Purpose: Pathogenic fungi must be able to survive and thrive in dynamic in vivo environments to successfully cause invasive disease. The ability to sense and utilize varying nutrient sources and to quickly respond to stress signals underpins Aspergillus fumigatus virulence. Typically, such environmental signals are transmitted to the cell machinery via various signal transduction pathways. We, and others, have shown that Ras networks are major contributors to stress and nutritional signaling. Ras guanine nucleotide exchange factors (GEFs) are known to be essential to the proper activation of Ras proteins. However, the signals to which fungal RasGEFs respond, how they become activated and how they regulate Ras in response to these signals are all largely unknown. Here, we sought to identify the RasGEF(s) responsible for Ras activation and delineate the importance of the RasGEF sub-family to A. fumigatus growth.
Methods: We utilized CRISPR-mediated gene editing to generate deletion, reconstituted and tetracycline-regulatable strains for each putative RasGEF in A. fumigatus. Germination, hyphal growth and stress analyses were performed using standard in vitro culture methods. Green fluorescence protein (GFP)-Ras chimeras and western blot analyses were utilized to examine the effect of loss of RasGEF function on Ras localization and protein abundance, respectively.
Results: We identified four putative RasGEFs by conducting a BLAST search of the A. fumigatus genome using the Saccharomyces cerevisiae Cdc25 protein sequence as a query. The putative A. fumigatus RasGEF homologs (GefA, GefB, GefC, and GefD) comprise three major classes of known RasGEFs: 1) SH3-class (GefA and GefB); 2) RasGRP-class (GefC) and; 3) LTE-class (GefD). Of these three classes, both the SH3- and the LTE-class are considered fungal-specific. Deletion and subsequent complementation of each RasGEF revealed important roles for gefA in germination, hyphal branching, and cell wall integrity. Although multiple attempts were made, double-deletion of the SH3-class GEFs was not achievable. Therefore, using the ΔgefA genetic background, we generated a strain in which gefB expression was regulatable by placing gefB under the control of a Tet-on promoter. Growth analyses, in the presence and absence of tetracycline, showed that loss of both gefA and gefB caused severely reduced hyphal growth, loss of RasA localization to the plasma membrane and reduction in RasA protein abundance.
Conclusions: Our results suggest that the SH3-class RasGEFs, gefA and gefB, likely play overlapping roles in the activation of Ras proteins. In addition, our data also suggest that the ability of Ras proteins to become activated is intricately entwined with Ras protein abundance. These findings support the hypothesis that a feedback loop exists in which RasGEFs, or active Ras proteins, send signals that either maintain Ras transcriptional levels or block Ras protein degradation. Further work will attempt to elucidate this feedback loop and potentially exploit it for therapeutic benefit.
Full conference title:
- AAA 8th (2018)