Green Fluorescent Protein as a Novel Indicator of Antimicrobial Susceptibility in Aureobasidium pullulans

Abstract

Presently there is no method available that allows noninvasive and real-time monitoring of fungal susceptibility to antimicrobial compounds. The green fluorescent protein (GFP) of the jellyfish Aequoria victoria was tested as a potential reporter molecule for this purpose. Aureobasidium pullulans was transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, BioTechniques 23:686-690, 1997). The transformed strain Ap1 gfp showed bright fluorescence that was amenable to quantification using fluorescence spectrophotometry. Fluorescence levels in Ap1 gfp blastospore suspensions were directly proportional to the number of viable cells determined by CFU plate counts (r 2 > 0.99). The relationship between cell viability and GFP fluorescence was investigated by adding a range of concentrations of each of the biocides sodium hypochlorite and 2-n-octylisothiozolin-3-one (OIT) to suspensions of Ap1 gfp blastospores (pH 5 buffer). These biocides each caused a rapid (<25-min) loss of fluorescence of greater than 90% when used at concentrations of 150 g of available chlorine ml ؊1 and 500 g ml ؊1 , respectively. Further, loss of GFP fluorescence from A. pullulans cells was highly correlated with a decrease in the number of viable cells (r 2 > 0.92). Losses of GFP fluorescence and cell viability were highly dependent on external pH; maximum losses of fluorescence and viability occurred at pH 4, while reduction of GFP fluorescence was absent at pH 8.0 and was associated with a lower reduction in viability. When A. pullulans was attached to the surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly than in cell suspensions, with >95% loss of fluorescence after 27 h. This technique should have broad applications in testing the susceptibility of A. pullulans and other fungal species to antimicrobial compounds.Since the cloning of wild-type green fluorescent protein (GFP) from the jellyfish Aequoria victoria (25), expression of GFP has been demonstrated in numerous organisms, including plants (31), animals (5), bacteria (3), yeasts (7), and filamentous fungi (13, 37). Most applications of GFP have been as a passive label of gene expression and protein localization (for a review, see reference 36). However, GFP and selected mutants are now increasingly used as active sensors of physiological events within cells. In this role, GFP fluorescence is influenced posttranslationally by its chemical environment. For example, the pH sensitivity of GFP has recently been exploited to measure intracellular (16,18,27) and organellar (20) pH, and GFP-based systems have been developed to monitor intracellular calcium (21), microviscosity (34), and protease activity (15).One application of GFP that has not been explored with fungi is its use as an indicator of antimicrobial susceptibility. GFP has several properties that are desirable for this purpose, including simplicity and versatility for in vitro use (9). GF...

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