Cell Wall Changes in Amphotericin B-Resistant Strains from Candida tropicalis and Relationship with the Immune Responses Elicited by the Host

Abstract

We have morphologically characterizedCandida tropicalisisolates resistant to amphotericin B (AmB). These isolates present an enlarged cell wall compared to isolates of regular susceptibility. This correlated with higher levels of β-1,3-glucan in the cell wall but not with detectable changes in chitin content. In line with this, AmB-resistant strains showed reduced susceptibility to Congo red. Moreover, mitogen-activated protein kinases (MAPKs) involved in cell integrity were already activated during regular growth in these strains. Finally, we investigated the response elicited by human blood cells and found that AmB-resistant strains induced a stronger proinflammatory response than susceptible strains. In agreement, AmB-resistant strains also induced stronger melanization ofGalleria mellonellalarvae, indicating that the effect of alterations of the cell wall on the immune response is conserved in different types of hosts. Our results suggest that resistance to AmB is associated with pleiotropic mechanisms that might have important consequences, not only for the efficacy of the treatment but also for the immune response elicited by the host.

FIG 1

Electron microscopy of ultrathin sections from C. tropicalis strains with different antifungal susceptibility profiles. Shown are representative fields of yeast cell wall from strains ATCC 750 (A), CL-7099 (B), CL-7119 (C), TP13650 (D), CL-6835 (E), and ATCC 200956 (F). In each panel, a picture of the whole cell (left part) and a magnification of the cell wall (right part) are shown. The scale bars correspond to 0.2 μm.

FIG 2

Measurement of the cell wall thickness of AmB-resistant C. tropicalis strains. The width (A) and percentage of the volume of the cell wall relative to the total volume of the cell (B) were calculated from the TEM images. The mean value and the standard deviation are plotted as bar graphs. Asterisks indicate statistically significant difference (P < 0.05) between each strain and the ATCC 750 strain. Gray bars correspond to the strain resistant to azoles and black bars to the strains resistant to AmB.

FIG 3

Detection of β-1,3-glucans at the cell wall. Immunofluorescent detection of β-1,3-glucans in the cell wall of C. tropicalis strains ATCC 750 (A), CL-7099 (B), TP-13650 (C), CL-6835 (D), and ATCC 200956 (E) after heat treatment. The right panel corresponds to the same samples in a bright field. (F) Quantification of the fluorescent signal obtained in the cells from panel A. Images were processed with ImageJ, and the quantification of the fluorescent signal was obtained as arbitrary units in each case. Asterisks indicate statistically significant difference (P < 0.05) between each strain and the ATCC 750 strain.

FIG 4

Growth of C. tropicalis strain in Congo red. Candida tropicalis strains with different susceptibility profiles were incubated overnight in Sabouraud liquid medium, and after being washed with PBS, different amounts of cells were placed on Sabouraud medium plates or on the same plates containing Congo red (100 μg/ml). The plates were incubated at 37°C, and pictures were taken after 48 h.

FIG 5

Cytokines produced by PBMCs after exposure to C. tropicalis. PBMCs from healthy human donors were exposed to different C. tropicalis strains, and the concentrations of IL-1β (A), TNF-α (B), IL-10 (C), IFN-γ (D), IL-22 (E), and IL-17 (F) in the supernatants were determined by ELISA after 24 h (IL-1β, TNF-α, and IL-10) or 7 days (IFN-γ, IL-22, and IL-17). Significant statistical differences (P < 0.05) between AmB-resistant and AmB-susceptible strains are shown by asterisks.

FIG 6

Quantification of melanin in the hemolymph of G. mellonella larvae infected with C. tropicalis. Galleria mellonella larvae were infected with 10⁶, 2 × 10⁶, or 4 × 10⁶ C. tropicalis cells from strain ATCC 750 or ATCC 200956. The larvae were incubated at 37°C, and after 1 h, the hemolymph was isolated and melanin was estimated as described in Materials and Methods. The results correspond to the average ± standard deviation of the OD values obtained from each larva in each group. The asterisks indicate significant differences (P < 0.05).

FIG 7

Activation of MAPKs in C. tropicalis strains by Western blotting. The activation of the Mkc1, Hog1, and Cek1 MAPKs was detected by Western blotting in C. tropicalis strains with different susceptibility profiles. (A) Cells were grown in YEPD (left panel) and then exposed to tunicamycin (TM; 5 μg/ml [right panel]). Western blotting was performed to detect the activated forms of Mkc1, Cek1, and Hog1 with specific MAbs. The detection of nonphosphorylated form of Hog1 was used as a loading control. (B) The cells were grown as described above, but they were treated with AmB (1 μg/ml) for 60 or 120 min. Detection of activated forms of the MAPs was performed as in panel A.