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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.

SUBMITTER: Champion MM

PROVIDER: S-EPMC3434784 | biostudies-other | 2012 Sep

REPOSITORIES: biostudies-other

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Direct detection of bacterial protein secretion using whole colony proteomics.

Champion Matthew M MM Williams Emily A EA Kennedy George M GM Champion Patricia A DiGiuseppe PA

Molecular & cellular proteomics : MCP 20120510 9

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 implemen ...[more]

PMID: 22580590

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Project description:BackgroundThe cornea is a specialized transparent connective tissue responsible for the majority of light refraction and image focus for the retina. There are three main layers of the cornea: the epithelium that is exposed and acts as a protective barrier for the eye, the center stroma consisting of parallel collagen fibrils that refract light, and the endothelium that is responsible for hydration of the cornea from the aqueous humor. Normal cornea is an immunologically privileged tissue devoid of blood vessels, but injury can produce a loss of these conditions causing invasion of other processes that degrade the homeostatic properties resulting in a decrease in the amount of light refracted onto the retina. Determining a measure and drift of phenotypic cornea state from normal to an injured or diseased state requires knowledge of the existing protein signature within the tissue. In the study of corneal proteins, proteomics procedures have typically involved the pulverization of the entire cornea prior to analysis. Separation of the epithelium and endothelium from the core stroma and performing separate shotgun proteomics using liquid chromatography/mass spectrometry results in identification of many more proteins than previously employed methods using complete pulverized cornea.ResultsRabbit corneas were purchased, the epithelium and endothelium regions were removed, proteins processed and separately analyzed using liquid chromatography/mass spectrometry. Proteins identified from separate layers were compared against results from complete corneal samples. Protein digests were separated using a six hour liquid chromatographic gradient and ion-trap mass spectrometry used for detection of eluted peptide fractions. The SEQUEST database search results were filtered to allow only proteins with match probabilities of equal or better than 10-3 and peptides with a probability of 10-2 or less with at least two unique peptides isolated within the run along with default Xcorr values. These parameters resulted in the identification of over 350 proteins, including over 225 new proteins not previously detected in the cornea by mass spectrometry. In addition, corneal layer separation resulted in identification of nearly every protein that was identified in the complete cornea assay. The epithelium and endothelium each revealed many unique proteomes specific to each layer. In the endothelium, the protein olfactomedin-like 3 was identified for the first time in the cornea by this analysis. Olfactomedin-3 is a neuronal expressed protein also known as optimedin that stimulates formation of cell adherent and cell-cell tight junctions and its expression modulates cytoskeleton organization and cell migration. However, the function of this protein in rabbit corneal endothelium is currently unknown.ConclusionThis manuscript presents a description of a more comprehensive proteomic profile for mammalian cornea compared to past methods. The use of simple dissection procedures of the tissue and the application of long chromatographic gradients, many more proteins can be identified.

Dataset Information

Direct detection of bacterial protein secretion using whole colony proteomics.

Publications

Direct detection of bacterial protein secretion using whole colony proteomics.

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