Adaptation of Aspergillus Niger to Multiple Agents Whose Action Mechanisms Are Different
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Jpn. J. Med. Mycol. Vol. 36, 19-24, 1995 ISSN 0916-4804 Original Article Adaptation of Aspergillus niger to Multiple Agents Whose Action Mechanisms are Different Hideaki Matsuoka1, Jong-Chul Park1, Yasuyuki Nemoto1, Satoru Yamada2, Weimin Jing3, Yuansong Chen3, Kosuke Takatori4, Hiroshi Kurata5 1Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology, 2-24-16, Nakamachi, Koganei, Tokyo 184, Japan 2Research and Development Division, Bio-Liken Inc., 1-5-8, Iwamotocho, Chiyoda-ku, Tokyo 101, Japan 3Research and Development Division, Hidan Co. Ltd., 627, Hananoi, Kashiwa, Chiba 277, Japan 4Hatano Research Institute, Food & Drug Safety Center, 729-5, Ochiai, Hadano, Kanagawa 257, Japan 5The Tokyo Kembikyoin Foundation, 4-8-32, Kudanminami, Chiyoda-ku, Tokyo 102, Japan (Received: 9, August 1994. Accepted: 9, September 1994] Abstract Adaptation of Aspergillus niger to amphotericin B (AMPH) and two imidazoles (miconazole (MCZ) and ketoconazole (KCZ)) was observed at a single hypha level with a continuous measurement system. It was found that a test hypha adapted to MCZ or KCZ was also adapted to AMPH but that a hypha adapted to AMPH was not adapted either to MCZ or KCZ. These adaptation phenomena to respective agents did not occur after incubation in the medium supplemented with ergosterol. The cross adaptation phenomena were suspected to be due to modification of the synthesis pathway of ergosterol and related derivatives. Key words: adaptation, hyphal growth rate, Aspergillus niger, amphotericin B, miconazole, ketoconazole. rienced by many mycologists and microbiologists Introduction 5-9). The newly developed BCT system has dem- A single hypha-based microbioassay has recently onstrated those adaptation phenomena much more been proposed and applied to the evaluation of clearly by tracing the same hypha throughout. antifungal activity of antimycotics against filamen- In the present study, we examined the responses tous fungi1-37). This microbioassay is based on the of a test hypha to the repeated actions of different continuous measurement of the growth of a test agents for the purpose of clarifying whether the hypha under a microscope. The movement of the hypha is addpted to respective agents exclusively. hyphal apex can be precisely traced automatically Materials and Methods by a computer-aided system, which is called Bio- Cell Tracer (BCT). BCT enables the continuous Microorganism measurement of the responses to repeated actions Aspergillus niger IFO 6661 was maintained at 4 of agents to the same hypha. Such a continuous C on slants of potato dextrose agar (PDA, Difco measurement of the same hypha is impossible by Laboratories, Detroit, Mich., USA). The mould conventional assay methods dealing with massive was precultured on PDA slants at 28CCfor 7 days hyphae. prior to use. The authors previously examined the responses Antifungal agents and ergosterol of a test hypha of Aspergillus niger to the repeated Amphotericin B (AMPH) and miconazole (MCZ) actions of salt and observed adaptation phenomena were purchased from Sigma (St. Louis, Mo., USA), 4), which were similar to those formerly expe- and ketoconazole (KCZ) was purchased from 20 真 菌 誌 第36巻 第1号 平 成7年 Biomol Research Laboratories, Inc. (Plymouth to confirm that a test hypha met this criterion, Meeting, Pa., USA). Stock solutions of these the following criteria were introduced: agents were prepared at 10 mg/ml in dimethyl 1. The growth of a test hypha is inhibited sulphoxide (DMSO) and stored at -20C. These remarkably by the first action of an agent, stock solutions were used within 7 days. Test and the growth rate initially drops to zero. solutions of AMPH, MCZ and KCZ were prepared 2. The growth recovers during the first action by diluting the stock solutions with potato dextrose or after removal of the agent. broth (PDB, Difco Laboratories) before use. 3. The second action of the same agent at the Final concentrations of DMSO in the test solu- same concentration as the first action causes tions were less than 0.25% (v/v). Ergosterol no remarkable inhibition effect on the growth purchased from Sigma was dissolved in DMSO of the same hypha. and 2-propanol (2:1, v/v) to prepare 10 mg/ml When these criteria were satisfied, another stock solution immediately prior to use. This stock agent was applied in order to check whether the solution was diluted with PDB and applied to adaptation was exclusive. the assays. Results System for measuring hyphal growth Hyphal growth was measured with a BCT system Pre-exposure growth rate (Hidan Co., Ltd., Chiba, Japan). The system A test hypha was arbitrarily selected on the TV consists of a microscope, a charge coupled device monitor and its growth rate was measured for (CCD) camera, an image analysing system, and 30 min. If the rate was not stable and variation a microscope stage controller1,4). A reaction vessel exceeded 20%, another hypha was selected as a was set on the microscope stage. The inner bottom test hypha and the growth rate was again meas- surface of the reaction vessel was coated with a ured. Once stable, the average of the growth rate solution of 0.01% (w/v) filter-sterilized poly-L- during a 30 min measurement was defined as lysine (poly-L-lysine hydrobromide, MW 70,000- the pre-exposure growth rate. The average and 150,000, Sigma). In the image obtained through standard deviation of the pre-exposure growth rate a microscope, an appropriate hypha was selected were determined as 3.96 0.82 pm/min from the and its growth was traced automatically. Its data obtained with 81 test hyphae. growth rate was calculated from the distance the Optimum concentrations of agents for the hypha lengthened during the measuring interval adaptation experiment (30 sec). AMPH was applied to hyphae at various Assay procedure concentrations in the range of 0.01-1.0 ig/ml. The spores from a 7-day-old culture grown on When the concentration was 0.075 pg/ml, the a PDA slant at 2 8C were harvested in sterile growth rate once became zero and then returned saline containing 0.05% (v/v) Tween 80. After to the initial level within 30 min as shown in washing three times with sterile distilled water, one Fig. 1-A. After washing with fresh medium, the microlitre of the spore suspension at a concentra- same concentration of AMPH was applied again tion of 2X108 spores/ml was placed onto the poly- to the hypha. No response was obtained within L-lysine-coated glass plate of the reaction vessel 30 min. When the concentration was higher, the and left standing for 30 min. Then the suspension first action of AMPH caused the zero growth was removed, and one millilitre PDB was poured rate more rapidly than did 0.075 pg/mi. The into the reaction vessel. The reaction vessel was second action, however, still caused the same incubated for 24h at 28C to form a colony growth inhibition. On the other hand, at lower about 5 mm in diameter. The reaction vessel was concentrations, the growth rate did not become then mounted on the microscope stage, and the zero within 30 min during the first action. medium was replaced by one millilitre fresh PDB Therefore, the optimum concentration of AMPH and left to stand for 30 min at 28C. An appro- was determined to be 0.075 ig/ml. priate hypha was selected on the TV monitor In the same manner, the effects of MCZ and and its growth rate was measured for 30 min. KCZ were examined in the range 0.001-1.0 ug After confirming that the growth rate was stable, /ml and 0.01-5.0 ag/ml, respectively. The the medium was replaced by a PDB containing optimum concentrations of MCZ and KCZ were an antifungal agent. determined as 0.01 g/ml and 0.5 ig/ml, Confirmation of adaptation to an anti- respectively. These response curves are shown in fungal agent Fig. 1-B, C. A test hypha needs to be adapted to an agent before being exposed to another agent. In order Jpn. J. Med. Mycol. Vol. 36 (No. 1), 1995 21 A A B B C Fig. 2. Effects of (A) MCZ or (B) KCZ on the hyphal growth of A, niger previously adapted to AMPH. Refer to the legend for Fig. 1. in response to MCZ was identical with that of Fig. 1-B; therefore, the acquisition of adaptation to AMPH had no effect on the sensitivity to MCZ. Acquisition of adaptation to AMPH also had no effect on the sensitivity to KCZ, as shown in Fig. 2-B. In contrast, a hypha adapted either to 0.01 ig/ml MCZ or to 0.5 eg/ml KCZ beforehand was insensitive to the other two agents, as shown in Figs. 3 and 4. Fig, 1. Adaptation of A. niger hyphal growth to anti- fungal agents. (A) AMPH; (B) MCZ; (C) KCZ Effect of ergosterol on the elimination of At the arrows marked AMPH, MCZ, and KCZ, adaptation PDB was replaced by PDB containing 0.075 ug/ml After confirming the adaptation to 0.07ug/ AMPH, 0.01 ug/ml MCZ, and 0.5 ug/ml KCZ, ml AMPH, the PDB containing 10.0 ag/ml respectively. At the arrows marked "Washing", PDB ergosterol was applied to the test hypha. Then, containing an antifungal agent was replaced by anti- after 60 min incubation, 0.075 g/ml AMPH fungal-free PDB. Experiments were done in triplicate. was applied to the hypha. As shown in Fig. 5-A, The data are representative results obtained with respective experiments. the growth rate decreased sharply, indicating the adaptation had been eliminated. The treatment Responses of an adaptation-acquired hypha with PDB containing ergosterol was also effective to different agents in eliminating the adaptation to MCZ and KCZ After confirming that a test hypha was adapted as demonstrated in Fig.