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.

A Novel ESX-1 Locus Reveals that Surface-Associated ESX-1 Substrates Mediate Virulence in Mycobacterium marinum

“…The M. marinum strain 14 was constructed by introduction of the pMB272 suicide plasmid which carries the Mariner transposon (Tn) into the M strain (43). Mycobacterial transformation was performed using electroporation as previously described (43,44). Following introduction of the plasmid, counter-selection was applied by adding sucrose to the agar, and growth was at 32°C to promote plasmid loss and transposon insertion into the genome as described previously (44).…”

Automated Mass Spectrometry–Based Functional Assay for the Routine Analysis of the Secretome

“…48,55,56 The principles of the concept and high-throughput methods are shown in Figure 1. In general, cells grown under identical or similar conditions differ by only one parameter such as (growth factor) unstimulated and stimulated or cells grown with serum-containing medium or with serum-deprived medium.…”

“…[45][46][47] However, even if the differences detected are not at the "naked" protein level, the differences observed may still be detected and the proteins responsible for them can be identified. For example, in a very recent study, Champion et al 48 investigated secreted protein from intact biosafety level 2 pathogenic mycobacterial colonies using a modified version of whole-colony MALDI-time-of-flight (TOF)-MS. In their studies, they spotted onto a 384-well Gold MALDI target 1 µL of filtrates of Mycobacterium marinum and analyzed the spots by MALDI-TOF.…”

DisconnectingIn VitroESX-1 Secretion from Mycobacterial Virulence

“…In M. smegmatis, ESX-1 secretion is induced by growth in Sauton's broth in vitro but not by growth in Middlebrook 7H9 broth, a defined medium for mycobacterial growth (9). We have adapted this approach to induce ESX-1 on solid Sauton's agar to detect ESX-1 export (26). Thus far, the means of induction by growth in Sauton's medium or on agar remains unknown.…”