Study and characterization of azole resistance in aspergillus section nigri

Resumen

Several species of Aspergillus display clinical relevance, since they are opportunistic pathogens associated to a broad variety of human conditions. Among these, invasive aspergillosis is the most severe in terms of morbidity and mortality. Although Aspergillus fumigatus is the most common causal agent of invasive disease, elevated prevalence of species from other sections, such as section Nigri members, has been reported lately in the clinical field. Despite the current treatment of choice for aspergillosis is voriconazole (VRC), azole resistance events have alarmingly increased in the last years,critically impairing disease management. In this context, whereas azole resistance mechanisms in A. fumigatus have been extensively studied, azole resistance in non-fumigatus species remains poorly characterized. On this basis, the main objective of this work is to characterize azole resistance in species of Aspergillus section Nigri. With this purpose, we have determined azole susceptibility in section Nigri strains and we have carried out molecular and genetic analyses of azole target Cyp51 proteins, encoded by the cyp51 genes. Our results showed good activity of the triazoles itraconazole (ITC), posaconazole (PSC) and VRC against the strains we tested of Aspergillus section Nigri, with PSC exerting the best in vitro activity. Regarding the molecular analyses, we observed that the Cyp51A protein leads azole response in Aspergillus section Nigri in contrast to Cyp51B, as evidenced by the higher expression of the cyp51A gene and its upregulation upon VRC exposure. Moreover, we also concluded that amino acid substitutions in the Cyp51A protein are not clearly correlated to azole resistance in Aspergillus section Nigri, whereas it has been reported to occur in A. fumigatus. In addition, ergosterol content and properties of the plasma membrane and the cell wall were investigated. In this sense, basal ergosterol content or its depletion upon VRC exposure showed no correlation with azole susceptibility. Interestingly, deletion of either cyp51A or cyp51B genes impacted azole susceptibility by reducing VRC MICs and decreasing ergosterol levels, but the cyp51A deletion was associated to the most notable impact in VRC susceptibility. Furthermore, composition and properties of the fungal plasma membrane and cell wall appear to be important for azole resistance. Finally, potential resistance mechanisms related to drug degradation by fungal enzymes and the association between azole resistance and in vitro macrophage phagocytosis were evaluated. Altogether our results slightly diverge from those obtained from A. fumigatus, which evidence the complexity behind azole resistance, especially in non-fumigatus species of Aspergillus.