Investigation of Azole Resistance Involving cyp51A and cyp51B Genes in Clinical Aspergillus flavus Isolates

Aspergillus flavus is a filamentous fungus, with worldwide distribution (Hedayati et al. 2007). After Aspergillus fumigatus this species is the second most important causative agent of invasive and non-invasive aspergillosis in patients (Baranyi et al. 2017). Three classes of antifungal agents form current treatments of invasive aspergillosis (IA): polyenes (amphotericin B (AMB)), azoles (voriconazole (VOR), itraconazole (IT), posaconazole (POS)) and more recently semi-synthetic echinocandins (caspofungin, micafungin, rezafungin and anidulafungin). Among these, azoles are the first choice drugs in managing and prophylaxis of aspergillosis (Patterson et al. 2016). The resistance to azoles has increased, especially in Aspergillus species. The overall azole resistance rate of A. fumigatus could reach 27.8%, according to Vermeulen et al. (2013). Prevalence rate variation between studies could be due to regional and disease differences (Vermeulen et al. 2013). Recently, Rhodes et al. (2022) explored 218 isolates collected from patients with different aspergillosis manifestations and from the environment. These A. fumigatus isolates were collected from different hospital centers (England, Wales, Scotland, and Ireland). They found that 49% showed resistance to at least one of the tested antifungal drugs. For specific azoles, 52% (n = 104) exceeded minimal inhibitory concentration (MIC) breakpoints to itraconazole (≥ 2 mg/l), 31% (n = 64) to voriconazole (≥ 2 mg/l), and 25% (n=44) to posaconazole (≥ 0.5 mg/l). They also found that 26 isolates (12%) exceeded MIC breakpoints to two or more azole drugs from both clinical (n = 23) and environmental (n = 3) sources (Rhodes et al. 2022). Shelton et al. (2023) explored 1,894 air samples collected from England, Wales, Scotland, and Northern Ireland. They identified 99 TEB-resistant A. fumigatus isolates. These isolates were resistant to itraconazole (86%), to voriconazole (64%), to posaconazole (18%) and to isavuconazole (83%). Thus, there was clear cross-resistance between the agrochemical TEB and the medical azole antifungals (Shelton et al 2023).

Knowing that antifungal susceptibility testing is not routine testing in many centers around the world, the azole resistance in A. flavus still needs to be fully explored. In addition, the molecular mechanism of azole resistance concerning this species of filamentous mold has yet to be fully explained.

Several studies have shown that azoles inhibit the ergosterol biosynthetic pathway by interacting with the two CYP51 proteins (CYP51A and CYP51B) and alter the metabolism of sterols in cells (Alcazar-Fuoli et al. 2008). However, a risk of decisive infection may occur if resistance is acquired (Howard and Arendrup 2011; Hadrich et al. 2012a). Various studies focus on azole-resistant Aspergillus species, with a particular interest in A. fumigatus isolates (Verweij et al. 2016; Chowdhary et al. 2017). Studying molecular mechanisms of resistance is an essential step and complementary to the in vitro identification of antifungal resistance (Buil et al. 2018). Over the last decade, there has been some progress in identifying genes responsible for antifungal resistance in A. fumigatus, but studies have been very rare in A. flavus (Hagiwara et al. 2016). A duplication of several genes (cyp51A and cyp51B) involved in the sterol biosynthesis pathway was noted in A. fumigatus (Mellado et al. 2001; Da Silva Ferreira et al. 2005; Nierman et al. 2005). In A. flavus, there are three CYP51 proteins: CYP51A, CYP51B, and CYP51C. Recently, rare studies have analyzed genetic alterations and/or overexpression of azole target genes and efflux pump genes (EPs) that reduce intracellular drug concentrations in azole-resistant isolates (Krishnan-Natesan et al. 2008; Liu et al. 2012; Paul et al. 2015). Furukawa et al. (2020) constructed a 484-member transcription factor null mutant library to provide a systematic evaluation of regulators that contribute to azole resistance in A. fumigatus. They identified 12 regulators that have a demonstrable role in itraconazole susceptibility. The loss of the negative cofactor 2 complex leads to resistance to itraconazole, amphotericin B, and terbinafine.

This study evaluated the mRNA expression levels of cyp51 (A and B) genes in clinical Aspergillus flavus isolates determined by RT-qPCR. A PCR sequencing of cyp51A and cyp51B genes was used to detect the presence of gene mutations.

Several antifungal drugs can be used against Aspergillus spp. such as amphotericin B, triazoles and echinocandins. In patients with aspergillosis who require long-term treatment, triazoles become the most effective because they are essential therapeutic options and are of particular significance due to the availability of oral formulations (Denning and Perlin 2011; Arikan-Akdagli et al. 2018).

The triazole antifungals present a broad spectrum for the management of invasive aspergillosis. The treatment of A.flavus infection is similar to that caused by other species of Aspergillus (Meena et al. 2021).

Over the past two decades, many researchers have studied and identified that resistance mechanisms to azoles vary depending on the country and the studies of clinical and environmental strains of A.flavus (Sharma et al. 2018). However, most recently published studies refer to alterations in the structure of cyp51A, cyp51B, or cyp51C, or of these expression levels, as the primary resistance mechanisms of this fungus, which leads to the failure of the treatment in patients with aspergillosis infected with azole-resistant strains. It should be noted that there are two reported pathways for developing azole resistance: the first environmental, corresponding to the use of fungicides to protect crops, and a second medicine due to long-term azole therapies (Gonzalez-Jimenez et al. 2020).

A. flavus has three different enzymes, which are successfully inhibited by azole drugs, suggesting that these enzymes can play a role in azole resistance (Mellado et al. 2001; Howard et al. 2011). However, other Aspergillus spp. and most filamentous fungi possess two CYP51 proteins, CYP51A and CYP51B, although some species have only one (Hawkins et al. 2014). A CYP51A, A2, and B paralog pattern was observed in the azole-resistant species Aspergillus fumigatiaffinis. A new CYP51 paralog, CYP51D, was recently described in fungi, which predominantly occurs in genomes of intrinsically azole-resistant Eurotiomycetes (Van Rhijn et al. 2021). Celia-Sanchez et al. (2022) found four major CYP51 groups: CYP51, CYP51A, CYP51B, and CYP51C. They confirm that all filamentous Ascomycota had a CYP51B paralog, while only 50% had a CYP51A paralog. They also suggest that CYP51 is present in all fungi, including CYP51C, which appears to have diverged from the progenitor of the CYP51A and CYP51B groups. Several synonymous and non-synonymous point mutations were found in cyp51C gene of A.flavus among both susceptible and non-susceptible strains such as M54T, S240A, D254N, I285V, and N423D (Liu et al. 2012; Paul et al. 2015; Sharma et al. 2018), D254G, P276T, S399I, and a mutation responsible for a stop codon (R250ST) (Lucio et al. 2020). The point mutation (Y319H) in A. flavus cyp51C suggests a relation to azole resistance although its role still needs further confirmation (Paul et al. 2015; Sharma et al. 2018).

However, there is minimal data on A. flavus isolates in Tunisia, and less is known of their resistance mechanisms, despite being the most commonly isolated mold species from patients and their environment (Hadrich et al. 2010). Therefore, our study evaluated the quantification of expression and number of copies for the two genes (cyp51A and cyp51B) in clinical A. flavus isolates IT-resistant and POS-resistant.

Our findings showed overexpression in two IT/POS resistant A. flavus isolates for cyp51A with the highest level of 16.8-fold and in one IT/POS resistant isolate (TN-31) for cyp51B, suggesting that these two genes contribute to resistance to azoles. According to Fattahi et al. (2015) a significant up-regulation of cyp51A (6.89 to 7.93-folds) (p < 0.001) in four A. flavus VOR-resistant isolates was found. Several authors have agreed that the up-regulation of the cyp51A gene is most commonly associated with azole resistance in A. fumigatus and Aspergillus lentulus (Howard et al. 2010; Mellado et al. 2011). In addition, CYP51B has been shown to be a functional biosynthetic enzyme that is inhibited by azoles. However, Liu et al. indicated that the expression levels of the three genes cyp51A, cyp51B, and cyp51C were not associated with azole resistance in A. flavus (Liu et al. 2012).

However, Sharma et al. (2018), found that cyp51A and cyp51B were upregulated (> 3-fold) for a single VOR-resistant strain (VPCI195/P/10). Contrary to what was expected, the only strain with cross-resistance to antifungals did not show up-regulation of any target genes analyzed.

In this study, the option of overexpression in sensitive profile strains may be explained by the vast clinical use of this antifungal in our country, particularly in patients with chronic diseases. This suggests that the cyp51A and B gene overexpression does not necessarily result in azole resistance in this fungus.

Furthermore, the mechanism of azole resistance in most of the previously published studies is associated with changes that are often corresponding to mutations in the cyp51A gene (Nascimento et al. 2003; Chen et al. 2005; Garcia-Effron et al. 2005; Buil et al. 2018). Several publications have clearly described single amino acid substitutions at G54, P216, F219, M220, and G448 in the CYP51A protein to confer azole resistance in A. fumigatus (Mann et al. 2003; Mellado et al. 2004; Camps et al. 2012; Krishnan Natesan et al. 2012). Over time, besides these ‘hot spots’, amino acid changes at other sites (Y121, G138, and Y431) have also been found in the CYP51A protein of resistant strains (Albarrag et al. 2001; Lescar et al. 2014). Prolonged azole treatment in chronic aspergillosis is a significant cause of these resistance mutations (Howard et al. 2009), as is environmental exposure to azole fungicides (Snelders et al. 2008). Typically, combinations of tandem repeats and point mutations of the cyp51A gene (TR34/L98H and TR46/Y121F/T289A) have been observed in azoleresistant A. fumigatus isolated from patients regardless of their azole treatment history (Snelders et al. 2008).

Here, we described for A. flavus, three new point mutations observed in Cyp51A (a synonymous substitution (P61P) and a non-synonymous (G206L)) and Cyp51B (a non-synonymous mutation in cyp51B (L177G). To our knowledge, these point mutations have not been described before. In a recent report, susceptible and non-susceptible A. flavus strains presented several synonymous and nonsynonymous point mutations in cyp51A and cyp51C genes. However, two amino acid mutations were noted in only two resistant strains: one strain contained a P214L substitution in cyp51A, and another was H349R in cyp51C (Gonzalez-Jimenez et al. 2020).

As stated previously, Krishnan-Natesan et al. (2008) were among the first to identify different mutations of cyp51A and cyp51B in spontaneous mutant strains obtained after exposure to voriconazole. These mutations were found in both cyp51A (K197N, D282E, M288L, Y132N, and T469S) and cyp51B (H399P, D411N, T454P, and T486P). However, these substitutions were recently revealed in azoles-sensitive A. flavus strains, making their implication in drug resistance highly unlikely (Hagiwara et al. 2016; Lucio et al. 2020). Similarly, Lucio et al. (2020) found that cyp51B showed no amino acid changes in A. flavus strains, except for some polymorphisms responsible for synonymous mutations. In our study, synonym and nonsynonymous mutations of cyp51A (P61P and G206L) were detected in azole-sensitive and non-sensitive A. flavus strains, excluding the possibility that they are associated with azole resistance. Similarly, Lucio et al. (2020) reported in susceptible and resistant strains the presence of three synonymous and nonsynonymous point mutations.

In previous studies, the crystallographic structures of the CYP51 proteins of Saccharomyces cerevisiae complexed with the substrates lanosterol, itraconazole, voriconazole, and fluconazole have been described (Monk et al. 2014; Sagatova et al. 2015). So also, the structure of Candida albicans CYP51 complexed with posaconazole (5FSA) and the tetrazole-based antifungal drug candidate VT1161 was analyzed (Hargrove et al. 2017a). Hargrove et al. (2017b) have described the structure of CYP51B in complex with the NIV derivative and the tetrazole-based VT-1598 inhibitor for A. fumigatus. Upon the recently resolved crystal structure of the CYP51B protein of A. fumigatus, a new 3D structural model of A. flavus CYP51A and CYP51B was built. Docking analysis of three azole molecules to their target proteins, CYP51A and CYP51B was performed based on this structural model.

According to the models provided by the PatchDock software, three new mutations, a synonym and a nonsynonym in cyp51A (P61P and G206L) and a nonsynonymous mutation in cyp51B (L177G) do not affect the interaction between the drug and protein CYP51A or CYP51B. The resistance of our strains could be mainly due to the overexpression of the cyp51A and cyp51B genes. However, an in-depth study of azole resistance-related genes in a large population of clinical isolates will expand our knowledge.

Therefore, further examination of other resistance mechanisms is necessary, like the CCAAT-binding domain complex CBC (a heterotrimer comprising HapB, HapC, and HapE, which is a negative regulator of sterol biosynthesis directly binding the promoters of 14 ergosterol biosynthetic genes), overexpression of MFS (major facilitator superfamily) transporters or overexpression of ABC transporters (ATP binding cassette superfamily). A deeper understanding of azole resistance mechanisms will facilitate the development of new therapeutic drugs against azole-resistant strains of Aspergillus.