Control of Ras-mediated signaling in Aspergillus fumigatus.

Pathogenic fungi employ numerous mechanisms to flourish in the stressful environment encountered within their mammalian hosts. Central to this arsenal for filamentous fungi is invasive growth within the host microenvironment, mediated by establishment and maintenance of polarized hyphal morphogenesis. In Aspergillus fumigatus, the RasA signal transduction pathway has emerged as a significant regulator of hyphal morphogenesis and virulence, among other processes. The factors contributing to the regulation of RasA itself are not as thoroughly understood, although proper temporal activation of RasA and spatial localization of RasA to the plasma membrane are known to play major roles. Interference with RasA palmitoylation or prenylation results in mislocalization of RasA and is associated with severe growth deficits. In addition, dysregulation of RasA activation results in severe morphologic aberrancies and growth deficits. This review highlights the relationship between RasA signaling, hyphal morphogenesis, and virulence in A. fumigatus and focuses on potential determinants of spatial and temporal RasA regulation.

The Aspergillus fumigatus farnesyltransferase ß-subunit, RamA, mediates growth, virulence, and antifungal susceptibility.

Post-translational prenylation mechanisms, including farnesylation and geranylgeranylation, mediate both subcellular localization and protein-protein interaction in eukaryotes. The prenyltransferase complex is an αß heterodimer in which the essential α-subunit is common to both the farnesyltransferase and the geranylgeranyltransferase type-I enzymes. The ß-subunit is unique to each enzyme. Farnesyltransferase activity is an important mediator of protein localization and subsequent signaling for multiple proteins, including Ras GTPases. Here, we examined the importance of protein farnesylation in the opportunistic fungal pathogen Aspergillus fumigatus through generation of a mutant lacking the farnesyltransferase ß-subunit, ramA. Although farnesyltransferase activity was found to be non-essential in A. fumigatus, diminished hyphal outgrowth, delayed polarization kinetics, decreased conidial viability, and irregular distribution of nuclei during polarized growth were noted upon ramA deletion (ΔramA). Although predicted to be a target of the farnesyltransferase enzyme complex, we found that localization of the major A. fumigatus Ras GTPase protein, RasA, was only partially regulated by farnesyltransferase activity. Furthermore, the farnesyltransferase-deficient mutant exhibited attenuated virulence in a murine model of invasive aspergillosis, characterized by decreased tissue invasion and development of large, swollen hyphae in vivo. However, loss of ramA also led to a Cyp51A/B-independent increase in resistance to triazole antifungal drugs. Our findings indicate that protein farnesylation underpins multiple cellular processes in A. fumigatus, likely due to the large body of proteins affected by ramA deletion.

A Fungus-Specific Protein Domain Is Essential for RasA-Mediated Morphogenetic Signaling in Aspergillus fumigatus.

Ras proteins function as conserved regulators of eukaryotic growth and differentiation and are essential signaling proteins orchestrating virulence in pathogenic fungi. Here, we report the identification of a novel N-terminal domain of the RasA protein in the filamentous fungus Aspergillus fumigatus. Whereas this domain is absent in Ras homologs of higher eukaryotes, the N-terminal extension is conserved among fungi and is characterized by a short string of two to eight amino acids terminating in an invariant arginine. For this reason, we have termed the RasA N-terminal domain the invariant arginine domain (IRD). Through mutational analyses, the IRD was found to be essential for polarized morphogenesis and asexual development, with the invariant arginine residue being most essential. Although IRD truncation resulted in a nonfunctional Ras phenotype, IRD mutation was not associated with mislocalization of the RasA protein or significant changes in steady-state RasA activity levels. Mutation of the RasA IRD diminished protein kinase A (PKA) activation and resulted in decreased interaction with the Rho-type GTPase, Cdc42. Taken together, our findings reveal novel, fungus-specific mechanisms for Ras protein function and signal transduction. IMPORTANCEAspergillus fumigatus is an important fungal pathogen against which limited treatments exist. During invasive disease, A. fumigatus hyphae grow in a highly polarized fashion, forming filaments that invade blood vessels and disseminate to distant sites. Once invasion and dissemination occur, mortality rates are high. We have previously shown that the Ras signaling pathway is an important regulator of the hyphal growth machinery supporting virulence in A. fumigatus. Here, we show that functional Ras signaling in A. fumigatus requires a novel, fungus-specific domain within the Ras protein. This domain is highly conserved among fungi, yet absent in higher eukaryotes, suggesting a potentially crucial difference in the regulation of Ras pathway activity between the human host and the fungal pathogen. Exploration of the mechanisms through which this domain regulates signaling could lead to novel antifungal therapies specifically targeting fungal Ras pathways.