title: Direct detection of bacterial protein secretion using whole colony proteomics.
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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Champion
authors.forename: Emily A
authors.initials: EA
authors.lastname: Williams
authors.forename: George M
authors.initials: GM
authors.lastname: Kennedy
authors.forename: Patricia A DiGiuseppe
authors.initials: PA
authors.lastname: Champion
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D058947:Bacterial Secretion Systems; D019910:Mycobacterium marinum / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D011550:Pseudomonas aeruginosa / Q000166:cytology / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; C093276:ESAT-6 protein, Mycobacterium tuberculosis