Efficient labeling of pathogenic fungal chitin with bioorthogonal probes.

The fungal cell wall plays a large role in pathogenicity and is essential to the survival and virulence of fungi such as Candida albicans. Filamentous growth and its causes have been studied, however specific cell wall features that contribute to this morphology have yet to be investigated thoroughly. Understanding how the makeup of cell wall polymers changes during hyphal growth is therefore critical to developing novel targets for therapeutics. Bio-orthogonal chemistry has emerged as a powerful tool to profile critical cellular processes without interfering with canonical machinery. This chapter describes multiple methods for utilizing bio-orthogonal probes to characterize chitin in the commensal and pathogenic forms of C. albicans. This metabolic labeling approach is based on the native uptake and incorporation pathways in Candida species and allows for selective labeling of the cell wall polymer chitin. Further, mass spectrometry validation protocols have been developed to isolate carbohydrate components of the fungal cell wall without deacetylation of the polymers. Detailed, up-to-date protocols are outlined in this chapter and comprise all steps of the workflow, including cell growth conditions, sample processing, and data analysis.