RNA Enrichment Method for Quantitative Transcriptional Analysis of Pathogens In Vivo

In vivo transcriptional analyses of microbial pathogens are often hampered by low proportions of pathogen biomass in host organs, hindering the coverage of full pathogen transcriptome. Researchers at University Hospital Lausanne, aimed to address the transcriptome profiles of Candida albicans, the most prevalent fungal pathogen in systemically infected immunocompromised patients, during systemic infection in different hosts. They developed a strategy for high-resolution quantitative analysis of the C. albicans transcriptome directly from early and late stages of systemic infection in two different host models, mouse and the insect Galleria mellonella.

Their results show that transcriptome sequencing (RNA-seq) libraries were enriched for fungal transcripts up to 1,600-fold using biotinylated bait probes to capture C. albicans sequences. This enrichment biased the read counts of only ~3% of the genes, which can be identified and removed based on a priori criteria. This allowed an unprecedented resolution of C. albicans transcriptome in vivo, with detection of over 86% of its genes. The transcriptional response of the fungus was surprisingly similar during infection of the two hosts and at the two time points, although some host- and time point-specific genes could be identified. Genes that were highly induced during infection were involved, for instance, in stress response, adhesion, iron acquisition, and biofilm formation. Of the in vivo-regulated genes, 10% are still of unknown function, and their future study will be of great interest. The fungal RNA enrichment procedure used here will help a better characterization of the C. albicans response in infected hosts and may be applied to other microbial pathogens.

Identification of C. albicans genes differentially expressed in vivo versus in vitro using a meta-analytical approach. (a) Meta-analysis strategy. Limma contrast statistics were converted to z scores (see Materials and Methods). z scores were then combined meta-analytically as illustrated. (b) Scatter plot of mouse and Galleria z scores obtained meta-analytically. Mouse and Galleria z scores are further combined into one in vivo z score. The 1,169 genes for which this combined z score is significant (Bonferroni P value ≤ 0.05) are indicated in brown. Genes for which the combined z score is not significant are indicated in blue if the mouse z score is significant or in green if the G. mellonella z score is significant. TLO1 is the only gene with significant and anti-correlated z scores in mouse and G. mellonella. r, Pearson correlation coefficient with confidence interval. (c) Heat map of the 1,169 significant genes by meta-analysis. For each sample, a log fold change versus the average in vitro expression was computed. The log fold change values were variance scaled and are represented on the heat map. Hierarchical clustering tree of the samples is indicated at the top. Gm, G. mellonella; Mm, Mus musculus.