Elongation factor Tu is a multifunctional and processed moonlighting protein

Widjaja, Michael; Harvey, Kate Louise; Hagemann, Lisa; Berry, Iain J.; Jarocki, Veronica M.; Raymond, Benjamin B. A.; Tacchi, Jessica L.; Gründel, Anne; Steele, Joel R.; Padula, Matthew P.; Charles, Ian G.; Dumke, Roger; Djordjevic, Steven P.

doi:10.1038/s41598-017-10644-z

Cited by 95 publications

(86 citation statements)

References 148 publications

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“…Trypsin shaving of M. pneumoniae cells was carried out as described previously 12 . Shaving was for 5 minutes at 37 °C and released peptides were trypsin digested a second time before analysis by LC-MS/MS.…”

Section: Liquid Chromatography Tandem Mass Spectrometry (Lc-ms/ms) Anmentioning

confidence: 99%

Cell surface processing of the P1 adhesin of Mycoplasma pneumoniae identifies novel domains that bind host molecules

Widjaja

Berry

Jarocki

et al. 2020

Sci Rep

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Mycoplasma pneumoniae is a genome reduced pathogen and causative agent of community acquired pneumonia. The major cellular adhesin, P1, localises to the tip of the attachment organelle forming a complex with P40 and P90, two cleavage fragments derived by processing Mpn142, and other molecules with adhesive and mobility functions. LC-MS/MS analysis of M. pneumoniae M129 proteins derived from whole cell lysates and eluents from affinity matrices coupled with chemically diverse host molecules identified 22 proteoforms of P1. Terminomics was used to characterise 17 cleavage events many of which were independently verified by the identification of semitryptic peptides in our proteome studies and by immunoblotting. One cleavage event released 1597 tSAAKpGApRppVppKpGApKppVQppKKpA 1627 from the C-terminus of P1 and this peptide was shown to bind to a range of host molecules. A smaller synthetic peptide comprising the C-terminal 15 amino acids, 1613 pGApKppVQppKKpA 1627 , selectively bound cytoskeletal intermediate filament proteins cytokeratin 7, cytokeratin 8, cytokeratin 18, and vimentin from a native A549 cell lysate. Collectively, our data suggests that ectodomain shedding occurs on the surface of M. pneumoniae where it may alter the functional diversity of P1, Mpn142 and other surface proteins such as elongation factor Tu via a mechanism similar to that described in Mycoplasma hyopneumoniae. The attachment organelle is a structurally and functionally sophisticated component of the M. pneumoniae cell that is responsible for the assembly of proteins essential for motility and adherence 1-8. An extensive list of host molecules including fibronectin 9-13 , fibrinogen 10-14 , plasminogen 11-13,15-17 , lactoferrin 10-12 , laminin 10-12 , and vitronectin 10-13 interact with surface accessible adhesins in M. pneumoniae. Other less well-defined host molecules include sialylated molecules 18 , oligosaccharides 19 , glycolipids 20 , and glycoproteins 21. The gene mpn141 encoding the major adhesin P1 is located in the same operon along with mpn140 and mpn142 and these three genes constitute a polycistronic transcriptional unit 22,23. mpn140 encodes for a 28 kDa putative phosphoesterase 24 and while it has been shown to degrade nanoRNA and dephosphorylate 3′-phosphoadenosine 5′-phosphate to AMP 25 , no role in adherence has been assigned for this protein. mpn142 generates a 130 kDa product (Mpn142) that is cleaved into two fragments of 40 kDa (P40) and 90 kDa (P90) immediately after or concurrent with translation 26,27. The cleavage event in Mpn142, first described over 25 years ago, was the first in what is now known to be a highly processed molecule on the surface of M. pneumoniae 28. P1 is a remarkably versatile molecule and the subject of numerous studies over the past 30 years. The only cleavage event that has been accurately assigned to P1 is the removal of the N-terminal 59 amino acids as a leader peptide 29. Molecular cross-linking and immunogold-labelling studies indicated that P1 forms a complex with P30, P40, and P90 ...

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Section: Liquid Chromatography Tandem Mass Spectrometry (Lc-ms/ms) Anmentioning

confidence: 99%

Cell surface processing of the P1 adhesin of Mycoplasma pneumoniae identifies novel domains that bind host molecules

Widjaja

Berry

Jarocki

et al. 2020

Sci Rep

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“…They harbour a specific OMP for their export and one among the best-studied is TolC from E. coli [54]. Elongation factor TU is a GTPase also known to perform moonlighting functions on the surface of human pathogens acting as a multifunctional adhesin [55].…”

Section: Degs Pertaining To Virulence Factorsmentioning

confidence: 99%

Integrative analysis of outer membrane vesicles proteomics and whole-cell transcriptome analysis of eravacycline induced Acinetobacter baumannii strains

Kesavan

Vasudevan

et al. 2020

BMC Microbiol

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Background: Acinetobacter baumannii is a multidrug-resistant (MDR) hazardous bacterium with very high antimicrobial resistance profiles. Outer membrane vesicles (OMVs) help directly and/or indirectly towards antibiotic resistance in these organisms. The present study aims to look on the proteomic profile of OMV as well as on the bacterial transcriptome upon exposure and induction with eravacycline, a new synthetic fluorocycline. RNA sequencing analysis of whole-cell and LC-MS/MS proteomic profiling of OMV proteome abundance were done to identify the differential expression among the eravacycline-induced A. baumannii ATCC 19606 and A. baumannii clinical strain JU0126. Results: The differentially expressed genes from the RNA sequencing were analysed using R package and bioinformatics software and tools. Genes encoding drug efflux and membrane transport were upregulated among the DEGs from both ATCC 19606 and JU0126 strains. As evident with the induction of eravacycline resistance, ribosomal proteins were upregulated in both the strains in the transcriptome profiles and also resistance pumps, such as MFS, RND, MATE and ABC transporters. High expression of stress and survival proteins were predominant in the OMVs proteome with ribosomal proteins, chaperons, OMPs OmpA, Omp38 upregulated in ATCC 19606 strain and ribosomal proteins, toluene tolerance protein, siderophore receptor and peptidases in the JU0126 strain. The induction of resistance to eravacycline was supported by the presence of upregulation of ribosomal proteins, resistance-conferring factors and stress proteins in both the strains of A. baumannii ATCC 19606 and JU0126, with the whole-cell gene transcriptome towards both resistance and stress genes while the OMVs proteome enriched more with survival proteins.Conclusion: The induction of resistance to eravacycline in the strains were evident with the increased expression of ribosomal and transcription related genes/proteins. Apart from this resistance-conferring efflux pumps, outer membrane proteins and stress-related proteins were also an essential part of the upregulated DEGs. However, the expression profiles of OMVs proteome in the study was independent with respect to the whole-cell RNA expression profiles with low to no correlation. This indicates the possible role of OMVs to be more of back-up additional protection to the existing bacterial cell defence during the antibacterial stress.

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“…These results suggest that the NF-jB activation may be related to the anti-apoptotic activity of LAMPs. Some reports have shown that mycoplasma can activate NF-jB through TLRs [18,32]. It could be presumed that M. genitalium lipoproteins could inhibit apoptosis by activating NF-jB through TLR2.…”

Section: Comparative Analysismentioning

confidence: 99%

Mycoplasma genitaliumlipoproteins inhibit tumour necrosis factor α-induced apoptosis in HeLa cells

Zuo

Sun

et al. 2018

Biotechnology & Biotechnological Equipment

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This aim of this study was to investigate the effects of Mycoplasma genitalium lipoproteins on the tumour necrosis factor a (TNF-a)-induced apoptosis in HeLa cells. We separated the M. genitalium lipid-associated membrane proteins (LAMPs) from M. genitalium and used them to treat HeLa cells. Apoptosis was induced by TNF-a treatment. Cell cycle and apoptosis were detected by flow cytometry. Pro-inflammatory cytokine contents were determined by enzymelinked immunosorbent assay (ELISA). Mitochondrial membrane potential and caspase-3 protease activity were also measured. There was no significant difference in the viable cell percentage in HeLa cells treated with M. genitalium LAMPs at 0-20 lg/mL. However, when treated with 40 lg/mL LAMPs, cytocidal activity was observed during the first 24 h. M. genitalium LAMPs induced cell cycle arrest at the G 1 phase in HeLa cells. When apoptosis was induced by TNF-a in HeLa cells, M. genitalium LAMPs significantly reduced the percentage of apoptotic cells. In addition, M. genitalium LAMPs significantly reduced the loss of mitochondrial membrane potential and caspase-3 protease activity in TNF-a-treated HeLa cells. However, the blocking of NF-jB significantly increased the mitochondrial membrane potential and caspase-3 protease activity in TNF-a-induced HeLa cells treated with M. genitalium LAMPs. The obtained results suggest that M. genitalium LAMPs could inhibit TNF-a-induced apoptosis in HeLa cells, which would contribute to the understanding of the pathogenesis of M. genitalium. ARTICLE HISTORY

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Elongation factor Tu is a multifunctional and processed moonlighting protein

Cited by 95 publications

References 148 publications

Cell surface processing of the P1 adhesin of Mycoplasma pneumoniae identifies novel domains that bind host molecules

Cell surface processing of the P1 adhesin of Mycoplasma pneumoniae identifies novel domains that bind host molecules

Integrative analysis of outer membrane vesicles proteomics and whole-cell transcriptome analysis of eravacycline induced Acinetobacter baumannii strains

Mycoplasma genitaliumlipoproteins inhibit tumour necrosis factor α-induced apoptosis in HeLa cells

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