Abstract
Inhibition of Δ24-sterol methyltransferase (24-SMT) in Sporothrix schenckii sensu stricto and Sporothrix brasiliensis was investigated in vitro. The effects on fungal growth and sterol composition of the 24-SMT inhibitor 22-hydrazone-imidazolin-2-yl-chol-5-ene-3β-ol (H3) were compared to those of itraconazole. MIC and MFC analysis showed that H3 was more effective than itraconazole against both species in both their filamentous and yeast forms. H3 showed fungistatic activity in a time-kill assay, with inhibitory activity stronger than that of itraconazole. GC analysis of cell sterol composition showed that sterols present in control cells (ergosterol and precursors) were completely replaced by 14α-methylated sterols after H3 exposure. Itraconazole only partially inhibited ergosterol synthesis but completely arrested synthesis of other sterols found in control cells, promoting accumulation of nine 14α-methyl sterols. Based on these results, we propose a schematic model of sterol biosynthesis pathways in S. schenckii and S. brasiliensis. Effects on cell morphology due to 24-SMT inhibition by H3 as analyzed by SEM and TEM included irregular cell shape, reduced cytoplasmic electron-density, and reduced thickness of the microfibrillar cell wall layer. Moreover, 24-SMT inhibition by H3 promoted mitochondrial disturbance, as demonstrated by alterations in MitoTracker® Red CMXRos fluorescence intensity evaluated by flow cytometry. When used in conjunction with itraconazole, H3 enhanced the effectiveness of itraconazole against all tested strains, reducing at least half (or more) the MIC values of itraconazole. In addition, cytotoxicity assays revealed that H3 was more selective toward these fungi than was itraconazole. Thus, 24-SMT inhibition by H3 was an effective antifungal strategy against S. schenckii and S. brasiliensis. Inhibition of the methylation reaction catalyzed by 24-SMT has a strong antiproliferative effect via disruption of ergosterol homeostasis, suggesting that this enzyme is a promising target for novel antifungal therapies against sporotrichosis, either as sole treatments or in combination with itraconazole.
Highlights
In the last decade, the incidence of sporotrichosis, a subcutaneous mycosis with worldwide distribution, has increased in Brazil (Chakrabarti et al, 2015), in the state of Rio de Janeiro (Silva et al, 2012)
Itraconazole was less effective against S. brasiliensis than it was against S. schenckii (p = 0.0015 and 0.008 for the filamentous and yeast forms, respectively)
We investigated the effect of the 24-sterol methyltransferase (24-SMT) inhibitor H3 on the sterol biosynthesis pathway in S. schenckii and S. brasiliensis
Summary
The incidence of sporotrichosis, a subcutaneous mycosis with worldwide distribution, has increased in Brazil (Chakrabarti et al, 2015), in the state of Rio de Janeiro (Silva et al, 2012). Sporotrichosis is caused by dimorphic fungi from the Sporothrix schenckii complex (Marimon et al, 2007), with Sporothrix schenckii sensu stricto and Sporothrix brasiliensis representing the most common clinically isolated forms in Brazil (Rodrigues et al, 2014b) and the most virulent species infecting animal models (Arrigala-Moncrieff et al, 2009; Fernandes et al, 2013). S. brasiliensis is epidemic and responsible for human and feline sporotrichosis cases in the state of Rio de Janeiro, Brazil (Rodrigues et al, 2013). Itraconazole is the first-choice treatment for cutaneous and lymphocutaneous sporotrichosis (Kauffman et al, 2007). This azole compound inhibits ergosterol biosynthesis at the C14α-demethylation stage (Odds et al, 2003) that is catalyzed by the cytochrome P-450-dependent 14α-demethylase. The resulting ergosterol depletion, with the accumulation of 14α-methylated sterols, interferes with the architecture and fluidity of fungal membranes, which can no longer act as permeability barriers (Odds et al, 2003)
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