Fungal cell wall is a dynamic organelle that plays a vital role, particularly in cellular growth, elongation and division. Any modification or disruption of the wall leads to lysis and cell death, hence serving an excellent target for anti-fungal drugs. Chemically, the cell wall architecture in fungi is complex comprising mainly polysaccharides (glucan, mannan and chitin) and a small proportion of glycoproteins. Traditionally, electron microscopy and biochemical extraction methods were employed while recently immunocytochemical analysis is used to understand its structure. The former lacks chemical specificity requiring genetically modified cells to study different structures in detail while the later involves development of fluorescent monoclonal antibodies specific to glycosidic linkages among cell wall polysaccharides. Hence we aim to develop a label-free method based on confocal Raman microscopy to visualize distribution of various polysaccharide components of fungal cell and spore wall. Fission yeast <i>Schizosaccharomyces pombe </i>is used<i> </i>as a model to demonstrate our method. First, space-resolved Raman spectra from lipid droplets, cytoplasm and cell wall were obtained to identify marker bands for individual components (Figure 1A) followed by imaging. Then, by employing multivariate curve resolution (MCR) analysis, we successfully separated Raman spectra of several pure bio-macromolecular components and their detailed chemical images are shown in Figure 1B [1]. We then studied pharmacokinetics and pharmacodynamics of antifungal drug terbinafine at the single cell level in yeast model. We believe that our method will help in understanding the complex fungal spore wall architecture and eventually lead to advancements in drug discovery and development in the future.

Label-free visualization of fungal cell and spore wall architecture by confocal Raman microscopy

ACS Spring Meeting 2021

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