These authors contributed equally to this work.
Analysis of SecA2-dependent substrates in Mycobacterium marinum identifies protein kinase G (PknG) as a virulence effector
Version of Record online: 6 NOV 2013
© 2013 John Wiley & Sons Ltd
Volume 16, Issue 2, pages 280–295, February 2014
How to Cite
van der Woude, A. D., Stoop, E. J. M., Stiess, M., Wang, S., Ummels, R., van Stempvoort, G., Piersma, S. R., Cascioferro, A., Jiménez, C. R., Houben, E. N. G., Luirink, J., Pieters, J., van der Sar, A. M. and Bitter, W. (2014), Analysis of SecA2-dependent substrates in Mycobacterium marinum identifies protein kinase G (PknG) as a virulence effector. Cellular Microbiology, 16: 280–295. doi: 10.1111/cmi.12221
- Issue online: 13 JAN 2014
- Version of Record online: 6 NOV 2013
- Accepted manuscript online: 9 OCT 2013 08:35PM EST
- Manuscript Accepted: 24 SEP 2013
- Manuscript Revised: 14 SEP 2013
- Manuscript Received: 1 DEC 2012
- Netherlands Organization for Scientific Research
- Smart Mix Programme of the Netherlands Ministry of Economic Affairs
- Netherlands Ministry of Education, Culture and Science
- Human Frontiers Science Program long term fellowship
- Canton Basel
- Optimus Foundation
- Swiss National Science Foundation
|cmi12221-sup-0001-figs1.tif||1418K||Fig. S1. Coomassie stained gel used for nanoLC-MS/MS analysis. Detergent extraction of cell envelopes of M. marinum wild type (wt) and secA2 mutant in duplo (sec_I and sec_II). Fractions detergent pellet and detergent supernatant in a 1:1 ratio are loaded on SDS-PAGE and stained by Coomassie.|
Fig. S2. Expression levels of HA-tagged constructs.
A. Immunoblots of total lysates from M. marinum wild type (wt), secA2 mutant (sec) and complemented secA2 mutant (c) expressing IipA-HA behind hsp60 promoter in liquid culture.
B. Immunoblots of total lysates obtained directly from plate-grown strains expressing HA-tagged constructs with dppA, iipA and MMAR_3060 behind their own promoter. Note that the expression of Mm3060 in the plate-grown bacteria seems higher than in culture (Fig. 2). Blots were probed with an antiserum against the HA epitope. Anti-GroEL2 is used as loading control.
|cmi12221-sup-0003-figs3.tif||156K||Fig. S3. Quantification: three putative substrates are dependent on SecA2 for stability and/or processing. Relative staining levels of different fractions containing HA-labelled proteins, quantified by densitometric analysis after immunoblotting with an antiserum directed against the HA epitope, expressed as a fraction of wild type total lysate levels. The following fractions of M. marinum E11 wild type (wt), secA2 mutant (SecA2) and complemented mutant (comp) were loaded on a polyacrylamide gel: total lysate (T), cytosol (cyt), cell envelopes (CE), detergent-treated CE pellet (DP) and detergent-treated CE supernatant (DS). Strains contained HA-labelled (A) DppA, (B) MMAR_3060 or (C) IipA respectively. Quantification of the DppA and MMAR_3060 signal was performed on the observed band, whereas for IipA the whole lane was quantified. Results represent the mean of three independent experiments with standard deviation.|
|cmi12221-sup-0004-figs4.tif||58K||Fig. S4. Quantification: SecA2-dependent cell wall localization of PknG. Relative staining levels of PknG (upper band), quantified by densitometric analysis after immunoblotting with an antiserum directed PknG, expressed as a fraction of wild type total lysate levels. The following fractions of M. marinum E11 wild type (wt), secA2 mutant (secA2) and complemented mutant (comp) were loaded on a polyacrylamide gel: total lysate (T), cytosol (cyt) and cell envelopes (CE). Results represent the mean of three independent experiments with standard deviation.|
|cmi12221-sup-0005-figs5.tif||957K||Fig. S5. Effects of SecA2 on antibiotic susceptibility. Meropenem E-test result of a 1:1 mix of M. marinum wild type (wt) containing the pSMT3-eGFP vector and the secA2 mutant (secA2) containing the pSMT3- mCherry vector monitored by fluorescent microscopy. At increasing meropenem concentration (top), growth of the secA2 mutant (right panel) is more restricted than that of wild type bacteria (middle panel). This result is representative for 2 independent experiments.|
Fig. S6. A. Fractionation of M. bovis BCG. Total lysate (T), cytosol (cyt) and cell envelopes (CE) were loaded on a polyacrylamide gel in 1:1:1 ratio, transferred to a nitrocellulose membrane and immunostained using antisera against EccC5, PknG and GroEL2.
B. Intracellular replication of secA2 mutant. J774 macrophages were infected at an moi of 0.1 with M. marinum wild type (wt + pMV), M. marinum WT overexpressing PknGBCG (wt + pMV::PknG), the secA2 mutant (secA2 + pMV) and the secA2 mutant overexpressing PknGBCG (secA2 + pMV::PknG). The number of cfu was determined by lysing infected J774 cells and plating lysates at the indicated time-points. Graph represents mean ± SEM from three independent experiments.
C. Quantification of infection of embryos with wild type M. marinum (wt + pMV) and wild type M. marinum overexpressing PknGBCG (wt + PknG) at 5 dpi. Each data point represents the number of cfu per embryo, as determined by plating and the bars indicate means after log transformation. Mean ± standard deviation of inocula were 134 ± 19 and 127 ± 28 cfu respectively. The graphs represent data of 4 to 5 independent experiments.
|cmi12221-sup-0007-tables1.xls||883K||Table S1. Proteins identified by nanoLC-MS/MS analysis. Supplied separately as Microsoft Excel file.|
|cmi12221-sup-0008-tables2.doc||36K||Table S2. Primers used in this study.|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.