Resistance mechanisms of Aspergillus fumigatus to antifungals

Aspergillus fumigatus is a universally distributed opportunistic fungal pathogen with a significant global incidence and extremely high mortality. The widespread and extensive use of azole antifungals has led to the emergence of A. fumigatus azole resistant, resulting in serious consequences for patients infected with these isolates, who are left with limited therapeutic options.       
Initially, the emergence of resistant strains was very sporadic and showed point mutations in key areas of the Cyp51A enzyme (G54, G138, F219, M220, G448S) in strains isolated from patients undergoing long-term treatment with azoles. This clinical pathway is due to the selective pressure that azoles exert on A. fumigatus within the patient. However, since 2014, resistance has grown significantly, and almost all azole-resistant A. fumigatus strains have a combined mechanism of modifications in the promoter and the coding portion of cyp51A (TR34/L98H or TR46/Y121F/T289A). Both resistance mechanisms are frequently detected in strains from patients who have never been exposed to antifungal therapy. In these cases, the involvement of an environmental route is raised, in which the unintentional exposure of A. fumigatus to DMIs (imidazole and triazole) in the field would be favoring the resistance emergence.

Origin and Evolution of A. fumigatus Resistance to Antifungals

Nowadays, the isolation of A. fumigatus strains resistant to antifungals is an increasing global emergence. The continuous exposure of A. fumigatus to environmental fungicides, used for crop protection against other fungal species that cause agricultural damage, is believed to be selecting multi drug resistant strains. The main azole resistance mechanisms in A. fumigatus are strains with modifications of the azole target (cyp51A gene), mainly the TR34/L98H, followed by TR46/Y121F/A289T. Both types of mechanisms are responsible for panazole resistance and cross resistance to DMIs used for crop protection (imidazoles and triazoles). More recently, resistance to several fungicide classes such as, Bencimidazoles (MBC), Estrobilurinas (QoIs), sucinato deshidrogenase inhibitors (SDHIs) and  Dicarboximides, has also been acknowledged.

Genomic characterization (NGS) of strains from both clinical and environmental sources allows linking genomic differences with the acquisition of resistance to different fungicides. Adding data on susceptibility to non-azole antifungals provides a more precise picture of the phylogenetic relationships among strains, as distinct subclades are formed in which strains multi-resistant to non-azole antifungals grouped with azole-resistant strains with TRs resistance mechanisms. This formation of specific clades with strains that differ in geographic origin and year of isolation suggests the existence of a common link, an evolutionary origin according to which the strains have developed under similar circumstances that converge in a series of multi-resistance mechanisms to fungicides from different families. The resistance of A. fumigatus to non-azole fungicides, that are exclusively used in the environment, confirms that the strains with TRs resistance mechanisms are selected and developed in the environment where they are exposed to the selective pressure of multiple fungicides.

Tolerance and Persistence to Azole Antifungals in Aspergillus fumigatus

Tolerance and persistence are two phenomena by which pathogenic organisms can survive the microbicidal action of antimicrobials that should kill them over an extended period. In our laboratory, we investigate the ability of certain A. fumigatus isolates to exhibit tolerance and persistence to azoles, which are the first-line antifungal treatment for aspergillosis infections.

We are developing methodologies to detect and study tolerance and persistence, both in the laboratory and in clinical diagnosis. Using these methods, we are exploring the underlying molecular and genomic mechanisms that enable these phenomena. In addition, we are investigating the potential relevance of tolerance and persistence in the efficacy of antifungal treatment.

Differential Modulation of Persulfidation in the Fungus and Host as a Novel Antifungal Strategy

Persulfidation is a post-translational modification in which an activated sulfur group (S₂-), through the action of an enzyme, performs a specific nucleophilic attack on thiol (-SH) groups of cysteine residues in target proteins, forming a persulfide group (-SSH). This modification has been shown to modulate the intrinsic activity of proteins, playing a crucial role in various cellular mechanisms and physiological functions.

In our previous research, we demonstrated that correct levels of persulfidation are important both for A. fumigatus virulence and for orchestrating an adequate immune response in the host. Based on this, our research explores the hypothesis that differential modulation of persulfidation could constitute a novel antifungal treatment strategy.

We are investigating the ability of compounds to inhibit fungal enzymes responsible for persulfidation, aiming to reduce persulfidation levels and thereby decrease A. fumigatus virulence. Additionally, we are studying the use of sulfur donors as a potential means to enhance persulfidation in pulmonary host cells, with the goal of strengthening the immune response.

Evolution of Cross-Resistance to the New Antifungals Olorofim and Manogepix

Azole resistance is already present worldwide. Studies have shown that the most common resistance mechanisms—tandem repeats in the promoter of the gene encoding the azole target—have developed in agricultural settings due to the indiscriminate use of pesticides from the same family as clinical azoles.

Currently, two new clinical antifungals with novel molecular mechanisms of action have been introduced: olorofim and manogepix. However, analogous compounds with the same mechanism of action, ipflufenoquin and aminopyrifen, have also been developed for use as pesticides. This situation puts us at risk of repeating the same mistake made with azoles.

In this international collaborative project, we study the evolution of resistance and cross-resistance to these clinical and environmental antifungals. Our goal is to design strategies to minimize the emergence of resistance in the environment and develop early detection methods for antifungal resistance.