Synergy, pharmacodynamics, and time-sequenced ultrastructural changes of the interaction between nikkomycin Z and the echinocandin FK463 against Aspergillus fumigatus

Abstract

We investigated the potential synergy between two cell wall-active agents, the echinocandin FK463 (FK) and the chitin synthase inhibitor nikkomycin Z (NZ), against 16 isolates of filamentous fungi. Susceptibility testing was performed with a broth macrodilution procedure by NCCLS methods. The median minimal effective concentration (MEC) of FK against all Aspergillus species was 0.25 microg/ml (range, 0.05 to 0.5 microg/ml). For Fusarium solani and Rhizopus oryzae, MECs of FK were >512 microg/ml. The median MEC of NZ against Aspergillus fumigatus was 32 microg/ml (range, 8 to 64 microg/ml), and that against R. oryzae was 0.5 microg/ml (range, 0.06 to 2 microg/ml); however, for the other Aspergillus species, as well as F. solani, MECs were >512 microg/ml. A checkerboard inhibitory assay demonstrated synergy against A. fumigatus (median fractional inhibitory concentration index = 0.312 [range, 0.15 to 0.475]). The effect was additive to indifferent against R. oryzae and indifferent against other Aspergillus spp. and F. solani. We further investigated the pharmacodynamics of hyphal damage by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and examined the time-sequenced changes in hyphal ultrastructure. Significant synergistic hyphal damage was demonstrated with the combination of NZ (2 to 32 microg/ml) and FK (0.03 to 0.5 microg/ml) over a wide range of concentrations (P < 0.001). The synergistic effect was most pronounced after 12 h of incubation and was sustained through 24 h. Time-sequenced light and electron microscopic studies demonstrated that structural alterations of hyphae were profound, with marked transformation of hyphae to blastospore-like structures, in the presence of FK plus NZ, while fungi treated with a single drug showed partial recovery at 24 h. The methods used in this study may be applicable to elucidating the activity and interaction of other cell wall-active agents. In summary, these two cell wall-targeted antifungal agents, FK and NZ, showed marked time-dependent in vitro synergistic activity against A. fumigatus.

FIG. 1

Percent hyphal damage measured by checkerboard MTT assays after 4 and 24 h of incubation of A. fumigatus (isolates 4215, 972025, and 972350) with FK (0.03, 0.25, and 0.5 μg/ml) and NZ (2 to 32 μg/ml) alone and in combination.

FIG. 2

Percent hyphal damage of A. fumigatus (isolates 4215, 972025, and 972350) in MTT checkerboard assay in the presence of NZ alone (32 μg/ml) (□), FK alone (0.5 μg/ml) (×), or the combination of NZ and FK (○) over time. ⊡, control. Error bars indicate standard error of the mean.

FIG. 3

Time-sequenced light microscopic changes in hyphal structure of A. fumigatus (isolate 4215) with FK and NZ alone and in combination after 4 h of incubation. (A) Drug-free control; (B) NZ alone (32 μg/ml); (C) FK alone (0.5 μg/ml); (D) FK (0.5 μg/ml) plus NZ (32 μg/ml).

FIG. 4

Time-sequenced light microscopic changes in hyphal structure of A. fumigatus (isolate 4215) with FK and NZ alone and in combination after 24 h of incubation. (A) Drug-free control; (B) NZ alone (32 μg/ml); (C) FK alone (0.5 μg/ml); (D) FK (0.5 μg/ml) plus NZ (32 μg/ml).

FIG. 5

Time-sequenced electron microscopic changes of A. fumigatus (isolate 4215) after 4 h of incubation with NZ (32 μg/ml) and FK (0.5 μg/ml) alone and in combination. L, lower magnifications; H, higher magnifications. (A) Normal growth control of A. fumigatus hyphae.Magnifications, ×1,100 and ×3,100. (B) NZ alone. Scattered reticular aggregates appear on the outer surface of the fungal cell wall (arrows). Magnifications, ×620 and ×2,900. (C) FK alone. These photomicrographs demonstrate an increase in electron-dense aggregates on the outer fibrillar layer of the cell wall (arrows). Magnifications, ×620 and ×2,900. (D) NZ plus FK. The cell wall has increased reticular aggregations on the outer fibrillar layer (arrows). Magnifications, ×760 and ×2,900.

FIG. 6

Time-sequenced electron microscopic changes of A. fumigatus (isolate 4215) after 8 h of incubation with NZ (32 μg/ml) and FK (0.5 μg/ml) alone and in combination. L, lower magnifications; H, higher magnifications. (A) Normal growth control of A. fumigatus hyphae. Magnifications, ×1,100 and ×3,100. (B) NZ alone. The cell membrane has undergone significant changes, with loss of the electron-dense lipid bilayer structure. Small microvesicles may be seen in the area of the cell membrane. The inner fibrillar layer of the cell wall has lost its characteristic distinct, fine granularity. This layer is also much wider than that of controls (thin arrows). The outer fibrillar layer of the cell wall is aggregated and thicker than in the controls (thick arrows). In other areas, the outer fibrillar layer appears to be detached from the cell itself. Magnifications, ×900 and ×3,100. (C) FK alone. The outer fibrillar layer is focally thicker than that of the controls, and there are aggregates similar to those seen in the NZ-treated cells. The granularity of the inner fibrillar layer is relatively preserved, but it is not as fine as in the controls (arrows). Magnifications, ×620 and ×4,800. (D) NZ plus FK. The outer fibrillar layer has a lattice-like structure that is loose and thready (thick arrows). The inner fibrillar layer is not uniformly visible at these magnifications (thin arrow). Magnifications, ×620 and ×4,800.

FIG. 7

Time-sequenced electron microscopic changes of A. fumigatus (isolate 4215) after 12 h of incubation with NZ (32 μg/ml) and FK (0.5 μg/ml) alone and in combination. L, lower magnifications; H, higher magnifications. (A) Normal growth control of A. fumigatus hyphae. Magnifications, ×1,100 and ×3,100. (B) NZ alone. The lower magnification (×620) shows a substantial disruption and loss of cellular organelles. The higher magnification (×4,800) shows that the cell membrane has lost its characteristic electron-dense structure. The architecture of the inner fibrillar layer has been obliterated. In its place there is an irregular distended structure that appears to be pulling away from the cell interior (thin arrows). There also appear to be some microvesicles (along the cell membrane) (arrowheads). The outer fibrillar layer is irregularly thickened and aggregated (thick arrows). (C) FK alone. The lower magnification (×620) demonstrates an increased number of cytoplasmic vacuoles. The higher magnification (×5,200) shows that the double-layer structure of the cytoplasmic membrane has been obliterated (thick arrow) and the granular architecture of the inner fibrillar layer is absent (thin arrow). The width of cell wall is increased, and the outer fibrillar layer is now thickened. (D) NZ plus FK. The internal structure is completely obliterated, with no sign of any distinct organelles. The cytoplasm is filled with vacuoles of various sizes. The cell membrane has become irregular, with loss of the bilayer structure and microvesicles budding from its outer surface. The severely disrupted and widened inner fibrillar layer lacks inner architecture and contains microvesicles (thin arrows). The outer fibrillar layer of the cell wall is uniformly thickened along the circumference of the cells with focal reticular aggregates (thick arrows). Magnifications, ×620 and ×4,800.

FIG. 8

Time-sequenced electron microscopic changes of A. fumigatus (isolate 4215) after 24 h of incubation with NZ (32 μg/ml) and FK (0.5 μg/ml) alone and in combination. L, lower magnifications; H, higher magnifications. (A) Normal growth control of A. fumigatus hyphae. Magnifications ×1,100 and ×3,100. (B) NZ alone. The fine granularity of the inner fibrillar layer appears to have been partially reestablished in comparison to the 12-h time point (arrows). The outer fibrillar layer is again irregularly thickened and aggregated, while microvesicles are visible at various points along the cell surface. Magnifications, ×620 and ×3,100. (C) FK alone. There are numerous small cytoplasmic vacuoles and small electron-dense vacuoles. There is some restoration of the normal inner fibrillar layer, as evidenced by new fine granular electron-dense particles between the inner and outer layers (arrows). Magnifications, ×620 and ×3,100. (D) NZ plus FK. The lower magnification (×620) shows that the internal cytoplasmic architecture is obliterated. The higher magnification (×4,800) shows that the cell wall architecture is markedly disrupted, with loss of the inner layer and marked attenuation of the outer fibrillar layer.