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Quantitative proteomic analysis of Burkholderia pseudomallei Bsa type III secretion system effectors using hypersecreting mutants.

Vander Broek CW, Chalmers KJ, Stevens MP, Stevens JM - Mol. Cell Proteomics (2015)

Bottom Line: Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hypersecreting mutants of B. pseudomallei with the isogenic parent strain and a bsaZ mutant incapable of effector protein secretion.Our study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors.Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei.

View Article: PubMed Central - PubMed

Affiliation: From the ‡The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland, UK.;

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Western blot analysis of the expression and secretion of BopE and BipD proteins by wild-type and mutant B. pseudomallei strains. 10276 WT, 10276 bsaZ::pDM4, 10276 bipD::pDM4, 10276 ΔbsaP, and 10276 ΔbsaP pbsaP were cultured for 6 h at 37 °C. Equal quantities of protein from the whole cell lysates or cell-free supernatants were separated by denaturing SDS-PAGE and proteins transferred to a nitrocellulose membrane for protein detection using polyclonal rabbit α-BopE or α-BipD (26) essentially as described under “Experimental Procedures.”
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Figure 1: Western blot analysis of the expression and secretion of BopE and BipD proteins by wild-type and mutant B. pseudomallei strains. 10276 WT, 10276 bsaZ::pDM4, 10276 bipD::pDM4, 10276 ΔbsaP, and 10276 ΔbsaP pbsaP were cultured for 6 h at 37 °C. Equal quantities of protein from the whole cell lysates or cell-free supernatants were separated by denaturing SDS-PAGE and proteins transferred to a nitrocellulose membrane for protein detection using polyclonal rabbit α-BopE or α-BipD (26) essentially as described under “Experimental Procedures.”

Mentions: Bacteria-free culture supernatants were collected from B. pseudomallei 10276 WT, 10276 bsaZ::pDM4, 10276 bipD::pDM4, 10276 ΔbsaP, and 10276 ΔbsaP (pbsaP) and analyzed by SDS-PAGE followed by Coomassie staining. The secreted protein profile of B. pseudomallei LB-culture supernatant is highly complex with few obvious differences between the strains (supplemental Fig. S3). The bacterial supernatants and whole cell lysates were also analyzed by Western blot using antibodies to detect the BopE effector protein and the BipD translocon protein (Fig. 1). The absence of both BopE and BipD in the supernatants of the 10276 bsaZ::pDM4 insertion mutant indicates that there was no detectable cell lysis at the time when the secretome was sampled. When compared with the 10276 WT strain, both 10276 bipD::pDM4 and 10276 ΔbsaP strains demonstrated increased secretion of BopE. Interestingly, the levels of BipD secreted into the supernatant was reduced in 10276 ΔbsaP compared with 10276 WT. This pattern of increased levels of the effector BopE and reduced secretion of the translocator BipD provides the first evidence of a role for BsaP as a “gatekeeper” protein, controlling a switch between the secretion of translocators and effectors. In the case of both BopE and BipD, the phenotype of 10276 ΔbsaP was partially restored by plasmid-mediated complementation, indicating the effects are unlikely to be caused by polar or secondary defects. Differences in the levels of BopE and BipD across the different strains sampled are not caused by differences in the number of bacteria at six hours subculture, because the growth rates of all strains were shown to be identical by viable count assessment (data not shown).


Quantitative proteomic analysis of Burkholderia pseudomallei Bsa type III secretion system effectors using hypersecreting mutants.

Vander Broek CW, Chalmers KJ, Stevens MP, Stevens JM - Mol. Cell Proteomics (2015)

Western blot analysis of the expression and secretion of BopE and BipD proteins by wild-type and mutant B. pseudomallei strains. 10276 WT, 10276 bsaZ::pDM4, 10276 bipD::pDM4, 10276 ΔbsaP, and 10276 ΔbsaP pbsaP were cultured for 6 h at 37 °C. Equal quantities of protein from the whole cell lysates or cell-free supernatants were separated by denaturing SDS-PAGE and proteins transferred to a nitrocellulose membrane for protein detection using polyclonal rabbit α-BopE or α-BipD (26) essentially as described under “Experimental Procedures.”
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4390269&req=5

Figure 1: Western blot analysis of the expression and secretion of BopE and BipD proteins by wild-type and mutant B. pseudomallei strains. 10276 WT, 10276 bsaZ::pDM4, 10276 bipD::pDM4, 10276 ΔbsaP, and 10276 ΔbsaP pbsaP were cultured for 6 h at 37 °C. Equal quantities of protein from the whole cell lysates or cell-free supernatants were separated by denaturing SDS-PAGE and proteins transferred to a nitrocellulose membrane for protein detection using polyclonal rabbit α-BopE or α-BipD (26) essentially as described under “Experimental Procedures.”
Mentions: Bacteria-free culture supernatants were collected from B. pseudomallei 10276 WT, 10276 bsaZ::pDM4, 10276 bipD::pDM4, 10276 ΔbsaP, and 10276 ΔbsaP (pbsaP) and analyzed by SDS-PAGE followed by Coomassie staining. The secreted protein profile of B. pseudomallei LB-culture supernatant is highly complex with few obvious differences between the strains (supplemental Fig. S3). The bacterial supernatants and whole cell lysates were also analyzed by Western blot using antibodies to detect the BopE effector protein and the BipD translocon protein (Fig. 1). The absence of both BopE and BipD in the supernatants of the 10276 bsaZ::pDM4 insertion mutant indicates that there was no detectable cell lysis at the time when the secretome was sampled. When compared with the 10276 WT strain, both 10276 bipD::pDM4 and 10276 ΔbsaP strains demonstrated increased secretion of BopE. Interestingly, the levels of BipD secreted into the supernatant was reduced in 10276 ΔbsaP compared with 10276 WT. This pattern of increased levels of the effector BopE and reduced secretion of the translocator BipD provides the first evidence of a role for BsaP as a “gatekeeper” protein, controlling a switch between the secretion of translocators and effectors. In the case of both BopE and BipD, the phenotype of 10276 ΔbsaP was partially restored by plasmid-mediated complementation, indicating the effects are unlikely to be caused by polar or secondary defects. Differences in the levels of BopE and BipD across the different strains sampled are not caused by differences in the number of bacteria at six hours subculture, because the growth rates of all strains were shown to be identical by viable count assessment (data not shown).

Bottom Line: Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hypersecreting mutants of B. pseudomallei with the isogenic parent strain and a bsaZ mutant incapable of effector protein secretion.Our study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors.Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei.

View Article: PubMed Central - PubMed

Affiliation: From the ‡The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland, UK.;

Show MeSH
Related in: MedlinePlus