Quantitative proteomic analysis of Burkholderia pseudomallei Bsa type III secretion system effectors using hypersecreting mutants.
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.
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
Mentions: Putative T3SS effector proteins were identified in the data sets primarily on the calculated iTRAQ protein abundance ratios. The protein abundances obtained in each of the WT or mutant strain samples were each expressed as a ratio of the 10276 bsaZ::pDM4 sample and proteins with an iTRAQ ratio higher than the 1.5 × interquartile range (IQR), together with at least two unique peptides with a MASCOT peptide score ≥21, were taken to be T3SS effector protein candidates. The 1.5 × IQR values were calculated and the cut-offs set at 1.56 for the 10276 WT/10276 bsaZ::pDM4 data set, 1.79 for the 10276 bipD::pDM4/10276 bsaZ::pDM4 data set, and 1.46 for the 10276 ΔbsaP/10276 bsaZ::pDM4 data set, based on the calculation described under “Experimental Procedures.” When the data was plotted as a boxplot, the data showed a trend toward more proteins with larger ratios in the 10276 bipD::pDM4/10276 bsaZ::pDM4 and 10276 ΔbsaP/10276 bsaZ::pDM4 data sets when compared with the 10276 WT/10276 bsaZ::pDM4 data set (Fig. 2A). A total of 26 proteins met the selection criteria and can be considered putative effectors (Table I). Of the 26 proteins there were strain-specific subsets, with eight proteins with high ratios in two of the data sets and three proteins with high ratios in all three (Fig. 2B). The three proteins that displayed high ratios in all three data sets, BopC, BopA, and BopE, are all known B. pseudomallei Type III secreted (T3S) effector proteins or predicted to be T3S proteins based on their T3SS-dependent secretion in a surrogate host system (25, 26, 31) (Fig. 2B). Of the 26 proteins, 20 were only found in the hypersecreting mutant strain samples and would not have been identified had our study focused solely on the study of the secreted proteins of the 10276 WT strain (Fig. 2B). Plotting the iTRAQ ratios of the 26 putative effector proteins against their B. pseudomallei K96243 genetic locus number, it is clear that there is a concentration of eight proteins with especially high ratios encoded within the annotated bsa T3SS locus on Chromosome 2 compared with that of the rest of the genome (Table I, Fig. 2C).
Affiliation: From the ‡The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland, UK.;