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

Abstract

Protein 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 αsubunit, 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 thermosensitivity, 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.

Loss of Septation Initiation Network (SIN) kinases blocks tissue invasion and unlocks echinocandin cidal activity against Aspergillus fumigatus

“…To examine the impacts of hyphal septation loss on virulence, we next compared the CEA10 parent and SIN kinase mutant strains in two well-described mouse models of invasive aspergillosis, representing chemotherapeutic and corticosteroid-induced immune suppression [ 56 ]. Mice (n = 8 / arm) were immune suppressed with injections of cyclophosphamide and triamcinolone acetonide or with triamcinolone acetonide alone for the chemotherapeutic and corticosteroid model, respectively.…”

“…For survival studies, two different models of invasive pulmonary aspergillosis were employed, as previously described [ 56 ]. Each model utilized CF-1 female mice weighing approximately 25 g. For the corticosteroid model, mice were immunosuppressed with 40 mg / kg of triamcinolone acetonide (TA) (Kenalog, Bristol-Myers Squibb, Princeton, NJ, USA), given subcutaneously the day prior to the infection.…”

Inhibiting Fungal Echinocandin Resistance by Small-Molecule Disruption of Geranylgeranyltransferase Type I Activity

“…Its ability to potentiate the antifungal activity of echinocandins makes L-269289 plus an echinocandin an ideal broad-spectrum drug combination to attack the fungal cell wall in pathogenic yeasts. A recent report showed that the expression of CDC43 was required for C. albicans pathogenesis (29), suggesting that, in addition to enhancing echinocandin activity, L-269289 alone can reduce C. albicans virulence by inhibiting GGTase I. This may explain why a fairly high concentration of L-269289 (Ͼ35 M) was required to significantly reduce the MIC of caspofungin in vitro (Fig.…”

The Lysine Deacetylase RpdA Is Essential for Virulence in Aspergillus fumigatus

“…The employed xylP system therefore appears to be a suitable new tool to conduct virulence studies as an interesting alternative to gene deletion mutants or expression systems that are induced or repressed by tetracycline/doxycycline (Tet-On/Tet-Off promoter systems). The latter systems have been introduced for A. fumigatus in several axenic approaches (Vogt et al, 2005; Helmschrott et al, 2013; Kalb et al, 2015; Macheleidt et al, 2015; Wanka et al, 2016) and there are four studies employing these promoters for control of A. fumigatus gene expression during infection: three for Tet-On (Lin et al, 2015; Sasse et al, 2016; Souza et al, 2019) and one for the Tet-Off system (Peng et al, 2018). A disadvantage of administration of tetracycline-type antibiotics might be their potential side effects in host mitochondria and on the animal microbiome (Ahler et al, 2013; Chatzispyrou et al, 2015; Moullan et al, 2015).…”

“…Different conditional gene expression systems have been established in A. fumigatus based on different promoters, e.g., the nitrate-inducible A. fumigatus niiA promoter, the alcohol-inducible A. nidulans alcA promoter, the xylose-inducible Penicillium chrysogenum xylP promoter (Zadra et al, 2000; Romero et al, 2003; Hu et al, 2007; Hartmann et al, 2010), and tetracycline-inducible/repressible Tet-On/Off systems exploiting the Escherichia coli tetracycline-resistance operon (Vogt et al, 2005). Of these, the Tet-On/Off systems have been applied for both up- and downregulation, while the niiA promoter was employed for downregulation of gene expression during infection (Hu et al, 2007; Lin et al, 2015; Sasse et al, 2016; Peng et al, 2018; Souza et al, 2019). The promoter of the P. chrysogenum β-1,4-endoxylanase-encoding xylP gene has been shown previously to allow xylose-mediated activation of gene expression (Figure 1A), even in the presence of glucose, during axenic growth in several molds including A. nidulans , A. fumigatus , P. chrysogenum , and Penicillium marneffei (Zadra et al, 2000; Pongsunk et al, 2005; Hartmann et al, 2010; Sigl et al, 2010; Tribus et al, 2010; Yasmin et al, 2012; Bauer et al, 2016; Vaknin et al, 2016; Gsaller et al, 2018; Misslinger et al, 2018).…”