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. 2012 Sep;11(9):596-604.
doi: 10.1074/mcp.M112.017533. Epub 2012 May 10.

Direct Detection of Bacterial Protein Secretion Using Whole Colony Proteomics

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Free PMC article

Direct Detection of Bacterial Protein Secretion Using Whole Colony Proteomics

Matthew M Champion et al. Mol Cell Proteomics. .
Free PMC article

Abstract

Bacteria use a variety of secretion systems to transport proteins beyond their cell membrane to interact with their environment. For bacterial pathogens, these systems are key virulence determinants that transport bacterial proteins into host cells. Genetic screens to identify bacterial genes required for export have relied on enzymatic or fluorescent reporters fused to known substrates to monitor secretion. However, they cannot be used in analysis of all secretion systems, limiting the implementation across bacteria. Here, we introduce the first application of a modified form of whole colony MALDI-TOF MS to directly detect protein secretion from intact bacterial colonies. We show that this method is able to specifically monitor the ESX-1 system protein secretion system, a major virulence determinant in both mycobacterial and Gram-positive pathogens that is refractory to reporter analysis. We validate the use of this technology as a high throughput screening tool by identifying an ESAT-6 system 1-deficient mutant from a Mycobacterium marinum transposon insertion library. Furthermore, we also demonstrate detection of secreted proteins of the prevalent type III secretion system from the Gram-negative pathogen, Pseudomonas aeruginosa. This method will be broadly applicable to study other bacterial protein export systems and for the identification of compounds that inhibit bacterial protein secretion.

Figures

Fig. 1.
Fig. 1.
Direct detection of ESX-1 protein secretion. a, Western blot analysis demonstrating the production (P, pellet) and secretion (S, supernatant) of CFP-10 in the wild-type M. marinum strain (lanes 3 and 4). CFP-10 is produced in but not secreted from strains bearing mutations in eccCb (lanes 5 and 6) and espB (lanes 9 and 10). CFP-10 is not detectable in strains bearing a deletion in esxA (lanes 1 and 2) or in RD1 (lanes 5 and 6). b, schematic of the ESX-1 locus in M. marinum. The genes covered by the RD1 deletion are indicated by the line below the genes. The labeled genes (black) specify strains used in this investigation. c, MALDI-TOF spectra obtained from wild-type M. marinum M (WT), M. marinum bearing a deletion in RD1 (ΔRD1), eccCb::TnKan, and espB::TnKan. Peaks labeled 1, 2, and 3 correspond to ESAT-6 (9913 Da), acetylated ESAT-6 (9960 Da), and CFP-10 (10609 Da).
Fig. 2.
Fig. 2.
Identification of an ESX-1-deficient strain using whole colony MALDI-TOF. Spectra generated by whole colony MALDI-TOF for wild-type M. marinum are shown. The panels show wild-type M. marinum (WT), M. marinum bearing a deletion in RD1 (ΔRD1), and the 6B10 strain of M. marinum. Peaks labeled 1, 2, and 3 correspond to ESAT-6 (9913 Da), acetylated ESAT-6 (9960 Da), and CFP-10 (10609 Da), respectively.
Fig. 3.
Fig. 3.
The 6B10 mutant is defective for ESX-1 secretion. a, sRBC lysis assay. Although wild-type M. marinum lyses sRBC, strains bearing deletions in RD1 or the transposon insertion 6B10 fail to lyse sRBCs. The error bars represent standard deviation. b, Western blot analysis demonstrating that 6B10 fails to export ESAT-6 and CFP-10. GroEL was used as a lysis control, and Mpt32, a substrate of the Sec secretion system, was used as a loading control. CFP-10 is produced in the cell pellet (P) and secreted into the supernatant (S) by wild-type M. marinum (lanes 1 and 2) but not seen in the pellet or supernatant of ΔRD1 (lanes 3 and 4) and 6B10 (lanes 5 and 6). ESAT-6 is produced at reduced levels but is not secreted by 6B10. c, the point of transposon insertion was mapped (between bases 6584232 and 6584233 in the M. marinum genome, reverse orientation) and corresponds to 254 base pairs into the open reading frame of MMAR_5444, eccB1. The potential operon including eccB1 is indicated by a bracket below the genes, including MMAR_5440–5445 (espF-eccCa1).
Fig. 4.
Fig. 4.
Direct detection of type III secretion from P. aeruginosa using whole colony MALDI-TOF. Spectra generated from whole colony washates of wild-type PA103 (WT), exsA::Ω, and ΔexsD, pscC::Ω in the presence (top, + EGTA) or absence (bottom) of EGTA. The major peak at 31,198 m/z is specific to EGTA induction and absent in a type III mutant strains. This mass corresponds to PopD (±0.1%). LC/MS/MS was used to confirm the identity of this protein. PopD was not identified from LC/MS/MS analysis of uninduced PA103 or the exsA::Ω and ΔexsD, pscC::Ω strains under any condition.

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