Proteo-genetic analysis reveals clear hierarchy of ESX-1 secretion in
            <i>Mycobacterium marinum</i>

Cronin, Rachel M.; Ferrell, Micah J.; Cahir, Clare W.; Champion, Matthew M.; Champion, Patricia A.

doi:10.1073/pnas.2123100119

Cited by 29 publications

(60 citation statements)

References 85 publications

(201 reference statements)

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“…While the reason for the observed differences in requirements for EsxA secretion remains unclear, these might be caused by a variation in in vitro growth conditions, similarly as previously observed for the importance of the conserved cytosolic component EccA1 in secretion (44). Notably, a recent proteo-genetic study also revealed the importance of PPE68 and MMAR_2894 for ESX-1 mediated secretion in M. marinum (64), fully supporting our results.…”

Section: Discussionsupporting

confidence: 91%

The ESX-1 substrate PPE68 has a key function in the ESX-1 mediated secretion inMycobacterium marinum

Damen¹,

Meijers

Keizer³

et al. 2022

Preprint

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Mycobacteria use specialized type VII secretion systems (T7SSs) to secrete proteins across their diderm cell envelope. One of the T7SS subtypes, named ESX-1, is a major virulence determinant in pathogenic species such as Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. ESX-1 secretes a variety of substrates, called Esx, PE, PPE and Esp proteins, at least some of which as folded heterodimers. Investigations into the functions of these substrates is problematic, because of the intricate network of co-dependent secretion between several ESX-1 substrates. Here, we describe that the ESX-1 substrate PPE68 is essential for secretion of the highly immunogenic substrate EsxA and EspE via the ESX-1 system in M. marinum. While secreted PPE68 is processed on the cell surface, the majority of cell-associated PPE68 of M. marinum and M. tuberculosis is present in a cytosolic complex with its PE partner and the EspG1 chaperone. Interfering with the binding of EspG1 to PPE68 blocked its export and the secretion of EsxA and EspE. In contrast, esxA is not required for the secretion of PPE68, revealing hierarchy in co-dependent secretion. Remarkably, the final ten residues of PPE68, a negatively charged domain, seem essential for EspE secretion, but not for the secretion of EsxA and PPE68 itself. This indicates that distinctive domains of PPE68 are involved in secretion of the different ESX-1 substrates. Based on these findings, we propose a mechanistic model for the central role of PPE68 in ESX-1 mediated secretion and substrate co-dependence.

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Section: Discussionsupporting

confidence: 91%

The ESX-1 substrate PPE68 has a key function in the ESX-1 mediated secretion inMycobacterium marinum

Damen¹,

Meijers

Keizer³

et al. 2022

Preprint

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“…First, we showed that PPE68 is exported in an ESX-1-dependent manner to the cell surface of M. marinum . While previous mass spectrometry analysis could also detect this protein in culture supernatants of M. marinum ( 33 , 46 , 62 ), we could not detect PPE68 in similar fractions by immunoblot analysis. Intriguingly, this protein is degraded upon secretion to the cell surface, as deletion of three Pec proteases (PecABC) increased the amount of PPE68 on the cell surface.…”

Section: Discussioncontrasting

confidence: 90%

The ESX-1 Substrate PPE68 Has a Key Function in ESX-1-Mediated Secretion in Mycobacterium marinum

Damen

Meijers

Keizer

et al. 2022

mBio

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Pathogenic mycobacteria, such Mycobacterium tuberculosis and Mycobacterium marinum , use a type VII secretion system (T7SS) subtype, called ESX-1, to mediate intracellular survival via phagosomal rupture and subsequent translocation of the mycobacterium to the host cytosol. Identifying the ESX-1 substrate that is responsible for this process is problematic because of the intricate network of codependent secretion between ESX-1 substrates.

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“…Lipid profiles have successfully been used to speciate bacteria using mass spectrometry − and even to detect strain level differences in antibiotic resistant bacteria . Typically, a mass spectrum can provide a metabolomic and lipidomic profile while MS/MS and collision-induced dissociation (CID) experiments provide structural information on specific features of interest. , Related methods involve the use of ion mobility coupled with MS, ,− rapid evaporative ionization mass spectrometry (REIMS), , matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), , direct analysis in real time mass spectrometry (DART-MS), , direct infusion mass spectrometry (DI-MS), and liquid chromatography coupled with mass spectrometry (LC-MS). , Many of these experiments are targeted at specific biomarkers, thus being limited to specific biological targets. Untargeted methods do not have the same limitations; however, they are time-consuming as many individual MS/MS spectra are required for each sample.…”

Section: Introductionmentioning

confidence: 99%

“…25 Typically, a mass spectrum can provide a metabolomic and lipidomic profile while MS/MS and collision-induced dissociation (CID) experiments provide structural information on specific features of interest. 24,26 Related methods involve the use of ion mobility coupled with MS, 24,26−28 rapid evaporative ionization mass spectrometry (REIMS), 29,30 matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), 31,32 direct analysis in real time mass spectrometry (DART-MS), 33,34 etry (DI-MS), 35 and liquid chromatography coupled with mass spectrometry (LC-MS). 36,37 Many of these experiments are targeted at specific biomarkers, thus being limited to specific biological targets.…”

Section: ■ Introductionmentioning

confidence: 99%

Metabolomic and Lipidomic Profiling of Bacillus Using Two-Dimensional Tandem Mass Spectrometry

et al. 2022

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Lipidomic and metabolomic profiles of sporulated and vegetative Bacillus subtilis and Bacillus thuringiensis from irradiated lysates were recorded using a quadrupole ion trap mass spectrometer modified to perform two-dimensional tandem mass spectrometry (2D MS/MS). The 2D MS/MS data domains, acquired using a 1.2 s scan of negative ions generated by nanoelectrospray ionization of microwave irradiated spores, showed the presence of dipicolinic acid (DPA) as well as various lipids. Aside from microwave radiation to extract DPA and lipids from spores, sample preparation was minimal. Characteristic lipid and metabolic profiles were observed using 107108 cells of the two Bacillus species. Major features of the lipid profiles observed for the vegetative states included sets of phosphatidylglycerol (PG) lipids. Product ion spectra were extracted from the 2D MS/MS data, and they provided structural information on the fatty acid components of the PG lipids. The study demonstrates the flexibility, speed, and informative power of metabolomic and lipidomic fingerprinting for identifying the presence of spore-forming biological agents using 2D MS/MS as a rapid profiling screening method.

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Proteo-genetic analysis reveals clear hierarchy of ESX-1 secretion in Mycobacterium marinum

Cited by 29 publications

References 85 publications

The ESX-1 substrate PPE68 has a key function in the ESX-1 mediated secretion inMycobacterium marinum

The ESX-1 substrate PPE68 has a key function in the ESX-1 mediated secretion inMycobacterium marinum

The ESX-1 Substrate PPE68 Has a Key Function in ESX-1-Mediated Secretion in Mycobacterium marinum

Metabolomic and Lipidomic Profiling of Bacillus Using Two-Dimensional Tandem Mass Spectrometry

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