Gel-free sample preparation techniques and bioinformatic enrichment analysis to in depth characterise the cell wall proteome of mycobacteria

Hermann, Clemens; Giddey, Alexander D.; Nel, Andrew J. M.; Soares, Nelson C.; Blackburn, Jonathan M.

doi:10.1016/j.mex.2018.05.013

Cited by 3 publications

(5 citation statements)

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“…Most importantly, future studies of the cell envelope proteome should assess the quality of the enrichment of the particular envelope fraction by either biochemical or bioinformatic methods either prior to or post proteomic measurement to provide robust data sets. This has already been done by a few studies reviewed in this Review 33,37,44,50,58,65 and should be the standard for all future studies. Also, studies should include sufficient details of the technical and biological reproducibility and should use stringent criteria for protein identification.…”

Section: ■ Common Approaches and Hurdles In Membrane Proteomics: Gene...mentioning

confidence: 93%

“…This postacquisition approach has been reported to greatly increase the number of proteins that have a signal sequence or transmembrane domains or are predicted lipoproteins and thus likely represent bona fide cell envelope proteins while at the same time minimizing the number of proteins that have no such predicted features in the cell-envelope fraction. This bioinformatically driven approach has been used to characterize changes in the cell envelope proteome after the sublethal treatment of M. smegmatis with the antibiotic rifampacin. , Other laboratories have used similar rationales to filter out proteins postacquisition that were identified in the respective enriched cell-envelope fraction but were present at a lower intensity compared with the cytosolic or other fraction. ,,, For example, van der Woude and colleagues identified n -octyl-β- d -glucopyranoside (OBG) as a unique detergent that selectively solubilized mycomembrane proteins in M. marinum and used this knowledge to guide their bioinformatic enrichment analysis postacquisition. Any protein that was more abundant in the OBG extract from the cell envelope compared with the respective pellet fraction was identified as a potential mycomembrane protein candidate.…”

Section: Approaches To Characterize the Cell Envelope Proteome Of Myc...mentioning

confidence: 99%

“…The efficiency of enrichment can be assessed by looking at the abundance of known subcellular markers such as GroEL for the cytosol, Mycp1 for the plasma membrane, and MspA for the outer membrane. 37,58 Whereas this approach is easy to use and allows the identification of proteins in the range of 1000−2200, it suffers from insufficient removal of cytosolic contaminants, which becomes problematic because the sensitivity of mass spectrometers has increased over the past decade, meaning that cytosolic contaminants, even present at very low levels, might be misidentified as the cell wall or plasma membrane components. A variation of this procedure is the use of sucrose gradient centrifugation, which has been applied by two elegant studies for M. smegmatis.…”

Section: ■ Common Approaches and Hurdles In Membrane Proteomics: Gene...mentioning

confidence: 99%

“…This bioinformatically driven approach has been used to characterize changes in the cell envelope proteome after the sublethal treatment of M. smegmatis with the antibiotic rifampacin. 37,58 Other laboratories have used similar rationales to filter out proteins postacquisition that were identified in the respective enriched cell-envelope fraction but were present at a lower intensity compared with the cytosolic or other fraction. 33,44,50,65 For example, van der Woude and colleagues 50 identified n-octyl-β-D-glucopyranoside (OBG) as a unique detergent that selectively solubilized mycomembrane proteins in M. marinum and used this knowledge to guide their bioinformatic enrichment analysis postacquisition.…”

Section: ■ Common Approaches and Hurdles In Membrane Proteomics: Gene...mentioning

confidence: 99%

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Cell Envelope Proteomics of Mycobacteria

et al. 2020

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The World Health Organization (WHO) estimates that Mycobacterium tuberculosis, the most pathogenic mycobacterium species to humans, has infected up to a quarter of the world's population, with the occurrence of multidrug-resistant strains on the rise. Research into the detailed composition of the cell envelope proteome in mycobacteria over the last 20 years has formed a key part of the efforts to understand host−pathogen interactions and to control the current tuberculosis epidemic. This is due to the great importance of the cell envelope proteome during infection and during the development of antibiotic resistance as well as the search of surface-exposed proteins that could be targeted by therapeutics and vaccines. A variety of experimental approaches and mycobacterial species have been used in proteomic studies thus far. Here we provide for the first time an extensive summary of the different approaches to isolate the mycobacterial cell envelope, highlight some of the limitations of the studies performed thus far, and comment on how the recent advances in membrane proteomics in other fields might be translated into the field of mycobacteria to provide deeper coverage.

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Section: ■ Common Approaches and Hurdles In Membrane Proteomics: Gene...mentioning

confidence: 93%

Section: Approaches To Characterize the Cell Envelope Proteome Of Myc...mentioning

confidence: 99%

Section: ■ Common Approaches and Hurdles In Membrane Proteomics: Gene...mentioning

confidence: 99%

Section: ■ Common Approaches and Hurdles In Membrane Proteomics: Gene...mentioning

confidence: 99%

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Cell Envelope Proteomics of Mycobacteria

et al. 2020

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“…Insoluble aggregates were removed by centrifugation at 22,000 RCF. See Supplementary Figure 1 for a graphical schema adapted from Hermann et al ( 27 ).…”

Section: Methodsmentioning

confidence: 99%

Cell Wall Proteomics Reveal Phenotypic Adaption of Drug-Resistant Mycobacterium smegmatis to Subinhibitory Rifampicin Exposure

Giddey

Ganief

et al. 2021

Front. Med.

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Despite the availability of effective drug treatment, Mycobacterium tuberculosis (Mtb), the causative agent of TB disease, kills ~1. 5 million people annually, and the rising prevalence of drug resistance increasingly threatens to worsen this plight. We previously showed that sublethal exposure to the frontline anti-TB drug, rifampicin, resulted in substantial adaptive remodeling of the proteome of the model organism, Mycobacterium smegmatis, in the drug-sensitive mc2155 strain [wild type (WT)]. In this study, we investigate whether these responses are conserved in an engineered, isogenic mutant harboring the clinically relevant S531L rifampicin resistance-conferring mutation (SL) and distinguish the responses that are specific to RNA polymerase β subunit- (RpoB-) binding activity of rifampicin from those that are dependent on the presence of rifampicin alone. We verified the drug resistance status of this strain and observed no phenotypic indications of rifampicin-induced stress upon treatment with the same concentration as used in WT (2.5 μg/ml). Thereafter, we used a cell wall-enrichment strategy to focus attention on the cell wall proteome and observed 253 proteins to be dysregulated in SL bacteria in comparison with 716 proteins in WT. We observed that decreased abundance of ATP-binding cassette (ABC) transporters and increased abundance of ribosomal machinery were conserved in the SL strain, whereas the upregulation of transcriptional machinery and the downregulation of numerous two-component systems were not. We conclude that the drug-resistant M. smegmatis strain displays some of the same proteomic responses observed in WT and suggest that this evidence supports the hypothesis that rifampicin exercises effects beyond RpoB-interaction alone and that mycobacteria recognise rifampicin as a signaling molecule in an RpoB-independent manner at sublethal doses. Taken together, our data indicates mixed RpoB-independent and RpoB-dependent proteomic remodeling in WT mycobacteria, with evidence for RpoB-independent ABC transporter downregulation, but drug activity-based transcriptional upregulation and two-component system downregulation.

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A conserved translation factor is required for optimal synthesis of a membrane protein family in mycobacteria

Fishbein

Wolf

Dulberger

et al. 2019

Preprint

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14Ribosomes require the activity of associated GTPases to synthesize proteins. Despite strong 15 evolutionary conservation, the roles of many of these remain unknown. For example, LepA (also known 16 as elongation factor 4) is a ribosome-associated GTPase found in bacteria, mitochondria, and 17 chloroplasts, yet its physiological contribution to cell survival is not clear. Recently, we found that loss of 18 lepA in Mycobacterium smegmatis (Msm) altered tolerance to rifampin, a drug that targets a non-19 ribosomal cellular process. To uncover the determinants of LepA-mediated drug tolerance, we 20 characterized the whole-cell proteomes and transcriptomes of a lepA deletion mutant relative to a wild-21 type strain. We find that LepA is important for the steady-state abundance of an outer membrane porin, 22which is integral to nutrient uptake and drug susceptibility. Loss of LepA leads to a decreased amount 23 of porin in the membrane, resulting in the drug tolerance phenotype of the lepA mutant. LepA control 24 requires a sequence motif in the 5' region of the porin transcript. Thus, LepA controls the abundance of 25 specific proteins, likely through its activity during translation. 27 Importance 28Our understanding of how ribosomes properly synthesis an entire cellular proteome, in all its 29 complexity, is still evolving. Ribosomal GTPases are often highly conserved, but the roles of many are 30 not well understood. For example, elongation factor 4, or LepA, is a ribosome-associated GTPase 31 conserved across bacteria, mitochondria, and chloroplasts. Using whole-cell proteomics and RNA-32 sequencing of wild type and a lepA deletion mutant, we find that LepA improves translation of 33 mycobacterial porins in a message-specific manner. As porins play a key role in cell wall permeability, 34 loss of LepA produces a plethora of phenotypic changes. These findings underline the problem of 35 building proteins into a complex cell wall, such as that of mycobacteria, and point to a solution in the 36 use of GTPases such as LepA, that have evolved to aid in specific protein synthesis. 37 38 Keywords 39 LepA; ribosome; mycobacteria; porin; drug susceptibility 40 41 45 to adaptive, cellular responses. Indeed, the ribosome machinery and its associated factors/RNA 46 species adapt the cell during periods of environmental transition [2] [3, 4]. Recent advances in 47 techniques such as quantitative proteomics, ribosome profiling, and cryo-electron microscopy (cryo-48 EM) have revealed an intricate balance of mRNA sequences and associating proteins at the ribosome 49 that are necessary for the homeostatic cellular proteome [5-7]. Regardless of growth environment,50bacterial cells are likely composed of a heterogenous population of ribosomes [8, 9], with some inactive 51 and others actively translating parts of the proteome [10, 11]. Associating factors and RNA species tune 52 each ribosome, creating a cell with a spatially-and temporally-regulated proteome [8, 12]. 53The successful synthesis of a given protein at the ribosome is ...

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Gel-free sample preparation techniques and bioinformatic enrichment analysis to in depth characterise the cell wall proteome of mycobacteria

Abstract: Graphical abstract

Cited by 3 publications

References 15 publications

Cell Envelope Proteomics of Mycobacteria

Cell Envelope Proteomics of Mycobacteria

Cell Wall Proteomics Reveal Phenotypic Adaption of Drug-Resistant Mycobacterium smegmatis to Subinhibitory Rifampicin Exposure

A conserved translation factor is required for optimal synthesis of a membrane protein family in mycobacteria

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