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

Damen, Merel P.M.; Meijers, Aniek S.; Keizer, E. M.; Piersma, Sander R.; Jiménez, Connie R.; Kuijl, Coenraad; Bitter, Wilbert; Houben, Edith N.G.

doi:10.1128/mbio.02819-22

Cited by 7 publications

(9 citation statements)

References 80 publications

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“…From this, we conclude that the PE/PPE pairs of the esx-5 locus are important for the secretion of the Esx substrates and at least one other PPE protein. This is in line with a recent observation for ESX-1 in M. marinum , where secretion of EsxA is dependent on the co-secretion of PPE68 encoded by the same gene cluster ( 34 , 35 ).…”

Section: Resultssupporting

confidence: 93%

“…In addition, we showed that secretion of the Esx heterodimers by ESX-5 is dependent on the locus-encoded PE/PPE proteins. This is in line with a recent observation for ESX-1 in Mycobacterium marinum , where secretion of EsxA is dependent on the co-secretion of PPE68 that is encoded by the same gene cluster ( 34 , 35 ). Notably, PPE68 was observed to be degraded on the cell surface upon export.…”

Section: Discussionsupporting

confidence: 92%

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Reconstitution of a minimal ESX-5 type VII secretion system suggests a role for PPE proteins in the outer membrane transport of proteins

Bunduc,

Ding,

Kuijl

et al. 2023

mSphere

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Mycobacteria utilize type VII secretion systems (T7SSs) to secrete proteins across their highly hydrophobic and diderm cell envelope. Pathogenic mycobacteria have up to five different T7SSs, called ESX-1 to ESX-5, which are crucial for growth and virulence. Here, we use a functionally reconstituted ESX-5 system in the avirulent species Mycobacterium smegmatis that lacks ESX-5, to define the role of each esx-5 gene in system functionality. By creating an array of gene deletions and assessing protein levels of components and membrane complex assembly, we observed that only the five components of the inner membrane complex are required for its assembly. However, in addition to these five core components, active secretion also depends on both the Esx and PE/PPE substrates. Tagging the PPE substrates followed by subcellular fractionation, surface labeling and membrane extraction showed that these proteins localize to the mycobacterial outer membrane. This indicates that they could play a role in secretion across this enigmatic outer barrier. These results provide the first full overview of the role of each esx-5 gene in T7SS functionality. IMPORTANCE Pathogenic mycobacteria, such as the notorious Mycobacterium tuberculosis , are highly successful as pathogens, in part due to their specific and diderm cell envelope, with a mycolic acid-containing outer membrane. The architecture of this highly impermeable membrane is little understood and the proteins that populate it even less so. To transport proteins across their cell envelope, mycobacteria employ a specialized transport pathway called type VII secretion. While recent studies have elucidated the type VII secretion membrane channel that mediates transport across the inner membrane, the identity of the outer membrane channel remains a black box. Here, we show evidence that specific substrates of the type VII pathway could form these channels. Elucidating the pathway and mechanism of protein secretion through the mycobacterial outer membrane will allow its exploitation for the development of novel mycobacterial therapeutics.

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Section: Resultssupporting

confidence: 93%

Section: Discussionsupporting

confidence: 92%

Reconstitution of a minimal ESX-5 type VII secretion system suggests a role for PPE proteins in the outer membrane transport of proteins

Bunduc,

Ding,

Kuijl

et al. 2023

mSphere

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“…Overall, how EsxAB, EsxGH and other substrates and components of the ESX secretion system interact during cell wall transport remains to be further explored. Nevertheless, we note that recent data on ESX-1 suggest a hierarchical model in which, rather than being shuttled or channeled through the cell wall, substrates such as EsxB support their own export out of the cell 55,64 and our data lend additional credence to this model. In summary protein tagging by APEX2 offers unprecedented accuracy in localizing proteins to subcellular compartments of M. tuberculosis, including the cell wall.…”

Section: Discussionsupporting

confidence: 79%

Cell wall proteomics in liveMycobacterium tuberculosisuncovers exposure of ESX substrates to the periplasm

Jaisinghani

Previti

Andrade

et al. 2023

Preprint

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The cell wall of mycobacteria plays a key role in interactions with the environment and its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites from ions to lipids to proteins. Accurately identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis enabled the accurate identification of the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the Mtb periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope.

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“…These analyses confidently identified a total of 1354 expressed proteins, including several proteins of the ESX-1, ESX-2 and ESX-5 secretion systems (Table S6). ESX-1 substrates and components detected included the major secreted M. tuberculosis antigen CFP-10 (F6B93_22245), the chaperone EspB (F6B93_22290), the PE protein chaperone EspG1 (F6B93_22210) and the ESX-1 secretion regulator EspI (F6B93_22255) (54–57). The ESX-5 secretion system has been shown to be essential to slow growing mycobacteria (51, 58) and several substrates of this system were detected, including a full-length version of the substrate EsxM (F6B93_11055), which has been suggested to promote dissemination in ancestral M. tuberculosis lineages (59).…”

Section: Resultsmentioning

confidence: 99%

A marine sponge-associated mycobacterium closely related toMycobacterium tuberculosis

Pidot,

Klatt,

Ates

et al. 2024

Preprint

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Reconstructing the evolutionary origins of Mycobacterium tuberculosis, the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M. tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens, M. spongiae is the most closely related species to the M. tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M. tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M. tuberculosis, together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M. tuberculosis challenge compared to BCG vaccination alone. The discovery of M. spongiae adds to our understanding of the emergence of the M. tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M. tuberculosis.

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

Cited by 7 publications

References 80 publications

Reconstitution of a minimal ESX-5 type VII secretion system suggests a role for PPE proteins in the outer membrane transport of proteins

Reconstitution of a minimal ESX-5 type VII secretion system suggests a role for PPE proteins in the outer membrane transport of proteins

Cell wall proteomics in liveMycobacterium tuberculosisuncovers exposure of ESX substrates to the periplasm

A marine sponge-associated mycobacterium closely related toMycobacterium tuberculosis

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