In C. albicans the product of the NRG1 gene, Nrg1p, functions as a key regulator of the yeast-to-hypha morphogenetic transition. Nrg1p is a DNA binding protein produced in yeast cells (blastospores) where it inhibits the transcription of hypha-specific genes. On receiving an appropriate signal (serum at 37 o C, pH, N-acetylglucosamine etc.), NRG1 is downregulated, relieving the inhibition on the hypha specific genes and facilitating filament formation. A large number of studies using C. albicans mutants locked in either the yeast (•••• efg1) or filamentous (•••• tup1) forms, have demonstrated the importance of the morphogenetic transition in various aspects of the infectious process. To ascertain the role of the yeast-to-hypha switch in the clinically important processes of invasion and biofilm formation, we have constructed a strain in which this developmental transition can be externally manipulated. This was achieved by placing one copy of the NRG1 gene under the control of a tetracycline regulatable promoter. Microscopic examination of this strain under several different hypha-inducing conditions demonstrated that in the absence of doxycycline (when Nrg1p is constitutively produced), the transition is inhibited and the cells grow in the yeast form. In the presence of doxycyline (when the modified copy of NRG1 is always inactive), the cells respond normally to the environmental cues stimulating hyphal development and grow as filaments. Using two different assays of invasion, the engineered strain was found to be unable to dig into an agar surface or penetrate an ECM matrigel membrane when grown in the absence of doxycycline. Biofilm formation by C.albicans is receiving much interest of late, especially in the medical setting, principally because of their inherent resistance to antifungal drugs. To assess the role of Nrg1p in biofilm formation, we grew our modified strain under conditions which normally favour biofilm growth in both the presence or absence of doxycycline. In the presence of the antibiotic, a mature biofilm composed of a dense network of yeast and hyphal cells developed, whereas those cells incubated without doxycycline were unable to form a biofilm. In summary, we have demonstrated a key role for Nrg1p in the clinically significant processes of invasion and biofilm formation.
Full conference title:
The 15 th Congress of the International Society for Human and Animal Mycology
- ISHAM 15th (2003)