Stimulated Raman scattering microscopy resolves amphotericin B orientation and accumulation of ergosteryl ester at single Candida albicans

Introduction: Ergosterol-targeting amphotericin B (AmB) is the first line of defense for life-threatening fungal infections. Two models have been proposed to illustrate AmB assembly in the cell membrane; one is the classical ion channel model in which AmB vertically forms transmembrane tunnel and the other is a recently proposed sterol sponge model where AmB is laterally adsorbed onto the membrane surface. To address this controversy, we use polarization-sensitive stimulated Raman scattering (SRS) from fingerprint C═C stretching vibration (1556 cm-1) to visualize AmB, ergosterol, and lipid in single fungal cells. Intracellular lipid droplet accumulation in response to AmB treatment is found. AmB is located in membrane and intracellular droplets. In the 16 strains studied, AmB residing inside cell membrane was highly ordered, and its orientation is primarily parallel to phospholipid acyl chains (2850 cm-1), supporting the ion channel model (Dong et al., Science Advances, 2021). Meanwhile, in the same spectral window (from 1500 cm-1 to 1700 cm-1), we found the aberrant ergosteryl ester accumulation in azole-resistant C. albicans compared to azole-susceptible species. Blocking ergosterol esterification by oleate and suppressing sterol synthesis by fluconazole synergistically suppressed the viability of C. albicans in vitro and limited the growth of biofilm on mouse skin in vivo (Zhang#, Dong# et al., Analytical Chemistry, 2023). Label-free imaging of AmB and chemical contents offers an analytical platform for developing low-toxicity, resistance-refractory antifungal treatments.

Materials and

Methods: A femtosecond solid-state laser provides two synchronized outputs as 80-MHz pulsed laser trains. The 1040-nm beam serves as the Stokes beam. The other beam is wavelength tunable (680 to 1300 nm) and serves as the pump beam (here, we chose 895 nm as the pump). An acousto-optic modulator is used to modulate the Stokes beam at 2.2 MHz with an efficiency of ~75%. The pump and Stokes beams were spatially aligned and sent to an upright microscope with two-dimensional galvo system for laser scanning. Half-wave plates at specific wavelength were used to control the polarization directions of these two beams. A spectral focusing approach was used to obtain spectral domain information. By delaying the pump pulses controlled by a motorized stage (each step is 10 um), a series of Raman shifts through polarization-sensitive SRS microscopy, we were able to visualize AmB and ergosteryl ester in their natural states. At a certain time delay, all the laser energy was spectrally focused to excite a narrow Raman band. The laser powers (pump ~10 mW and Stokes ~50 mW at the sample) were used to excite AmB (1556 cm-1) and ergosteryl esters (1603 cm-1) from the fungal cells. A 60× water immersion objective (NA=1.2) was used to focus pump and Stokes laser on the samples. An oil condenser (NA=1.4) was used to collect the laser light in the forward direction.

Results, Conclusions, and Discussions: Through polarization-sensitive SRS microscopy, we were able to visualize AmB in its natural state at a single-cell level. Meanwhile, by imaging AmB and CH2 from ghost RBCs under the same laser polarization, we found that AmB molecules are orientated parallel to the phospholipids inside the cell membrane, thus supporting the traditional ion channel model. Meanwhile, through hyperspectral stimulated Raman scattering imaging of single fungal cells in the fingerprint window and pixel-wise spectral unmixing, we discovered the aberrant ergosteryl ester accumulation in azole-resistant C. albicans as compared with non-resistant species at a single-cell level. Such accumulation is found to arise from de novo glucose lipogenesis. According to lipid profiling analysis by mass spectrometry, ergosterol oleate storage significantly increases in azole-resistant C. albicans. Consequently, blocking ergosteryl ester accumulation by using azoles in combination with oleate synergistically suppressed C. albicans cell viability in vitro and the growth of biofilms on the wounds of mice in vivo. Collectively, our discoveries showcased how label-free chemical imaging technique enables us to make fundamental discoveries in respect to unraveling the mechanism of antifungal agents and future therapeutic development.

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