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

Abstract

Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. We constructed a mutant lacking bsaP, a homolog of the T3SS "gatekeeper" family of proteins that exert control over the timing and magnitude of effector protein secretion. Mutants lacking BsaP, or the T3SS translocon protein BipD, were observed to hypersecrete the known Bsa effector protein BopE, providing evidence of their role in post-translational control of the Bsa T3SS and representing key reagents for the identification of its secreted substrates. 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.

Fig. 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.”

Fig. 2.

Proteins with high iTRAQ protein abundance ratios are enriched in hypersecreting B. pseudomallei strains and encoded within the bsa T3SS locus. A, Boxplot generated by R software (www.r-project.org) showing the distribution and median values of the ratios of the protein abundances in the 10276 WT (WT), 10276 bipD::pDM4 (bipD), or 10276 ΔbsaP (bsaP) samples compared with the 10276 bsaZ::pDM4 (bsaZ) sample. The “cut-off” values (calculated using R software) are shown by the dotted lines. Points shown above the “cut-off” value are candidate T3SS effectors. B, A Venn diagram (constructed with Venny: www.bioinfogp.cnb.csic.es/tools/venny) depicting the number of strain-specific and shared candidate T3SS effector proteins identified in each of the strains 10276 WT (WT), 10276 bipD::pDM4 (bipD), and 10276 ΔbsaP (bsaP). C, Distribution of the candidate T3SS effector proteins plotted according to B. pseudomallei gene number across both Chromosomes and their ratios. Gene numbers refer to the B. pseudomallei K96243 reference strain. The bsa T3SS locus is indicated by the red box.

Fig. 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.