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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 putative B. pseudomallei Bsa T3SS effector proteins expressed as c-Myc tagged fusion proteins. The coding region of bopE, bopA, BPSS0860, BPSS1512, BPSS1916, bprD, and bapA were expressed with a c-terminal c-Myc tag in the IPTG-inducible expression vector pME6032 in B. pseudomallei strains 10276 WT, 10276 bsaZ::pDM4, 10276 bipD::pDM4 or 10276 ΔbsaP. Supernatant and whole cell fractions were collected after 6 h of incubation at 37 °C in the presence of 0.5 mm IPTG. Equal quantities of protein were separated by reducing SDS-PAGE, blotted onto nitrocellulose membranes and probed using polyclonal rabbit α-c-Myc antibody.
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Figure 3: Western blot analysis of the expression and secretion of putative B. pseudomallei Bsa T3SS effector proteins expressed as c-Myc tagged fusion proteins. The coding region of bopE, bopA, BPSS0860, BPSS1512, BPSS1916, bprD, and bapA were expressed with a c-terminal c-Myc tag in the IPTG-inducible expression vector pME6032 in B. pseudomallei strains 10276 WT, 10276 bsaZ::pDM4, 10276 bipD::pDM4 or 10276 ΔbsaP. Supernatant and whole cell fractions were collected after 6 h of incubation at 37 °C in the presence of 0.5 mm IPTG. Equal quantities of protein were separated by reducing SDS-PAGE, blotted onto nitrocellulose membranes and probed using polyclonal rabbit α-c-Myc antibody.

Mentions: To confirm a number of targets are secreted in a Bsa-dependent manner, a number of candidates were selected and cloned into an inducible expression vector as in-frame C-terminal c-Myc tagged fusion proteins. The targets chosen were BprD, BapA, BPSS0860, BPSS1512, BopE, and BopA, as well as BPSS1916, a protein with a very high iTRAQ ratio in all strains that did not meet the initial selection criteria because of being quantified with only a single unique peptide. Targets were chosen to represent proteins with both high and low protein scores, high and low numbers of peptides, and to be encoded inside and outside of the bsa T3SS locus. The plasmids were transformed into strains 10276 WT, 10276 bsaZ::pDM4, 10276 bipD::pDM4 and 10276ΔbsaP, and bacterial supernatants were collected and assessed by Western blot for the presence of the tagged proteins using a c-Myc tag-specific polyclonal antibody. Initial studies in which BopE was cloned as a c-Myc fusion in the widely used shuttle vector pBHR1 under a constitutive chloramphenicol resistance gene promoter indicated that BopE-c-Myc was secreted in a BsaZ-independent manner, possibly owing to the high level of expression (data not shown). We therefore elected to clone effector-c-Myc fusions under an IPTG-inducible promoter in pME6032 and select inducer levels at which strict BsaZ-dependent BopE-c-Myc secretion was observed (Fig. 3). BopA-c-Myc was absent in the supernatant of the 10276 bsaZ::pDM4 strain, but secreted by the parent strain and secreted at elevated levels by 10276ΔbsaP (Fig. 3), confirming for the first time in B. pseudomallei that it is a substrate of the Bsa T3SS, not just the locus of enterocyte effacement-encoded T3SS in EPEC (31). Furthermore, both of the putative effector proteins located within the bsa locus, BprD-c-Myc and BapA-c-Myc, were secreted in a BsaZ-dependent manner (Fig. 3), confirming that these are novel effectors of the Bsa apparatus.


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 putative B. pseudomallei Bsa T3SS effector proteins expressed as c-Myc tagged fusion proteins. The coding region of bopE, bopA, BPSS0860, BPSS1512, BPSS1916, bprD, and bapA were expressed with a c-terminal c-Myc tag in the IPTG-inducible expression vector pME6032 in B. pseudomallei strains 10276 WT, 10276 bsaZ::pDM4, 10276 bipD::pDM4 or 10276 ΔbsaP. Supernatant and whole cell fractions were collected after 6 h of incubation at 37 °C in the presence of 0.5 mm IPTG. Equal quantities of protein were separated by reducing SDS-PAGE, blotted onto nitrocellulose membranes and probed using polyclonal rabbit α-c-Myc antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Western blot analysis of the expression and secretion of putative B. pseudomallei Bsa T3SS effector proteins expressed as c-Myc tagged fusion proteins. The coding region of bopE, bopA, BPSS0860, BPSS1512, BPSS1916, bprD, and bapA were expressed with a c-terminal c-Myc tag in the IPTG-inducible expression vector pME6032 in B. pseudomallei strains 10276 WT, 10276 bsaZ::pDM4, 10276 bipD::pDM4 or 10276 ΔbsaP. Supernatant and whole cell fractions were collected after 6 h of incubation at 37 °C in the presence of 0.5 mm IPTG. Equal quantities of protein were separated by reducing SDS-PAGE, blotted onto nitrocellulose membranes and probed using polyclonal rabbit α-c-Myc antibody.
Mentions: To confirm a number of targets are secreted in a Bsa-dependent manner, a number of candidates were selected and cloned into an inducible expression vector as in-frame C-terminal c-Myc tagged fusion proteins. The targets chosen were BprD, BapA, BPSS0860, BPSS1512, BopE, and BopA, as well as BPSS1916, a protein with a very high iTRAQ ratio in all strains that did not meet the initial selection criteria because of being quantified with only a single unique peptide. Targets were chosen to represent proteins with both high and low protein scores, high and low numbers of peptides, and to be encoded inside and outside of the bsa T3SS locus. The plasmids were transformed into strains 10276 WT, 10276 bsaZ::pDM4, 10276 bipD::pDM4 and 10276ΔbsaP, and bacterial supernatants were collected and assessed by Western blot for the presence of the tagged proteins using a c-Myc tag-specific polyclonal antibody. Initial studies in which BopE was cloned as a c-Myc fusion in the widely used shuttle vector pBHR1 under a constitutive chloramphenicol resistance gene promoter indicated that BopE-c-Myc was secreted in a BsaZ-independent manner, possibly owing to the high level of expression (data not shown). We therefore elected to clone effector-c-Myc fusions under an IPTG-inducible promoter in pME6032 and select inducer levels at which strict BsaZ-dependent BopE-c-Myc secretion was observed (Fig. 3). BopA-c-Myc was absent in the supernatant of the 10276 bsaZ::pDM4 strain, but secreted by the parent strain and secreted at elevated levels by 10276ΔbsaP (Fig. 3), confirming for the first time in B. pseudomallei that it is a substrate of the Bsa T3SS, not just the locus of enterocyte effacement-encoded T3SS in EPEC (31). Furthermore, both of the putative effector proteins located within the bsa locus, BprD-c-Myc and BapA-c-Myc, were secreted in a BsaZ-dependent manner (Fig. 3), confirming that these are novel effectors of the Bsa apparatus.

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