning

confidence: 99%

The genetic proteome: Using genetics to inform the proteome of mycobacterial pathogens

et al. 2021

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Mycobacterial pathogens pose a sustained threat to human health. There is a critical need for new diagnostics, therapeutics, and vaccines targeting both tuberculous and nontuberculous mycobacterial species. Understanding the basic mechanisms used by diverse mycobacterial species to cause disease will facilitate efforts to design new approaches toward detection, treatment, and prevention of mycobacterial disease. Molecular, genetic, and biochemical approaches have been widely employed to define fundamental aspects of mycobacterial physiology and virulence. The recent expansion of genetic tools in mycobacteria has further increased the accessibility of forward genetic approaches. Proteomics has also emerged as a powerful approach to further our understanding of diverse mycobacterial species. Detection of large numbers of proteins and their modifications from complex mixtures of mycobacterial proteins is now routine, with efforts of quantification of these datasets becoming more robust. In this review, we discuss the “genetic proteome,” how the power of genetics, molecular biology, and biochemistry informs and amplifies the quality of subsequent analytical approaches and maximizes the potential of hypothesis-driven mycobacterial research. Published proteomics datasets can be used for hypothesis generation and effective post hoc supplementation to experimental data. Overall, we highlight how the integration of proteomics, genetic, molecular, and biochemical approaches can be employed successfully to define fundamental aspects of mycobacterial pathobiology.

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Section: Targeted and Comprehensive Proteomic Approaches Define The Mmentioning

confidence: 99%

The genetic proteome: Using genetics to inform the proteome of mycobacterial pathogens

et al. 2021

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“…were analyzed using liquid chromatography and mass spectroscopy, as previously described (31). Mass spectrometry data was assigned to E. coli sequences and MS1…”

Section: Liquid Chromatography Mass Spectroscopy and Label-free Quanmentioning

confidence: 99%

Ceragenins and antimicrobial peptides kill bacteria through distinct mechanisms

Mitchell

Silvis

Talkington

et al. 2020

Preprint

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Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally-occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here we report the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA131. Using transcriptomic and proteomic analyses, we found that Escherichia coli responds similarly to both CAMPs and ceragenins by inducing a Cpx envelope stress response. However, whereas E. coli exposed to CAMPs increased expression of genes involved in colanic acid biosynthesis, bacteria exposed to ceragenins specifically modulated functions related to phosphate transport, indicating distinct mechanisms of action between these two classes of molecules. Although traditional genetic approaches failed to identify genes that confer high-level resistance to ceragenins, using a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) approach we identified E. coli essential genes that when knocked down modify sensitivity to these molecules. Comparison of the essential gene-antibiotic interactions for each of the CAMPs and ceragenins identified both overlapping and distinct dependencies for their antimicrobial activities. Overall, this study indicates that while some bacterial responses to ceragenins overlap with those induced by naturally-occurring CAMPs, these synthetic molecules target the bacterial envelope using a distinctive mode of action.

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“…To understand how splicing regulatory proteins shape global innate immune gene expression, we first needed to identify factors most likely to play a privileged role in the macrophage innate immune response. Two recent publications identified a number of splicing factors that were differentially phosphorylated during bacterial infection (specifically during infection with the intracellular bacterial pathogen Mycobacterium tuberculosis) in a RAW 264.7 macrophage cell line [32] or in primary mouse macrophages [33]. We manually annotated these lists and prioritized a set of SRSF and hnRNPs for transcriptomics analysis.…”

Section: Salmonella-infected Macrophagesmentioning

confidence: 99%

“…SR proteins often work cooperatively and antagonistically with proteins in the heterogenous nuclear ribonucleoprotein (hnRNP) family, which also bind to conserved sequences in exons and introns to influence splicing decisions. We became interested in a role for SR and hnRNP proteins in regulating macrophage gene expression after several global phosphoproteomics studies revealed that proteins involved in mRNA processing are among the most differentially phosphorylated proteins in macrophages following infection with a bacterial [27,28] or fungal pathogen [29], despite showing no significant change in protein abundance. These data led us to speculate that splicing regulatory proteins are functionalized following pathogen sensing.…”

Section: Introductionmentioning

confidence: 99%

Global transcriptomics reveals specialized roles for splicing regulatory proteins in the macrophage innate immune response

West

Wagner

Scott

et al. 2020

Preprint

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Pathogen sensing via pattern recognition receptors triggers massive reprogramming of macro-phage gene expression. While the signaling cascades and transcription factors that activate these responses are well-known, the role of post-transcriptional RNA processing in modulating innate immune gene expression remains understudied. Recent phosphoproteomics analyses revealed that members of the SR and hnRNP families of splicing regulatory proteins are dynamically post-translationally modified in infected macrophages. To begin to test if these splicing factors play a privileged role in controlling the innate immune transcriptome, we analyzed steady state gene expression and alternatively spliced isoform production in ten SR/hnRNP knockdown RAW 264.7 macrophage cell lines following infection with the bacterial pathogen Salmonella enterica serovar Typhimurium (Salmonella). We identified thousands of transcripts whose abundance was increased or decreased by SR/hnRNP knockdown in macrophages. We observed that different SR/hnRNPs control the expression of distinct gene regulons in uninfected and Salmonella-infected macrophages, with several key innate immune genes (Nos2, Mx1, Il1a) relying on multiple SR/hnRNPs to maintain proper induction and/or repression. Knockdown of SR/hnRNPs promoted differential isoform usage (DIU) for a number of key immune sensors and signaling molecules and many of these splicing changes were again, distinct in uninfected and Salmonella-infected macrophages. Finally, after observing a surprising degree of similarity between the DEGs and DIUs in hnRNP K and U knockdown macrophages, we found that these cells are better able to restrict vesicular stomatitis virus replication than control cells, supporting a role for these hnRNPs in controlling infection outcomes in macrophages ex vivo. Based on these findings, we conclude that many innate immune genes have evolved to rely on one or more splicing regulatory factors to ensure the proper timing and magnitude of their induction, bolstering a model wherein pre-mRNA splicing is a critical regulatory node in the innate immune response.

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Dynamic post-translational modification profiling of Mycobacterium tuberculosis-infected primary macrophages

Cited by 50 publications

References 106 publications

The genetic proteome: Using genetics to inform the proteome of mycobacterial pathogens

The genetic proteome: Using genetics to inform the proteome of mycobacterial pathogens

Ceragenins and antimicrobial peptides kill bacteria through distinct mechanisms

Global transcriptomics reveals specialized roles for splicing regulatory proteins in the macrophage innate immune response

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