Abstract
ABSTRACTProtein prenylation is a crucial post-translational modification largely mediated by two heterodimeric enzyme complexes, farnesyltransferase and geranylgeranyltransferase type-I (GGTase-I), each composed of a shared α-subunit and a unique β-subunit. GGTase-I enzymes are validated drug targets that contribute to virulence in Cryptococcus neoformans and to the yeast-to-hyphal transition in Candida albicans. Therefore, we sought to investigate the importance of the α-subunit, RamB, and the β-subunit, Cdc43, of the A. fumigatus GGTase-I complex to hyphal growth and virulence. Deletion of cdc43 resulted in impaired hyphal morphogenesis and thermo-sensitivity, which was exacerbated during growth in rich media. The Δcdc43 mutant also displayed hypersensitivity to cell wall stress agents and to cell wall synthesis inhibitors, suggesting alterations of cell wall biosynthesis or stress signaling. In support of this, analyses of cell wall content revealed decreased amounts of β-glucan in the Δcdc43 strain. Despite strong in vitro phenotypes, the Δcdc43 mutant was fully virulent in two models of murine invasive aspergillosis, similar to the control strain. We further found that a strain expressing the α-subunit gene, ramB, from a tetracycline-inducible promoter was inviable under non-inducing in vitro growth conditions and was virtually avirulent in both mouse models. Lastly, virulence studies using C. albicans strains with tetracycline-repressible RAM2 or CDC43 expression revealed reduced pathogenicity associated with downregulation of either gene in a murine model of disseminated infection. Together, these findings indicate a differential requirement for protein geranylgeranylation for fungal virulence, and further inform the selection of specific prenyltransferases as promising antifungal drug targets for each pathogen.
Highlights
Protein prenylation is a post-translational modification that increases protein hydrophobicity, promoting protein–protein interactions and protein affinity for cell membranes [1]
To first examine the role of protein geranylgeranylation in A. fumigatus growth and virulence, we initiated this study by identifying the GGTase-I β-subunit
S. cerevisiae Cdc43p protein sequence performed through the Aspergillus genome database revealed a single homologous gene for the GGTase-I β-subunit, Afu6g06710 (29.9% amino acid identity), hereafter called cdc43
Summary
Protein prenylation is a post-translational modification that increases protein hydrophobicity, promoting protein–protein interactions and protein affinity for cell membranes [1]. This modification is critical for the subcellular localization and function of important proteins, being crucial in various cellular physiological processes. FTase and GGTase-I recognize a consensus sequence of four amino acids that is usually referred to as the CaaX motif (C = cysteine; aa = two aliphatic amino acids, usually; X = any amino acid) [1,2,3]. It has been demonstrated that proteins bearing a C-terminus CaaaX sequence may be subject to prenylation by Ftase enzymes [4]. Subcloning of 1.5kb upstream flanking region of cdc in pJW24; amplification of cdc deletion cassette
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