Single-Molecule Fluorescent In Situ Hybridization (smFISH) for RNA Detection in the Fungal Pathogen Candida albicans

Area of Science:

• Microbiology
• Molecular Biology
• Mycology

Background:

• Candida albicans is a major human fungal pathogen.
• Its pathogenicity is linked to reversible morphological changes (yeast, pseudohyphae, hyphae).
• Understanding the molecular regulation of these switches is crucial for combating infections.

Purpose of the Study:

• To develop and detail a single-cell, single-molecule RNA imaging protocol for Candida albicans.
• To enable absolute mRNA quantification in intact, morphologically distinct cells.
• To facilitate the study of gene expression during morphological transitions.

Main Methods:

• Optimization of single-molecule fluorescent in situ hybridization (smFISH) for C. albicans.
• Detailed protocol steps: cell growth, fixation, hybridization, imaging, segmentation, and mRNA spot analysis.
• Quantification of specific mRNAs (CLB2, EFG1) in different cell types and conditions.

Main Results:

• The smFISH protocol allows accurate mRNA quantification in individual C. albicans cells.
• Differential gene expression was observed between yeast, pseudohyphae, and hyphal forms.
• Expression changes of CLB2 and EFG1 mRNAs were successfully measured.

Conclusions:

• This single-cell RNA imaging approach provides a powerful tool for molecular characterization of C. albicans.
• The protocol can be applied to study other filamentous fungi, both pathogenic and nonpathogenic.
• It offers a pathway to better understand and potentially target C. albicans infections.