Soft X-ray tomography of phenotypic switching and the cellular response to antifungal peptoids in Candida albicans

Abstract

The opportunistic pathogen Candida albicans can undergo phenotypic switching between a benign, unicellular phenotype and an invasive, multicellular form that causes candidiasis. Increasingly, strains of Candida are becoming resistant to antifungal drugs, making the treatment of candidiasis difficult, especially in immunocompromised or critically ill patients. Consequently, there is a pressing need to develop new drugs that circumvent fungal drug-resistance mechanisms. In this work we used soft X-ray tomography to image the subcellular changes that occur as a consequence of both phenotypic switching and of treating C. albicans with antifungal peptoids, a class of candidate therapeutics unaffected by drug resistance mechanisms. Peptoid treatment suppressed formation of the pathogenic hyphal phenotype and resulted in striking changes in cell and organelle morphology, most dramatically in the nucleus and nucleolus, and in the number, size, and location of lipidic bodies. In particular, peptoid treatment was seen to cause the inclusion of lipidic bodies into the nucleus.

Fig. 1.

Schematic representation of the three phenotypes adopted by C. albicans. Blue, nucleus; dark gray, septum.

Fig. 2.

Soft X-ray tomographic reconstruction of phenotypically distinct C. albicans cells. A representative orthoslice from each tomographic reconstruction is shown for a yeast-like (A), germ-tube (C) and hyphal (E) cell. Volume rendered views of the same cells are shown in (B, D, and F) respectively showing selected organelles that have been segmented and color-coded for identification. Blue, nucleus; orange, nucleolus; gray, mitochondria; yellow, vacuole; green, lipid bodies. (Scale bar for A–D, 0.5 μm, for E and F, 2.0 μm.)

Fig. 3.

Soft X-ray tomography of C. albicans cells after treatment with peptides. Peptoid 1 treated cell (A–D) Representative orthoslice through the tomographic reconstruction (A). All segmented organelles (B). Segmented nucleus and nucleolus (C). Peptoid 1 induced structural changes in the nucleolus are shown in detail in the Inset. Segmented vacuoles (D). Peptoid 2 treated cell (E–G). Representative orthoslice through the tomographic reconstruction (E). All segmented organelles (F). Segmented nucleus, nucleolus and lipid bodies (G). Peptoid 2 induced structural changes in the nucleolus and the incorporation of a large lipid body into the nucleus are shown in detail in the Inset. Blue, nucleus; orange, nucleolus; gray, mitochondria; yellow, vacuole; green, lipid bodies. (Scale bar, 1.0 μm).

Fig. 4.

Effect of peptoid treatment on selected organelle volumes. (A) Average percentage volume of the nucleus relative to the cell volume in control and peptoid treated cells. (B) Volume of lipid droplets in control and peptoid treated cells. The percentage relative value was calculated from the volume of each organelle divided by its cell volume. The error bars represent SD.

Fig. 5.

Dimensions of nuclei, nucleoli, and a lipid-like structure shown in Fig. 3. Blue, nucleus; orange, nucleolus; green, lipid body.