These authors contributed equally to this work.
Molecular architecture of Streptococcus pneumoniae surface thioredoxin-fold lipoproteins crucial for extracellular oxidative stress resistance and maintenance of virulence
Version of Record online: 18 OCT 2013
© 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
EMBO Molecular Medicine
Volume 5, Issue 12, pages 1852–1870, December 2013
How to Cite
Saleh, M., Bartual, S. G., Abdullah, M. R., Jensch, I., Asmat, T. M., Petruschka, L., Pribyl, T., Gellert, M., Lillig, C. H., Antelmann, H., Hermoso, J. A. and Hammerschmidt, S. (2013), Molecular architecture of Streptococcus pneumoniae surface thioredoxin-fold lipoproteins crucial for extracellular oxidative stress resistance and maintenance of virulence. EMBO Mol Med, 5: 1852–1870. doi: 10.1002/emmm.201202435
See accompanying article 10.1002/emmm.201303482
- Issue online: 2 DEC 2013
- Version of Record online: 18 OCT 2013
- Manuscript Accepted: 10 SEP 2013
- Manuscript Revised: 15 AUG 2013
- Manuscript Received: 29 DEC 2012
- Deutsche Forschungsgemeinschaft. Grant Numbers: DFG HA3125/4-2, DFG AN746/3-1, BFU2011-25326, S2010/BMD-2457
- EU FP7 CAREPNEUMO. Grant Number: EU-CP223111
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Figure S1. Comparison of Etrx1 (SPD_0572) and Etrx2 (SPD_0886) protein sequences of S. pneumoniae D39, showing an identity of 39.4%.
Figure S2. Comparison of CcdA1 protein sequences of S. pneumoniae as deposited in databases for 12 pneumococcal strains.
Figure S3. Comparison of Etrx1 protein sequences of S. pneumoniae as deposited in databases for 12 pneumococcal strains.
Figure S4. Comparison of MsrAB2 protein sequences of S. pneumoniae as deposited in databases for 12 pneumococcal strains.
Figure S5. Conservation and distribution of etrx genes in pneumococci as analysed by PCR. Gene fragments of etrx1 and etrx2 were amplified by PCR using specific primer pairs etrx1_447/etrx1_448 and etrx2_486/etrx2_487, respectively.
Figure S6. Sequence alignment between pneumococcal cytoplasmic SpMsrAB1 and surface-exposed SpMsrAB2. Secondary structural elements are also drawn. Conserved residues are highlighted with asterisk. On the bottom, the putative three-dimensional model is drawn in a ribbon representation. The MsrA domain is shown in blue and the MsrB in green, the transmembrane and coiled coil regions are coloured in magenta. Catalytic cysteines are labelled and represented as spheres. Sequence homology for D39 SpMsrAB1 (SPD_1193) and SpMsrAB2 (SPD_0573) is 77%.
Figure S7. Comparison of CcdA2 protein sequences of S. pneumoniae as deposited in databases for 12 pneumococcal strains.
Figure S8. Comparison of Etrx2 protein sequences of S. pneumoniae as deposited in databases for 12 pneumococcal strains.
Figure S9. Sequence alignment between S. pneumoniae D39 CcdA1 (SPD_0571) and CcdA2 (SPD_0885). The conserved residues are marked with asterisk. Protein sequences showed an identity of 58.8%.
Figure S10. Sequence analysis between S. pneumoniae CcdA proteins and DsbD of N. meningitidis. CcdA1 (SPD_0571) (left) and CcdA2 (SPD_0885) (right) of S. pneumoniae D39 were aligned with the N. meningitidis DsbD protein. The conserved transmembrane regions are coloured. Hypothetical transfer cysteines are highlighted in red or orange in the S. pneumoniae and N. meningitidis proteins, respectively. The disposition of transmembrane segments is shown.
Figure S11. Abundance of Etrx and Sp MsrAB proteins after proteolytic treatment of pneumococci. Pneumococci were grown in THY medium, harvested at OD600 of 0.3 and resuspended in 1 ml PBS pH 7.4 supplemented with 1% choline chloride to avoid autolysis. The proteolytic digest of surface-exposed proteins was carried out by treatment of 1010 pneumococci with trypsin (Tryp; Sigma; 1 mg/ml) or pronase E (ProE; Merck; 1 mg/ml). The untreated whole pneumococcal cell lysate was used as control (WCL). After 1 h at 37°C the untreated and trypsin or pronase E treated bacteria were sedimented, resuspended in SDS sample buffer, and proteolytic enzymes were inactivated at 95°C, which also lysed the bacteria. Proteins were separated by SDS-PAGE and immunoblotting was performed. The cytoplasmic enolase and the surface-exposed lipoprotein PsaA were used as controls (Bergmann et al, 2003; Johnston et al, 2004). The results revealed that proteolytic treatment of intact pneumococci reduces the abundance of Etrx1, Etrx2 and PsaA, while the abundance of cytoplasmic proteins such as the enolase was not affected. This confirms that Etrx1 and Etrx2 are surface-exposed proteins.
Figure S12. Molecular analysis of ccdA-, etrx- and msrAB1-mutants. (A) PCR analysis of pneumococcal mutants generated by allelic replacement. The mutants were verified by the size of the PCR product, which was larger compared to the PCR product obtained from wild-type DNA. (B) Production of MsrAB2 (and MsrAB1) in ccdA-, and etrx-mutants was confirmed by immunoblot analysis using mouse anti-MsrAB specific antibodies. The deficiency of CcdA1 or Etrx1 resulted in a higher production of MsrAB2, irrespective of the direction of the antibiotic gene cassette in the gene locus.
Figure S13. Purification and immunoblot analysis of recombinant Etrx proteins. Etrx1 (A), Etrx2 (B), SpMsrAB2 (C), MsrA2 (D) and MsrB2 (D) protein expression was induced with IPTG and total protein lysates were subjected to SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining. The His6-tagged proteins (His6-Etrx1 and His6-Etrx2, His6-MsrAB2, His6-MsrA2, His6-MsrB2) were purified by affinity chromatography and the His6-tag was removed using TEV protease. The proteins were detected by immunoblot analysis using anti-Histidine, anti-Etrx or anti-MsrAB2 antibodies. NI, non-induced; IN, induced; rP, recombinant protein.
Figure S14. Topology diagrams of the Etrx1 and Etrx2 structural elements in comparison with domain 1 of PilB (NterPilB), ResA and the E. coli Trx proteins fold. Canonical thioredoxin-fold is shown in light grey in all cases while the approximate positions of catalytic and the resolving cysteine residues are shown as asterisks.
Figure S15. Influence of Etrx and MsrAB2 on pneumococcal growth in the presence of DL-methionine sulfoxide. The pneumococcal strain D39lux and its isogenic pneumococcal etrx-mutants and msrAB-mutants were cultured in THY supplemented with 6 mM DL-methionine sulfoxide (MetSO) and the OD600 was measured continuously. In the absence of both Etrx proteins or MsrAB2 protein MetSO impairs pneumococcal growth compared to the isogenic wild-type.
Figure S16. Growth curves of S. pneumoniae wild-type strains D39lux and its isogenic pneumococcal etrx- and msrAB-mutants. The wild-type D39lux and the mutants D39luxΔetrx1, D39luxΔetrx2, D39luxΔetrx1Δetrx2, D39luxΔmsrAB1, D39luxΔmsrAB2, D39luxΔmsrAB1ΔmsrAB2 were grown at 37°C and 5% CO2 in complex THY media with erythromycin (2.5 µg/ml) (A and C) or in chemical defined medium CDM (B and D), respectively. The growth curves in THY and CDM indicated similar growth behaviour of the D39luxΔetrx1Δetrx2 mutant compared to the wild-type and individual etrx-mutants. Similarly, growth of the msrAB2-mutant was not changed, while the deficiency of MsrAB1 severely affected bacterial fitness in CDM.
Figure S17. Influence of Etrx and MsrAB2 on pneumococcal colonization of the mouse nasopharynx. (A) Bacterial load in the nasopharynx. (B) Bacterial load in the bronchoalveolar lavage. Groups of CD-1 mice (n = 9) were infected intranasally with 1 × 106 CFU of D39lux or its isogenic etrx- and msrAB2 mutants, respectively. One, 3 and 5 days post-infection the bacteria were recovered by a nasopharyngeal (A) and bronchoalveolar (B) wash. The bacteria were plated on blood agar and after overnight incubation at 37°C, 5% CO2 the CFU were determined. Results are presented as single values and medians.
Figure S18. Stereo view of the catalytic core of pneumococcal Etrx proteins. (A) Etrx1 (light blue) and Etrx2 (white) superimposition. Relevant residues are labelled and shown as capped sticks. β4–α3 loop is coloured in orange while the active CXXC region is coloured in pink. (B) NterPilB (orange) and ResA (green) superimposition. Relevant residues are labelled and shown as capped sticks. Polar interactions are shown as dashed lines.
Table S1. Strain and plasmid list.
Table S2. Primer list.
|emmm201202435-SourceData-Fig1D.pdf||508K||Source data for Figure 1D|
|emmm201202435-SourceData-Fig2B.pdf||503K||Source data for Figure 2B|
|emmm201202435-SourceData-Fig5A.pdf||6906K||Source data for Figure 5A|
|emmm201202435-SourceData-Fig5C.pdf||2077K||Source data for Figure 5C|
|emmm201202435-sm-0003-SuppMovie-S1.mp4||8064K||Movie S1. Extracellular oxidative stress resistance mechanism of Streptococcus pneumoniae|
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