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Cited by 131Biochemical and Structural CharacterizationMicrobial infections and disease researchBacterial Genetics and Biotechnology

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Elongation factor Tu is a multifunctional and processed moonlighting protein

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Widjaja, Michael, Harvey, Kate Louise, Hagemann, Lisa, Berry, Iain James, Jarocki, Veronica Maria, Raymond, Benjamin Bernard Armando, Tacchi, Jessica Leigh, Gründel, Anne, Steele, Joel Ricky, Padula, Matthew Paul, Charles, Ian George, Dumke, Roger, Djordjevic, Steven Philip·2017·Scientific Reports
DOI 10.1038/s41598-017-10644-zPMC PMC5593925
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Abstract

Many bacterial moonlighting proteins were originally described in medically, agriculturally, and commercially important members of the low G + C Firmicutes. We show Elongation factor Tu (Ef-Tu) moonlights on the surface of the human pathogens Staphylococcus aureus (Sa Ef-Tu) and Mycoplasma pneumoniae (Mpn Ef-Tu), and the porcine pathogen Mycoplasma hyopneumoniae (Mhp Ef-Tu). Ef-Tu is also a target of multiple processing events on the cell surface and these were characterised using an N-terminomics pipeline. Recombinant Mpn Ef-Tu bound strongly to a diverse range of host molecules, and when bound to plasminogen, was able to convert plasminogen to plasmin in the presence of plasminogen activators. Fragments of Ef-Tu retain binding capabilities to host proteins. Bioinformatics and structural modelling studies indicate that the accumulation of positively charged amino acids in short linear motifs (SLiMs), and protein processing promote multifunctional behaviour. Codon bias engendered by an A + T rich genome may influence how positively-charged residues accumulate in SLiMs.

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Figures

Figure 1 Bioinformatic analysis of MpnEf-Tu, MhpEf-Tu, and SaEf-Tu. (a) Schematic of MpnEf-Tu, MhpEf-Tu, and SaEf-Tu highlighting putative heparin and fibronectin-binding domains and cleavage sites. ScanProsite144 was used to predict heparin-binding motifs (dark blue boxes) by searching clusters of basic residues with the “X-[HRK]-X(0,2)-[HRK]-X(0,2)-[HRK]-X” and “X-[HRK]-X(1,4)-[HRK]-X(1,4)-[HRK]-X” motifs. A putative transmembrane domain (score 505) was predicted in MhpEf-Tu using TMpred142 (yellow box). In MpnEf-Tu, two fibronectin-binding regions (salmon boxes) and two predicted transmembrane domains (scores) are depicted in Panels a and b89, 90. Key amino acids in MpnEf-Tu involved in binding fibronectin89, 90 are underlined. Cleavage sites identified in this study are shown as arrows above the black bar (blue indicates cleavage sites identified by dimethyl labelling and red indicates cleavage sites identified by the characterisation of semi-tryptic peptides by LC-MS/MS). (b) Amino acid sequence alignments of MpnEf-Tu, MhpEf-Tu, and SaEf-Tu. For consistency, features described in Fig. 1a are represented by the same colour scheme in Fig. 1b. Cleavage sites identified in this study are depicted by the symbol Ζ. Sequence alignments have been separated into the three domains and the nucleotide-binding motifs (boxed regions) and the two pattern recognition receptors (broken black outline grey box from Acidovorax avenae 65 and Brassica-specific receptors64) are shown.
Figure 1 Bioinformatic analysis of MpnEf-Tu, MhpEf-Tu, and SaEf-Tu. (a) Schematic of MpnEf-Tu, MhpEf-Tu, and SaEf-Tu highlighting putative heparin and fibronectin-binding domains and cleavage sites. ScanProsite144 was used to predict heparin-binding motifs (dark blue boxes) by searching clusters of basic residues with the “X-[HRK]-X(0,2)-[HRK]-X(0,2)-[HRK]-X” and “X-[HRK]-X(1,4)-[HRK]-X(1,4)-[HRK]-X” motifs. A putative transmembrane domain (score 505) was predicted in MhpEf-Tu using TMpred142 (yellow box). In MpnEf-Tu, two fibronectin-binding regions (salmon boxes) and two predicted transmembrane domains (scores) are depicted in Panels a and b89, 90. Key amino acids in MpnEf-Tu involved in binding fibronectin89, 90 are underlined. Cleavage sites identified in this study are shown as arrows above the black bar (blue indicates cleavage sites identified by dimethyl labelling and red indicates cleavage sites identified by the characterisation of semi-tryptic peptides by LC-MS/MS). (b) Amino acid sequence alignments of MpnEf-Tu, MhpEf-Tu, and SaEf-Tu. For consistency, features described in Fig. 1a are represented by the same colour scheme in Fig. 1b. Cleavage sites identified in this study are depicted by the symbol Ζ. Sequence alignments have been separated into the three domains and the nucleotide-binding motifs (boxed regions) and the two pattern recognition receptors (broken black outline grey box from Acidovorax avenae 65 and Brassica-specific receptors64) are shown.CC BY · PubMed Central
Figure 2 Binding to heparin by Ef-Tu and cleavage fragments of Ef-Tu. Panel a–c show MpnEf-Tu, MhpEf-Tu, and SaEf-Tu, and surface accessible cleavage fragments of these molecules retained during heparin-agarose chromatography respectively. Peptides identified (black boxes within coloured bars) by mass spectrometry within Ef-Tu fragments were obtained from 1D and 2D SDS-PAGE of bacterial whole cell lysates (grey bars), avidin affinity chromatography of biotinylated surface proteins (red bars), and heparin-agarose affinity chromatography (blue bars). Full length Ef-Tu molecules are represented as black bars. Cleavage products of Ef-Tu are also shown. Cleavage sites were identified by identifying dimethyl labelled peptides (blue arrows and broken lines) and by characterising semi-tryptic peptides generated after trypsin digestion (red arrows and broken lines). Exact cleavage sites are shown in the amino acid sequences above the black bar. For M. pneumoniae, the two fibronectin-binding regions (salmon boxes, Fn) and two putative transmembrane domains described by89, 90 were included. Bioinformatic tools such as ScanProsite144, TMpred142, COILS143 and Meta-Disorder145 were used to predict putative heparin-binding motifs (Hep, blue boxes), transmembrane domains (TmD, yellow box for M. hyopneumoniae), coiled-coils (Coil, orange boxes), and disordered regions (purple boxes in grey bar), respectively. Peptides released from trypsin shaving of cells are shown as the green boxes in the grey bar. Circles just above fragments denote amino acid positions that are predicted to be surface exposed and represent putative protein-protein interaction regions (visual cues can be seen on the right of the cleavage map, sites listed in Tables S2A, S2B, and S2C). Those marked with an additional star denote amino acid residues that fall within predicted putative heparin-binding domains. White circles mark evolutionary unconserved binding regions, whilst yellow circles are mildly conserved and pink are highly conserved. Amino acid positions marked by grey triangles depict predicted nucleic acid interaction regions.
Figure 2 Binding to heparin by Ef-Tu and cleavage fragments of Ef-Tu. Panel a–c show MpnEf-Tu, MhpEf-Tu, and SaEf-Tu, and surface accessible cleavage fragments of these molecules retained during heparin-agarose chromatography respectively. Peptides identified (black boxes within coloured bars) by mass spectrometry within Ef-Tu fragments were obtained from 1D and 2D SDS-PAGE of bacterial whole cell lysates (grey bars), avidin affinity chromatography of biotinylated surface proteins (red bars), and heparin-agarose affinity chromatography (blue bars). Full length Ef-Tu molecules are represented as black bars. Cleavage products of Ef-Tu are also shown. Cleavage sites were identified by identifying dimethyl labelled peptides (blue arrows and broken lines) and by characterising semi-tryptic peptides generated after trypsin digestion (red arrows and broken lines). Exact cleavage sites are shown in the amino acid sequences above the black bar. For M. pneumoniae, the two fibronectin-binding regions (salmon boxes, Fn) and two putative transmembrane domains described by89, 90 were included. Bioinformatic tools such as ScanProsite144, TMpred142, COILS143 and Meta-Disorder145 were used to predict putative heparin-binding motifs (Hep, blue boxes), transmembrane domains (TmD, yellow box for M. hyopneumoniae), coiled-coils (Coil, orange boxes), and disordered regions (purple boxes in grey bar), respectively. Peptides released from trypsin shaving of cells are shown as the green boxes in the grey bar. Circles just above fragments denote amino acid positions that are predicted to be surface exposed and represent putative protein-protein interaction regions (visual cues can be seen on the right of the cleavage map, sites listed in Tables S2A, S2B, and S2C). Those marked with an additional star denote amino acid residues that fall within predicted putative heparin-binding domains. White circles mark evolutionary unconserved binding regions, whilst yellow circles are mildly conserved and pink are highly conserved. Amino acid positions marked by grey triangles depict predicted nucleic acid interaction regions.CC BY · PubMed Central
Figure 3 Binding of rMpnEf-Tu to human A549 epithelial cells. (a) A549 cells (‘with cells’) were bound to wells of a 96-well mictrotitre plate and incubated with rEf-Tu. Bound rMpnEf-Tu was detected with antisera raised against rMpnEf-Tu. rPdhB and rP08 were used as a positive and negative control98, respectively. Bars represent standard deviation of eight replicates. (b) rMpnEf-Tu was incubated with either antisera raised against rMpnEf-Tu or pre-immune sera (PIS) and added to A549 cells in ELISA plates. rPdhB and rP08 and the corresponding antisera were used as a positive and negative control98, respectively. Bars represent standard deviation of eight replicates.
Figure 3 Binding of rMpnEf-Tu to human A549 epithelial cells. (a) A549 cells (‘with cells’) were bound to wells of a 96-well mictrotitre plate and incubated with rEf-Tu. Bound rMpnEf-Tu was detected with antisera raised against rMpnEf-Tu. rPdhB and rP08 were used as a positive and negative control98, respectively. Bars represent standard deviation of eight replicates. (b) rMpnEf-Tu was incubated with either antisera raised against rMpnEf-Tu or pre-immune sera (PIS) and added to A549 cells in ELISA plates. rPdhB and rP08 and the corresponding antisera were used as a positive and negative control98, respectively. Bars represent standard deviation of eight replicates.CC BY · PubMed Central
Figure 4 Microtitre plate binding assays depicting the interaction of rMpnEf-Tu with human proteins. Wells of microtitre plates were coated with rMpnEf-Tu and incubated with increasing concentrations of host proteins. Antisera against each of the host proteins was used to detect interaction with rMpnEf-Tu. M. pneumoniae cells and BSA were used as a positive and negative control, respectively. Bars represent standard deviation of eight replicates.
Figure 4 Microtitre plate binding assays depicting the interaction of rMpnEf-Tu with human proteins. Wells of microtitre plates were coated with rMpnEf-Tu and incubated with increasing concentrations of host proteins. Antisera against each of the host proteins was used to detect interaction with rMpnEf-Tu. M. pneumoniae cells and BSA were used as a positive and negative control, respectively. Bars represent standard deviation of eight replicates.CC BY · PubMed Central
Figure 5 Influence of ions and lysine analog ACA on binding of rMpnEf-Tu to plasminogen and degradation of human fibrinogen and vitronectin by activated plasminogen. (a) Microtitre plate wells were coated with rMpnEf-Tu and incubated with plasminogen and increasing concentrations of either NaCl (‘ionic interactions’) or ε-aminocaproic acid (‘ACA’). Bound plasminogen was detected with anti-plasminogen antibodies. In control experiments M. pneumoniae cells were coated onto microtitre plates and incubated with plasminogen and increasing concentrations of either NaCl or ACA. Bars represent standard deviation of eight replicates. (b) Fibrinogen or vitronectin was mixed with either urinary plasminogen activator (uPA) or tissue plasminogen activator (tPA) and added to microtitre plates previously coated with rMpnEf-Tu and plasminogen. Samples were separated by SDS-PAGE, blotted onto nitrocellulose membrane and probed with anti-vitronectin and anti-fibrinogen antisera. Lane 1 is at 0 hours, lane 2 is after over-night incubation without plasminogen, and lane 3 is after over-night incubation with plasminogen. Full length blots can be seen in Figure S9.
Figure 5 Influence of ions and lysine analog ACA on binding of rMpnEf-Tu to plasminogen and degradation of human fibrinogen and vitronectin by activated plasminogen. (a) Microtitre plate wells were coated with rMpnEf-Tu and incubated with plasminogen and increasing concentrations of either NaCl (‘ionic interactions’) or ε-aminocaproic acid (‘ACA’). Bound plasminogen was detected with anti-plasminogen antibodies. In control experiments M. pneumoniae cells were coated onto microtitre plates and incubated with plasminogen and increasing concentrations of either NaCl or ACA. Bars represent standard deviation of eight replicates. (b) Fibrinogen or vitronectin was mixed with either urinary plasminogen activator (uPA) or tissue plasminogen activator (tPA) and added to microtitre plates previously coated with rMpnEf-Tu and plasminogen. Samples were separated by SDS-PAGE, blotted onto nitrocellulose membrane and probed with anti-vitronectin and anti-fibrinogen antisera. Lane 1 is at 0 hours, lane 2 is after over-night incubation without plasminogen, and lane 3 is after over-night incubation with plasminogen. Full length blots can be seen in Figure S9.CC BY · PubMed Central

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Research works printed but not linked: 2

  1. Tacchi, J. L. et al. Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae. Open biology 6, 150210, doi:10.1098/rsob.150210 (2016). · DOI · Paper page
  2. P, H. Diploma thesis, Technische Universität München, (2012).

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Metadata

Authors
Widjaja, M., Harvey, K. L., Hagemann, L., Berry, I. J., Jarocki, V. M., Raymond, B. B. A., Tacchi, J. L., Gründel, A., Steele, J. R., Padula, M. P., Charles, I. G., Dumke, R., & Djordjevic, S. P.
Affiliations
  • University of Technology Sydney (AU)
  • Technische Universität Dresden (DE)
  • Norwich Research Park (GB)
Corresponding author
Djordjevic, Steven Philip
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2017-09-11
Volume
7
Issue
1
Pages
11227-11227
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-017-10644-z
PMC
PMC5593925
OpenAlex
W2750987002
Cited by
131
References
154
Citations by year
2026: 7 · 2025: 17 · 2024: 20 · 2023: 7 · 2022: 16 · 2021: 20 · 2020: 14 · 2019: 13 · 2018: 17
FWCI
4.92
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • UEA Digital Repository (University of East Anglia) (acceptedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • UTS ePRESS (University of Technology Sydney) (submittedVersion)
Funders
  • Bristol-Myers Squibb Foundation
  • Deutsche Forschungsgemeinschaft (1280/1-1, DU 1280/1-1)
  • University of Technology Sydney
  • NSW Department of Primary Industries
  • Australian Research Council (LE130100096)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
  • Life Sciences › Immunology and Microbiology › Microbiology › Microbial infections and disease research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
elongation factor Tu, moonlighting proteins, protein processing, Staphylococcus aureus, Mycoplasma pneumoniae, Mycoplasma hyopneumoniae, plasmin, short linear motifs, positively charged amino acids, codon usage bias, AT-rich genome, host-pathogen interaction, bacterial cell surface, N-terminomics
MeSH terms
Fibrinolysin, Membrane Proteins, Models, Molecular, Mycoplasma pneumoniae, Peptide Elongation Factor Tu, Plasminogen, Protein Binding, Staphylococcus aureus, Computational Biology, Virulence Factors, Mycoplasma hyopneumoniae, Host-Pathogen Interactions

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