Exploring the molecular mechanism of azole resistance in Aspergillus fumigatus

Journal Pre-proof Exploring the molecular mechanism of azole resistance in Aspergillus fumigatus Peiying Chen Juan Liu Meihua Zeng Hong Sang MD

PII:

S1156-5233(19)30298-7

DOI:

https://doi.org/doi:10.1016/j.mycmed.2019.100915

Reference:

MYCMED 100915

To appear in:

´ Journal de Mycologie Medicale

Received Date:

3 September 2019

Revised Date:

24 October 2019

Accepted Date:

24 November 2019

Please cite this article as: Chen P, Liu J, Zeng M, Sang H, Exploring the molecular ´ mechanism of azole resistance in Aspergillus fumigatus, Journal de Mycologie Medicale (2019), doi: https://doi.org/10.1016/j.mycmed.2019.100915

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Exploring the molecular mechanism of azole resistance in Aspergillus fumigatus Peiying Chen, Juan Liu, Meihua Zeng, Hong Sang* Department of Dermatology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, China * Corresponding Hong Sang, MD Department of Dermatology, Jinling Hospital, No.305, Zhongshan East Rd., Nanjing 210002, Jiangsu Province, China；Tel: +86-025-80860092, Fax: +86-025-84815775；E-mail:

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[email protected] Abstract

Aspergillus infections are increasingly recognized as a global health problem because of limited antifungal drugs and occurrence of azole resistance worldwide. More cyp51-mediated and non-cyp51-mediated mechanisms of azole resistance have been identified in clinical and

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laboratory studies in recent years with applications of molecular biotechnology including next-generation sequencing, reverse genetics and so on. In this review, current research on the

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molecular mechanisms of azole resistance in A. fumigatus were presented and summarized and meanwhile the putative clinical relevance of these findings from bench work were discussed. Important aims are to gain more insight to mechanism of azole resistance and

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provide some efficient lead for prevention strategy.

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Keywords: Pathogenic fungi; Aspergillus fumigatus; azole resistance; cyp51 mutation; noncyp51 mutation Introduction

Aspergillus fumigatus, the most common Aspergillus species, is a major opportunistic fungal pathogen and often causes disseminated infections in immunocompromised hosts, with a relatively high mortality, including invasive pulmonary aspergillosis, pulmonary aspergilloma, and allergic bronchopulmonary aspergillosis [1, 2]. Antifungal drugs are limited for treatment options, including polyenes (amphotericin B), azoles and echinocandins. To date, azoles are recommended for prophylaxis and treatment of aspergillosis [3, 4]. With the usage of azole in medicine and agricultural settings, reports of azole-resistant A. fumigatus have increased around the world since the first emergence of azole-resistant isolate in 1997 [5]. The treatment of invasive infections has proved to be difficult because of limited antifungal drugs and occurrence of antifungal-resistant strains worldwide. A. fumigatus have remarkable ability to grow in diverse environments. Strong adaptability of fungi to the environment facilitates persistence of the species and compromises treatment option, even therapy failure, in the clinic [6]. At present, Aspergillus infections are increasingly recognized as a global health problem, ranging from allergic conditions to acute invasive aspergillosis. 1

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Based on published works from laboratory and clinical sources, the molecular mechanisms of azole resistance can be divided into the two general categories: cyp51mediated azole resistance and non-cyp51-mediated azole resistance. A number of studies have focused on elucidating the genetic basis of azole resistance. The most common resistance mechanisms in A. fumigatus are various non-synonymous mutations referring with azole resistance in the cyp51 gene. However, except for cyp51-mediated azole resistance, a growing number of non-cyp51-mediated mechanisms of resistance have been identified. 1. Cyp51-mediated azole resistance Cytochrome P450 14α-sterol demethylase is a key enzyme in the ergosterol biosynthesis pathway. Azoles act by inhibiting the activity of cytochrome P450 14α-sterol demethylase,

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thereby blocking demethylation of C-14 of lanosterol, resulting in blockage of conversion of lanosterol to ergosterol, which is essential cell membrane component of filamentous fungi [7]. The cyp51gene produces two isoforms of the enzyme, CYP51A and CYP51B. The 14αdemethylase activity regulated mainly by the CYP51A. Cyp51B is a redundant gene and its role is either being functionally redundant or having an alternative function under particular

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conditions [8, 9]. Cyp51B-mediated azole resistance is rare; point mutations of cyp51A are the predominant mechanisms of resistance to azole drugs in A. fumigatus [10, 11]. To date, resistance development has primarily been associated with nonsynonymous mutations in

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CYP51A including amino acid substitution of hot spots G54, G138, M220, and G448. Except for them, more than 20 different amino acid substitutions (single or multiple amino acid

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change in the same strain) in the CYP51A protein have been found in clinic-isolated and laboratory-isolated azole-resistant strains（Figure 1） [12-15]. Genetic reconstitution experiments have verified that the mutations G54A, G54W, P216L, M220V/K/T and G448S

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are related to azole resistance [16-18]. These mutations may directly block the entry or modify the binding site of the drug, thereby reducing affinity of drug-enzyme interaction. Another resistance-involving genetic change is the combination of a mutation in the

cyp51A gene with a tandem repeat (TR) in the promoter region, causing significant overexpression of the cyp51A referring with azole resistance. Most environmentally induced azole-resistant isolates involve in TR-mediated mutations. Emergence of this resistance mechanism are closely related to the widely used azole fungicides in agriculture [5, 19]. Up to now, six tandem repeat mutations have been found. TR34/L98H and TR46/Y121F/T289A and TR53 have been found both from the environment and different patients, suggesting their rapid migration [19-22]. Recently, TR463 (three copies of 46bp TR; TR463/Y121F/M172I/T289A/G448S), TR464 (four copies of 46bp TR; TR464/Y121F/M172I/T289A/G448S), and TR120 (120bp TR) in the promoter region of cyp51A were identified from patient samples [23, 24]. These new findings challenge current understanding of azole resistance development in A. fumigatus. They suggest the occurrence of novel molecular resistance mechanism. Extensive investigations to determine azoleresistant A. fumigatus prevalence in both clinical and environmental samples is needed. 2

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Azole resistance development occurs through two distinct pathways: 1) in vivo, selection of resistance as a consequence of long-term treatment of Aspergillus diseases with medical azoles; and 2) in vitro, de novo acquisition of a resistant strain directly from the environment as the widespread use of azole fungicides in soil or crop protection. The underlying mechanisms are primarily linked to structural changes or upregulation of the azole target lanosterol 14 α-demethylase encoded by cyp51A. In clinical cases, among serially isolated several strains from one patient receiving azole treatment, the firstly isolated strains exhibited azole susceptibility without any cyp51A mutations, however, subsequent isolates showed azole resistance associated with cyp51A mutations [17, 25-27]. In field condition, clinical azole-resistant strain in A. fumigatus and its associated mutations in cyp51A could be induced by triazole fungicides, indicating that current fungicide application is not sustainable because

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the resistance mutations continue to emerge, thereby threatening the use of triazoles in medicine [19]. These reports from clinical setting and laboratory conditions provide evidence that mechanism of resistance selection in the long-term azole-exposed environment may easily lead to the accumulation of resistance mutations.

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2. Non-cyp51-mediated azole resistance

A number of A. fumigatus were resistant to azoles but had no mutation in cyp51A or its promoter. In 2011, Bueid et al., reported that forty-three percent of isolates did not carry a

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cyp51A mutation, indicating that there are other mechanisms of azole resistance other than cyp51-mediated azole resistance [28]. Non-cyp51-mediated azole resistance can be divided mediated azole resistance.

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into three general groups: efflux pumps-mediated, non-cyp51A mutations, stress adaptation-

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2.1 Efflux pumps-mediated azole resistance

Efflux pumps have important role in the overcoming intracellular toxin accumulation to help fungi to successfully infect or colonize human hosts. Overexpression of efflux pumps can pump the drugs out of the cell, resulting in reduction of drugs in the cell, thereby the emergence of azole resistance. Fraczek et al found that, compared with susceptible strains, azole-resistant strains showed >5-30-fold increased expression of azole transporter genes [29]. Many important transporters belong to ATP-binding cassette (ABC) transporters or members of major facilitator superfamily (MFS). ABC transporters depend on ATP hydrolysis for energy, and proteins of MFS cross the cell membrane by proton gradients [30]. Report from bench work identified that ABC transporters play the significant roles in azole resistance [31]. Reported transporters in A. fumigatus were AfuMDR1, AfuMDR2, AfuMDR3, AfuMDR4, AbcA-E, MfsA-C, AtrF [29, 32-34]. Recently, more new hypothesized drug pump transporters have been discovered gradually, but they still need to be verified by gene knockout [35]. In fungi, these transporter-deficient strains show multidrug sensitivity, whereas their overexpression showing increased multidrug resistance [36, 37]. Increased expression of transporter is associated with phenotypic resistance in A. fumigatus. In fact, a number of azole-resistant isolates showed azole-induced overexpression of efflux 3

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pumps solely under drug exposure in bench work [33]. It is not yet clear about the relationship between efflux transporter expression and clinical azole resistance. 2.2 Non-cyp51A mutations Non-cyp51A mutations are less characterized. In 2012, Camps et al., identified a mutation P88L in CCAAT-binding transcription factor HapE conferring azole resistance in isolates from one patient [38]. In 2014, Hagiwara D et al., reported the occurrence of genomic deletions and nonsynonymous in genes afyap1 and aldA in A. fumigatus from patients, however, genetic evidence for a link between mutations and drug resistance is lacking [2]. In 2017, Lu et al., found from bench work that a putative calcium-dependent protein (encoding gene algA) causes an increased frequency of non-cyp51A mutations conferring azole

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resistance in A. fumigatus and revealed a novel mutation R243Q in AfCox10 (a putative farnesyltransferase) referring azole resistance [39]. However, some of above identified mutations screened from in vitro experiment and is not actively screened for in azole-resistant isolates from patients or environment, thus their clinical relevance is unknown.

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2.3 Stress adaptation-mediated azole resistance

In order to successfully survive and reproduction in human hosts, fungi depend on the complex signaling pathway to overcome or adapt stress from environment including drug

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stimuli. The event which these signaling pathways include calcium signaling pathway, cell membrane homeostasis, iron balance, HOG-MAPK signaling pathway, cell wall integrity and

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Hsp90-calcineurin pathway are able to response to azole stress, has been established and could provide some interesting lead.

There are reports documenting that calcium signaling mediates antifungal activity of

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azole drugs in Aspergilli. Calcium signaling pathway have a number of components: plasma membrane component CchA/MidA, Golgi component PmrA, endoplasmic reticulum protein ClxA, Mitochondrial protein McuA, Vesicle protein VcxA/PmcA/ YvcA. Deletion strains of key genes referring calcium signaling pathway show hypersensitive to azoles. Combination with calcium inhibitor, azole efficacy is greatly increased in vitro and in vivo [40-42]. Recently, it is reported that azoles interfere with ergosterol synthesis pathway and further

affect cell membrane homeostasis. Hokken et al., revealed that erg6 showed a significant upregulation during the whole itraconazole-exposure time, as opposed to the rest members of the ergosterol biosynthetic pathway, which showed a general down-regulation or noregulation [35]. Null mutations of genes referring maintenance of sterol biosynthesis are able to an increased azole susceptibility in A. fumigatus. The SrbA null mutation (a sterol-regulator element binding protein) was highly susceptible to voriconazole and fluconazole in vitro because SrbA possibly regulates Cyp51A activity by binding it [43, 44]. Inactivation of both Erg4A and Erg4B (encoded two sterol C-24 reductases during the final step of ergosterol biosynthesis) results in hypersensitivity to the itraconazole and voriconazole, due to that double deletions of erg4A and erg4B completely block the last step of ergosterol synthesis 4

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[45]. DapA, DapB, and DapC (members of a cytochrome b5-like heme-binding damage resistance protein family) coordinately regulates the functionality of cytochrome P450 enzymes Erg5 and Erg11 and oppositely affects susceptibility to azoles. Overexpression of DapB and DapC causes dysfunction of Erg5 and Erg11 and further accentuating the sensitivity of ΔdapA strains to azoles. Inactivated DapA combined with activated DapB yields an A. fumigatus mutant that is easily treatable with azoles in an invasive pulmonary aspergillosis mouse model [46]. Further, Song et al., found that the damage resistance protein (Dap) contributes to azole resistance in a sterol-regulatory-element-binding protein SrbAdependent way [47]. Many lines of evidence have shown that iron balance, HOG-MAPK signaling pathway, cell wall integrity and Hsp90-calcineurin pathway are involved in azole stress response. Long

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et al., found that knockout of mitochondrial iron transporter mrsA was hypersensitive to itraconazole [48]. Hokken et al., reports that HOG-MAPK pathway members SakA and MpkC were significantly up-regulated after 60 minutes of drug exposure [35]. Loss of the wall anchoring protein PerA leads to defective wall integrity and increased susceptibility to voriconazole [49]. Heat shock protein 90 (Hsp90) is an important chaperone protein involved

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in fungal stress response. The combination of geldanamycin (Hsp90 inhibitor) with caspofungin or FK506 (calcineurin inhibitor) had the greatest antifungal activity against the

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azole-resistant strains, achieving a fungicidal activity, comparing to that they used alone [50]. It suggest that Hsp90-calcineurin axis is involved in azole stress response.

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Above studies have shown that aspergillus responds to or resists drug environments through multiple signaling pathways or cross-talk between signaling pathways. These pathways have specific potential roles for antifungal strategy and need more attention in

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clinical practice. The important molecules in these signaling pathways are potential targets of new drugs and provide important strategies for drug combinations. Conclusion

In this review, based on published work, the molecular mechanisms of azole resistance have been summarized into the two general categories: cyp51-mediated azole resistance and noncyp51-mediated azole resistance (Figure 1). Important aims are to gain more understanding to mechanism of azole resistance at the molecular level and develop effective strategies to combat resistance and control this public health problem. Finally, alternative therapeutic options are required for effective management of azole-resistant infection by Aspergillus in future. Disclosure of interest The authors declare that they have no competing interest. Acknowledgments

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This work was supported by grants from the National Natural Science Foundation of China (NSFC31500122). References

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Figure legends

Figure 1 Schematic representation of the molecular mechanism of azole resistance in

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Aspergillus fumigatus including mutations or overexpression of the target enzyme, increased

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drug efflux, non-cyp51A mutations, and stress adaptation-mediated azole resistance.

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