Imaging and Quantification of mRNA Molecules at Single-Cell Resolution in the Human Fungal Pathogen Candida albicans

Abstract

Tools to visualize and quantify transcripts at single-cell resolution have enabled the dissection of spatiotemporal patterns of gene expression in animal cells and tissues. Yet the accurate quantification of transcripts at single-cell resolution remains challenging for the much smaller microbial cells.

RFX transcription factor in the human‐associated yeast Candida albicans regulates adhesion to oral epithelium

“…As shown in Figure 3a–h, wild‐type fungal cells that had penetrated the keratinized layer at the surface of the tongue displayed multiple punctae corresponding to the RFX1 transcript whereas no signal was detectable in comparable sections of tissue infected with the C. albicans rfx1 deletion strain. Assuming that each fluorescent spot corresponds to a single mRNA molecule, there were 3–8 RFX1 transcripts per cell, which is in agreement with the number of mRNAs per cell observed in yeasts for other transcription regulator genes (Moreno‐Velásquez & Pérez, 2021). From this experiment we conclude that RFX1 is indeed expressed by fungal cells during infection, which is consistent with the genetics (Figure 2b) showing a phenotype for the rfx1 mutation in the oral epithelium.…”

“…To establish whether the RFX1 gene was actually expressed during Pérez, 2021). From this experiment we conclude that RFX1 is indeed expressed by fungal cells during infection, which is consistent with the genetics (Figure 2b) showing a phenotype for the rfx1 mutation in the oral epithelium.…”

“…To establish whether the RFX1 gene was actually expressed during infection, we sought to visualize RFX1 transcripts at the single‐cell level in C. albicans ‐infected mouse tongues. For this, we adapted hybridization chain reaction (HCR)—a method that we recently applied to Candida cells grown in planktonic cultures (Moreno‐Velásquez & Pérez, 2021)—to paraffin‐embedded tissues. Sections of infected tongues were hybridized with two sets of compatible HCR probes for fluorescent in situ mRNA visualization, one targeting the ribosomal RNA RDN25 ( CR_08810W ) to detect C. albicans cells in general and the other targeting the RFX1 mRNA.…”

“…Moreno-Velásquez & Pérez, 2021). Target DNA sequences for C. albicans RFX1 and RDN25 (CR_08810W) were obtained from the Candida Genome Database (CGD, http:// www.…”

“…org/ ). Split-initiator probe pairs were designed according to described parameters(Choi et al, 2018;Moreno-Velásquez & Pérez, 2021) using Serial Cloner (2.6.1) and validated by the CGD multi-genome NCBI BLAST+ tool to minimize potential off-target hybridization. Probe sets consisting of split-initiator oligonucleotides A and B (Supporting information, Table…”