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Cell wall proteomics in live Mycobacterium tuberculosis uncovers exposure of ESX substrates to the periplasm

ABSTRACT: 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. The mass spectrometry raw files and search results for the APEX strategy are included here.

INSTRUMENT(S): Q Exactive HF-X, Q Exactive

ORGANISM(S): Mycobacterium Tuberculosis (ncbitaxon:1773)

SUBMITTER: Beatrix Ueberheide

PROVIDER: MSV000092907 | MassIVE |

SECONDARY ACCESSION(S): PXD045524

REPOSITORIES: MassIVE

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Project description:Increasing experimental evidence supports that Mycobacterium tuberculosis (Mtb) has evolved strategies to survive within the lysosomes from activated macrophages, which may represent a reservoir for persistent mycobacteria. To further our knowledge in Mtb response to the lysosomal environment, we profiled the global transcriptional activity of Mtb in a lysosomal in vitro exposure (LivE) model. At inhibitory conditions of lysosomal SF (iLivE), which did not kill but arrested mycobacterial replication thereby mimicking persistence, Mtb expresses a unique transcriptome, where genes involved in general stress response, metabolic reprogramming, cell wall remodeling, respiration, oxidative stress and dormancy response were found to be significantly modulated. Genes encoding for protein families involved in Mtb virulence including ESAT-6, PE-PPE and Mce proteins were also distinctly regulated. Extensive meta-analysis of the Mtb transcriptomes from iLivE and previously reported ex vivo, in vivo and in vitro stress models revealed most significant overlap between iLivE and primary murine macrophage infection model. Our study also highlighted the α-glucan synthesis pathway and a distinct set of toxin-antitoxin systems as toxicity mechanisms that could be exploited as novel antimicrobial approaches for intra-macrophage persistent bacilli. The specificity in responses generated in the LivE model was supported by the significant number of iLivE genes that did not overlap with any of the previously reported in vitro stress models. Finally, to validate the relevance of the LivE model, rv1258c encoding for the efflux pump protein Tap, was selected from the iLivE Mtb transcriptome for further characterization. An Mtb Δrv1258c mutant was constructed and displayed increased susceptibility to killing by lysosomal SF and by the antimicrobial peptide LL-37 found in lysosomes. Furthermore, Δrv1258c Mtb was attenuated in its ability to survive in primary murine macrophages.

Project description:Background: Mtb's cell wall comprises peptidoglycan, arabinogalactan, and mycolic acids linked to capsule proteins and polysaccharides by noncovalent bonds. Cell division requires extensive remodeling by inserting cell wall-building subunits, which require multiple enzymes to ensure precision and accuracy during the addition and conjugation of biomolecules to the cell wall. Approximately 35% of division and cell wall cluster operon genes are involved in cell wall biosynthesis, 22% in cell division, and 43% are still unstudied. Results: Rv2166c was examined in M. smegmatis, a substitute for M. tuberculosis, using three strains: CRISPR-Cas12a-knockout ∆Ms_4236, complemented ∆Ms_4236::Rv2166c, and pALACE transformed Ms_Vec were grown at 370C in 7H9 or 7H10 to evaluate phenotypic, chemical stress, and antibiotic responses in normoxia and hypoxia. We also examined the whole transcriptome to identify genes associated with MSMEG_4236 deletion of the Rv2166c homolog under normoxia and hypoxia. Deletion and overexpression of Rv2166c affect mycobacterial biofilm formation, cell elongation, colony morphology, and sliding motility in normoxia but are alleviated in hypoxia. Overexpression showed resistance to nucleic acid-target antibiotics, but sensitivity to cell wall-target drugs, yet long-term expression arrests growth. According to transcriptome investigation, the deletion of MSMEG_4236 upregulated all division and cell wall cluster operon genes, several mycolic acids and arabinogalactan biosynthesis genes and downregulated numerous promoters outside of the dcw cluster operon through binding at AAAGTG[G/T] sequence motifs. Conclusion: This study shows that the mycobacterial Rv2166c represses the division and cell wall cluster operon as a transcriptional regulator. Thus, modulating this gene's expression affects many mycobacteria phenotypes and environmental stress resistance. Several AAAGTG[G/T] motifs containing promoters have been found, demonstrating that Rv2166c regulates genes other than its operon. These data suggest that the Rv2166c can be targeted as future antituberculosis medicines.

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Cell wall proteomics in live Mycobacterium tuberculosis uncovers exposure of ESX substrates to the periplasm

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