Holden WM et al. (2014), Nikkomycin Z is an effective inhibitor of the c...

XB-ART-60803

Fungal Biol 2014 Jan 01;1181:48-60. doi: 10.1016/j.funbio.2013.11.001.

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Nikkomycin Z is an effective inhibitor of the chytrid fungus linked to global amphibian declines.

Holden WM , Fites JS , Reinert LK , Rollins-Smith LA .

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Fungal infections in humans, wildlife, and plants are a growing concern because of their devastating effects on human and ecosystem health. In recent years, populations of many amphibian species have declined, and some have become extinct due to chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis. For some endangered amphibian species, captive colonies are the best intermediate solution towards eventual reintroduction, and effective antifungal treatments are needed to cure chytridiomycosis and limit the spread of this pathogen in such survival assurance colonies. Currently, the best accepted treatment for infected amphibians is itraconazole, but its toxic side effects reduce its usefulness for many species. Safer antifungal treatments are needed for disease control. Here, we show that nikkomycin Z, a chitin synthase inhibitor, dramatically alters the cell wall stability of B. dendrobatidis cells and completely inhibits growth of B. dendrobatidis at 250 μM. Low doses of nikkomycin Z enhanced the effectiveness of natural antimicrobial skin peptide mixtures tested in vitro. These studies suggest that nikkomycin Z would be an effective treatment to significantly reduce the fungal burden in frogs infected by B. dendrobatidis.

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Species referenced: Xenopus laevis

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	Fig 1. Coculture with nikkomycin Z impaired B. dendrobatidis (Bd) growth in vitro. (A) B. dendrobatidis was cultured in equal parts tryptone broth and HPLC water (‘Bd only’) or in equal parts tryptone broth with various concentrations of nikkomycin Z in HPLC water ranging from 0.02 to 200 μM (‘Bd + Nikkomycin Z’). Concentrations ≥ 0.3 μM nikkomycin Z showed significantly lower growth than the positive control (*p < 0.01). (B) At 2000 μM nikkomycin Z, B. dendrobatidis growth is not significantly different (p > 0.2) from the heat-killed B. dendrobatidis negative control. Data shown are representative of at least three similar experiments.
	Fig 2. Suboptimal nikkomycin Z (NZ) concentrations altered B. dendrobatidis cellular diameter. Populations treated with or without nikkomycin Z were stained with calcofluor white to visualize the cell walls; these populations included cells that had matured to germlings, thalli, and zoosporangia. (A) When B. dendrobatidis was cultured without nikkomycin Z, zoospore maturation into zoosporangia was unimpeded. Typical mature zoosporangia are shown here with a maximum diameter of approximately 15 μm. (B–E) Increasing concentrations of nikkomycin Z resulted in increasing cell diameters in the zoosporangia at all concentrations tested. (F) The largest cell diameters were observed with zoosporangia that developed in culture with 100 μM nikkomycin Z. Letters within the panel indicate groups that differ significantly by one-way ANOVA. (G) Increasing nikkomycin Z concentrations resulted in larger ranges of observed diameters and more heterogeneous cell populations. Scale bars (A–E) represent 20 μm.
	Fig 2. Suboptimal nikkomycin Z (NZ) concentrations altered B. dendrobatidis cellular diameter. Populations treated with or without nikkomycin Z were stained with calcofluor white to visualize the cell walls; these populations included cells that had matured to germlings, thalli, and zoosporangia. (A) When B. dendrobatidis was cultured without nikkomycin Z, zoospore maturation into zoosporangia was unimpeded. Typical mature zoosporangia are shown here with a maximum diameter of approximately 15 μm. (B–E) Increasing concentrations of nikkomycin Z resulted in increasing cell diameters in the zoosporangia at all concentrations tested. (F) The largest cell diameters were observed with zoosporangia that developed in culture with 100 μM nikkomycin Z. Letters within the panel indicate groups that differ significantly by one-way ANOVA. (G) Increasing nikkomycin Z concentrations resulted in larger ranges of observed diameters and more heterogeneous cell populations. Scale bars (A–E) represent 20 μm.
	Fig 3. Characterization of nikkomycin Z effects on B. dendrobatidis (Bd) growth. (A) Following plating on agar, cells that had been exposed to ≥ 250 μM were not viable. Thus, 250 μM is the MIC. (B) When cells adhering to microtiter plate wells following culture with nikkomycin Z were cultured an additional 7 d without nikkomycin Z, no significant growth (p > 0.2) was observed for cells previously exposed to ≥ 250 μM nikkomycin Z, confirming the MIC. (C) When cultured with 20 μM nikkomycin Z, cells exhibited significantly reduced cell replication (p < 0.001). (D) The percent of original zoospores cultured that matured past the zoospore stage was reduced after incubation for 3 d with 20 μM nikkomycin Z (p < 0.02). In panels A, C, and D, the mean of three experiments is graphed. The data shown in panel B is a single experiment representative of at least three similar assays.
	Fig 3. Characterization of nikkomycin Z effects on B. dendrobatidis (Bd) growth. (A) Following plating on agar, cells that had been exposed to ≥ 250 μM were not viable. Thus, 250 μM is the MIC. (B) When cells adhering to microtiter plate wells following culture with nikkomycin Z were cultured an additional 7 d without nikkomycin Z, no significant growth (p > 0.2) was observed for cells previously exposed to ≥ 250 μM nikkomycin Z, confirming the MIC. (C) When cultured with 20 μM nikkomycin Z, cells exhibited significantly reduced cell replication (p < 0.001). (D) The percent of original zoospores cultured that matured past the zoospore stage was reduced after incubation for 3 d with 20 μM nikkomycin Z (p < 0.02). In panels A, C, and D, the mean of three experiments is graphed. The data shown in panel B is a single experiment representative of at least three similar assays.
	Fig 4. Nikkomycin Z (NZ) exposure increases B. dendrobatidis (Bd) sensitivity to osmotic lysis. B. dendrobatidis alone (A) or with 50 μM NZ (B) are stable in isotonic conditions (APBS), but experienced cellular swelling followed by osmotic lysis in hypotonic conditions (dH2O). Swelling and subsequent lysis are indicated by an initial increase followed by a decrease in OD490 over a 210 min time course. In panels A and B, graphs are representative of three identical experiments for each condition. (C) The ratio of optical densities in dH2O compared to APBS indicates that osmotic lysis is significantly higher in NZ-treated cells (p < 0.05, p < 0.001 by unpaired, two-tailed Student's t tests). (D) Cell counts immediately prior to resuspension and 120 min after resuspension in APBS or dH2O indicate that cell numbers significantly decreased in the dH2O hypotonic environment (p < 0.05 by unpaired, two-tailed Student's t test), but were not significantly changed upon resuspension in isotonic APBS (p > 0.05). Panels C and D show the mean ± SEM of three identical experiments.
	Fig 4. Nikkomycin Z (NZ) exposure increases B. dendrobatidis (Bd) sensitivity to osmotic lysis. B. dendrobatidis alone (A) or with 50 μM NZ (B) are stable in isotonic conditions (APBS), but experienced cellular swelling followed by osmotic lysis in hypotonic conditions (dH2O). Swelling and subsequent lysis are indicated by an initial increase followed by a decrease in OD490 over a 210 min time course. In panels A and B, graphs are representative of three identical experiments for each condition. (C) The ratio of optical densities in dH2O compared to APBS indicates that osmotic lysis is significantly higher in NZ-treated cells (p < 0.05, p < 0.001 by unpaired, two-tailed Student's t tests). (D) Cell counts immediately prior to resuspension and 120 min after resuspension in APBS or dH2O indicate that cell numbers significantly decreased in the dH2O hypotonic environment (p < 0.05 by unpaired, two-tailed Student's t test), but were not significantly changed upon resuspension in isotonic APBS (p > 0.05). Panels C and D show the mean ± SEM of three identical experiments.
	Fig 5. Nikkomycin Z and amphibian AMPs inhibit B. dendrobatidis (Bd) growth in a cooperative manner. (A) B. dendrobatidis cells were cultured in equal parts tryptone broth and HPLC water either alone (‘Bd only’), with serial dilutions of R. sphenocephala AMPs in HPLC water (‘Bd + Peptides’), with 2 μM nikkomycin Z in HPLC water (‘Bd + NZ’), or with both serial dilutions of R. sphenocephala AMPs and 2 μM nikkomycin Z (‘Bd + Peptides + NZ’). A negative control of dead B. dendrobatidis cells (‘Heat-Killed Bd’) was also included in each assay. B. dendrobatidis growth in the presence of NZ and peptides was significantly reduced compared to the growth in the presence of either NZ or peptides alone (*p < 0.05). Similar assays replaced a natural mixture of R. sphenocephala peptides with (B) purified brevinin-1Sb or (C) purified brevinin-1Sc. Each panel is representative of at least three experiments.
	Fig 5. Nikkomycin Z and amphibian AMPs inhibit B. dendrobatidis (Bd) growth in a cooperative manner. (A) B. dendrobatidis cells were cultured in equal parts tryptone broth and HPLC water either alone (‘Bd only’), with serial dilutions of R. sphenocephala AMPs in HPLC water (‘Bd + Peptides’), with 2 μM nikkomycin Z in HPLC water (‘Bd + NZ’), or with both serial dilutions of R. sphenocephala AMPs and 2 μM nikkomycin Z (‘Bd + Peptides + NZ’). A negative control of dead B. dendrobatidis cells (‘Heat-Killed Bd’) was also included in each assay. B. dendrobatidis growth in the presence of NZ and peptides was significantly reduced compared to the growth in the presence of either NZ or peptides alone (*p < 0.05). Similar assays replaced a natural mixture of R. sphenocephala peptides with (B) purified brevinin-1Sb or (C) purified brevinin-1Sc. Each panel is representative of at least three experiments.
	Fig 5. Nikkomycin Z and amphibian AMPs inhibit B. dendrobatidis (Bd) growth in a cooperative manner. (A) B. dendrobatidis cells were cultured in equal parts tryptone broth and HPLC water either alone (‘Bd only’), with serial dilutions of R. sphenocephala AMPs in HPLC water (‘Bd + Peptides’), with 2 μM nikkomycin Z in HPLC water (‘Bd + NZ’), or with both serial dilutions of R. sphenocephala AMPs and 2 μM nikkomycin Z (‘Bd + Peptides + NZ’). A negative control of dead B. dendrobatidis cells (‘Heat-Killed Bd’) was also included in each assay. B. dendrobatidis growth in the presence of NZ and peptides was significantly reduced compared to the growth in the presence of either NZ or peptides alone (*p < 0.05). Similar assays replaced a natural mixture of R. sphenocephala peptides with (B) purified brevinin-1Sb or (C) purified brevinin-1Sc. Each panel is representative of at least three experiments.