An approach to analyze spatiotemporal patterns of gene expression at single-cell resolution in Candida albicans-infected mouse tongues

Abstract

Microbial gene expression measurements derived from infected organs are invaluable to understand pathogenesis. However, current methods are limited to "bulk" analyses that neglect microbial cell heterogeneity and the lesion's spatial architecture. Here, we report the use of hybridization chain reaction RNA fluorescence in situ hybridization (HCR RNA-FISH) to visualize and quantify Candida albicans transcripts at single-cell resolution in tongues of infected mice. The method is compatible with fixed-frozen and formalin-fixed paraffin-embedded tissues. We document cell-to-cell variation and intriguing spatiotemporal expression patterns for C. albicans mRNAs that encode products implicated in oral candidiasis. The approach provides a spatial dimension to gene expression analyses of host-Candida interactions.

Importance: Candida albicans is a fungal pathobiont inhabiting multiple mucosal surfaces of the human body. Immunosuppression, antibiotic-induced microbial dysbiosis, or implanted medical devices can impair mucosal integrity enabling C. albicans to overgrow and disseminate, causing either mucosal diseases such as oropharyngeal candidiasis or life-threatening systemic infections. Profiling fungal genes that are expressed in the infected mucosa or in any other infected organ is paramount to understand pathogenesis. Ideally, these transcript profiling measurements should reveal the expression of any gene at the single-cell level. The resolution typically achieved with current approaches, however, limits most gene expression measurements to cell population averages. The approach described in this report provides a means to dissect fungal gene expression in infected tissues at single-cell resolution.

Keywords: Candida albicans; Candida-host interactions; Candida-infected tissue; RNA-FISH; hybridization chain reaction; microbial transcript visualization; oropharyngeal candidiasis.

Fig 1

Fungal transcript visualization in C. albicans-infected mouse tongues. (A) PAS-stained sagittal section of murine tongue infected with C. albicans. Inset in left panel is enlarged to the right to show infection site structure. SC, stratum corneum; SEL, suprabasal epithelial layer; BEL, basal epithelial layer; infiltr, immune cell infiltrate (purple). Black arrowheads point to C. albicans cells. Dashed square depicts approximate area of HCR visualization and analysis. Scale bar in left panel, 500 µm; in right panel, 50 µm. (B and C) Fixed-frozen tongue sections collected 28 h after infection from mice inoculated with C. albicans SC5314 wild-type (B) or an isogenic hwp1 deletion strain (C). Candida hyphae (green) are detected with HCR probes and amplifiers that hybridize fungal rRNA. HWP1 transcripts (purple) are indicated with white arrowheads. Nuclei from epithelial and immune cells are stained with DAPI (blue). Insets in left panels are enlarged to the right. Scale bars in left panels, 10 µm; in the enlarged images, 2 µm. Numbers in parenthesis are the wavelengths of the laser lines used for imaging.

Fig 2

Localized expressions of ECE1, PRA1, and ZRT1 in C. albicans hyphae. High-resolution images of multiplex HCR targeting ECE1 (candidalysin), PRA1, and ZRT1 transcripts in formalin-fixed paraffin-embedded sections. Tissue collected 48 h after infection. Scale bars, 0.5 µm. Numbers in parenthesis are the wavelengths of the laser lines used for imaging.

Fig 3

Quantification and distribution of HWP1 transcript during infection. (A) HWP1 transcript quantification in C. albicans hyphae located in the stratum corneum. Shown are violin plots where each dot corresponds to a single C. albicans hypha (length > 5 µm in MaxZ projection). Tongues were collected 18 or 48 h after infection. N is the number of hyphae quantified at each timepoint. Statistical analysis by the Mann–Whitney U-test. (B and C) Representative images showing HWP1 transcript distribution and abundance in C. albicans hyphae laying in the tongue's stratum corneum. Formalin-fixed paraffin-embedded sections from tissues collected 18 h (B) or 48 h (C) after infection. Candida hyphae (green) are detected with HCR amplifiers and probes targeting fungal rRNA. HWP1 transcripts (purple) are indicated with white arrowheads. Nuclei from epithelial and immune cells are stained with DAPI (blue). Insets in left panels are enlarged to the right. Dashed square in panel C is enlarged in panel D. Scale bars in left panels, 10 µm; in the enlarged images, 2 µm. (D) Aberrant patterns of HWP1's fluorescent signal in C. albicans cells that had penetrated beyond the tongue's stratum corneum. White arrow points to HWP1 signal that did not colocalize with fungal rRNA. Asterisk indicates tightly clustered foci of intense fluorescent signal. Nuclei from epithelial and/or immune cells are stained with DAPI (blue). Scale bars, 2 µm. Numbers in parenthesis are the wavelengths of the laser lines used for imaging.

Fig 4

Visualizing host mRNA and C. albicans in infected tongues. Distribution of mouse keratin 13 mRNA (red) in dorsal and ventral tongue surfaces of control (not infected) and C. albicans-infected mice. Keratin 13 is a marker of the postmitotic suprabasal epithelial layer (SEL). Formalin-fixed paraffin-embedded sections were used. Mouse nuclei are stained with DAPI (blue). Notice keratin 13 signal abrogation in the area adjacent to C. albicans (green). BEL, proliferative basal epithelial layer; SC, stratum corneum. White arrowheads point to C. albicans cells, which are visualized with probes against RDN25. Scale bars, 20 µm.