Multiple regulators contribute to the transcriptional control of the Candidalysin encoding gene ECE1

Objectives:
Candida albicans hyphae are major contributors to fungal virulence. Not only do they mediate adherence to host cells as well as consequent invasion, but they are also crucial for further dissemination and nutrient acquisition in infected tissue. Recently, it was shown that hyphae secrete a toxic peptide called Candidalysin which is essential for host cell membrane destruction. This peptide derives from a heavily processed precursor protein which is encoded by the ECE1 gene. For many years, it is known that ECE1 is one of the most abundant transcripts in hyphae, but barely detectable in C. albicans yeast cells. Due to its importance for fungal virulence, we analyzed the mechanisms which control the efficient activation of the gene after the initiation of hyphal growth.

Methods:
A GFP reporter system for ECE1 was used to study the expression of the gene by fluorescence microscopy. The construct was transformed into wild type and strains lacking putative regulators. The resulting mutants were then examined under yeast promoting conditions (minimal medium at 37°C). Hyphal growth was induced by the addition of 10% human serum. We also analyzed the consequences of the overexpression of selected transcription factors on ECE1 transcription. Aside the GFP reporter system, expression of ECE1 was also examined by quantitative RT PCR.

Results:
At first, we used the GFP reporter system to screen mutants lacking either putative negative or positive regulators. This screening revealed a much more complex role for the global repressor Tup1 than initially thought. Under yeast growth conditions, it contributed clearly to repression of ECE1. In hyphae promoting media however, it is also involved in the activation of the gene. In this context, we also noticed that a certain threshold of expression must be passed to produce enough GFP for bright signals. We further identified the transcription factor Ahr1 as a crucial regulator of ECE1 transcription which is required for a full expression of the gene. Interestingly, a hyperactive version of AHR1 was able to induce high levels of ECE1 transcription without additional environmental stimuli and even bypassed the absence of such transcriptional activators like Cph1, Efg1, Tec1 and Ume6. Interestingly, it could not bypass the absence of the transcriptional regulator Tup1.

Conclusion:
Our data suggest that several regulators contribute to the transcriptional control of ECE1. This complex mode of regulation enables C. albicans to rapidly activate or repress the gene's expression depending on the environmental conditions. Our study also revealed new functions for the transcriptional regulators Ahr1 and Tup1 in the control of ECE1 transcription.