TheAspergillus fumigatusfarnesyltransferase β-subunit, RamA, mediates growth, virulence, and antifungal susceptibility

Abstract

Post-translational prenylation mechanisms, including farnesylation and geranylgeranylation, mediate both subcellular localization and protein-protein interaction in eukaryotes. The prenyltransferase complex is an αβ heterodimer in which the essential α-subunit is common to both the farnesyltransferase and the geranylgeranyltransferase type-I enzymes. The β-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 β-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.

Systems impact of zinc chelation by the epipolythiodioxopiperazine dithiol gliotoxin inAspergillus fumigatus: a new direction in natural product functionality

“…AlcC has been identified as the primary hypoxia-responsive ADH in A. fumigatus, with a potential role in pathogenesis, 51 while farnesyltransferase has a role in signalling and also contributes to disease. 52 Further investigation could elucidate the effect of DTG on the functionality of these proteins in A. fumigatus. Pre-incubation of DTG with excess Zn 2+ abrogated this effect, most likely through formation of Zn(DTG) complexes prior to addition to protein lysates, leading to reduced levels of protein aggregation.…”

SH3‐class Ras guanine nucleotide exchange factors are essential forAspergillus fumigatusinvasive growth

“…In addition, we have previously shown that Ras signalling is required for cell wall stability in A . fumigatus (Fortwendel et al, ; Norton, Al Abdallah, Hill, Lovingood, & Fortwendel, ). To determine potential involvement of A .…”

Differential requirements of protein geranylgeranylation for the virulence of human pathogenic fungi

“…All animal experiments were done according to protocols approved by the University of Tennessee Institutional Animal Care and Use Committee. For the virulence studies using A. fumigatus strains, six-week-old female CF-1 mice (Harlan Laboratories) were immunosuppressed using two different protocols previously described [33]. For both the non-neutropenic and neutropenic models of invasive aspergillosis, mice were subcutaneously injected with 40 mg/kg of triamcinolone acetonide one day prior to infection.…”

“…CDC43, which is the β-subunit of GGTase-I, is essential in S. cerevisiae [26], and mediates morphogenesis in C. albicans [31] and thermotolerance, morphology, and virulence in C. neoformans [32]. Recently, we characterized the β-subunit of the farnesyltransferase complex (RamA) in Aspergillus fumigatus and demonstrated that ramA null mutants display attenuated virulence, accompanied by growth defects and decreased conidial viability [33]. In this mutant, localization of RasA was only partially impaired, suggesting that lack of Ras farnesylation could be compensated by GGTase-I activity.…”