Study of the action and cellular mechanisms of resistance of antifungal drugs

Postgraduate Thesis uoadl:1712577 394 Read counter

Κατεύθυνση Μικροβιακή Βιοτεχνολογία
Library of the School of Science
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Kapetanakis George
Supervisors info:
Γεώργιος Διαλλινάς, Καθηγητής Μοριακής Μικροβιολογίας, Τμήμα Βιολογίας, ΕΚΠΑ
Original Title:
Μελέτη της δράσης και των κυτταρικών μηχανισμών ανθεκτικότητας αντιμυκητιακών φαρμάκων
Translated title:
Study of the action and cellular mechanisms of resistance of antifungal drugs
Transporters are the transmembrane proteins that transfer substances to and from the cell. In particular, drug transporters provide protection to the host organisms because they are the means which transport the antimicrobial compounds from the host microenvironment into the invader’s microorganisms cells. Sometimes, transporters are targets for the antimicrobial compounds and in other cases; they support the entry of the compounds into the target cells. While it is fundamental to design the drugs in order to serve as a connecting bond between the substance and the transporter, most of the times the selection of the desired antimicrobial compound is made according to its efficiency against a microbial disease with the minimum possible side effects to the host.

The current study offers an original analysis of the potential transporters which are involved in the accumulation of a group of antifungal compounds called Succinic Dehydrogenase Inhibitors (SDHIs) in Aspergillus nidulans cells. Generally, the substances belonging to this group are effective against several species of fungi that infect cultivated plants. After in depth investigation in several groups of substances, boscalid was chosen for extensive research. The paper analyzes its action and also focuses on the resistance mechanism of Aspergillus nidulans against boscalid. It is noted that the most common mechanisms of resistance against antifungal compounds are either overexpression or modification of the target while in other times it can be activation of efflux transporters in several strains like ABC or MFS transporters.

The toxicity of boscalid was tested in different species of the Aspergillus genus. The efficacy of the drug differed from species to species. At the same time, the phylogenetic analysis of the tested species according to the sequence of sdhB, the target protein, did not indicate any correlation between the protein sequence and the toxicity of boscalid against Aspergillus genus. It is noteworthy that the physiological conditions under which growth assays evolve, limit the efficacy of the drug against the target cell. It is important to underline the essential role of temperature in membrane fluidity and consequently to the diffusion of substances. Furthermore, pH has a basic role to the function of proton symporters connected to the availability of protons in the growth media. A test that conducted under different physiological conditions proved that at high pH, transporters mediate the accumulation of boscalid, while in lower pH is achieved by both transporters and diffusion.

In addition, it is already known that the available nitrogen source regulates the expression of the transporters. Specifically, in presence of ammonium ions, as the sole nitrogen source, suppress the expression of transporters that accumulate alternative nitrogen sources, such as purine and pyrimidine transporters, which have low expression levels. So, when ammonium ions were the sole nitrogen source, it was observed that boscalid was ineffective against the wild-type strain of Aspergillus nidulans. This experiment showed that nucleotide transporters are likely to be involved in the accumulation of boscalid.

Boscalid’s action mechanism was thoroughly investigated by using strains from the strain bank of Fungal Molecular Biology Laboratory; also, during this study, new strains were constructed with the desirable properties. In detail, growth assays were performed in strains with mutations in transcription factors, in presence of antifungal drugs. The areA30 strain, with point mutation (Leu683Met) in the transcription factor’s region, showed increased resistance against boscalid comparing to wild-type strain. At the same time, strain (Δ7) with 7 deleted transporters (UapA, UapC, AzgA, FcyB, FurD, CntA and FurA) showed similar resistance. In order to investigate which of these 7 transporters are involved in the accumulation of boscalid in fungal cells, plasmid transformations took place; Δ7 strain was used in order to have the same isogenetic background and the expression of the transporter was, each time, under the control of a strong promoter. The inversion of the persistent phenotype reveals the involvement of each of them in the accumulation of the pesticide. The results indicated that boscalid is accumulated by UapC, AzgA, FcyB, CntA and FurA. Conversely, UapA and FurD transporters are not involved in boscalid’s accumulation process. The current research has proved that the three basic transporters FurA, AzgA, UapC, tagged with GFP, are endocytosed by their substances. More importantly in this study, endocytosis took place because of boscalid’s presence.

In conclusion, the current research examined specific transporters in the accumulation of boscalid. Moreover, it focused on finding new genes that may be related to the resistance of the fungi to boscalid using the Minos transposon. At first, the current study optimized the insertion detection method of the Minos transposon. Then, a strain was isolated with an insertion of Minos transposon into the AN6927 gene promoter. There is a notion that gene AN6927 encodes a protein with role either as a sensor to the osmotic stress or/and the signaling transduction for gene activation associated with boscalid uptake; a process that needs further investigation.
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Other subject categories:
Fungi, nucleobase, pyrimidine, structure-function
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