Systematic gene overexpression in Candida albicans identifies a regulator of early adaptation to the mammalian gut.

Sadri Znaidi,Christophe D'Enfert,Marie-Elisabeth Bougnoux,Lasse Van Wijlick,Jean-Luc Desseyn,Arturo Hernández-Cervantes,Ralitsa Atanassova,Sophie Bachellier-Bassi,Thierry Jouault,Carol A Munro,Valérie Gouyer,Frédéric Dalle,Natacha Sertour,Frédéric Vincent

doi:10.1111/cmi.12890

Abstract

Candida albicans is part of the human gastrointestinal (GI) microbiota. To better understand how C. albicans efficiently establishes GI colonisation, we competitively challenged growth of 572 signature‐tagged strains (~10% genome coverage), each conditionally overexpressing a single gene, in the murine gut. We identified CRZ2, a transcription factor whose overexpression and deletion respectively increased and decreased early GI colonisation. Using clues from genome‐wide expression and gene‐set enrichment analyses, we found that the optimal activity of Crz2p occurs under hypoxia at 37°C, as evidenced by both phenotypic and transcriptomic analyses following CRZ2 genetic perturbation. Consistent with early colonisation of the GI tract, we show that CRZ2 overexpression confers resistance to acidic pH and bile salts, suggesting an adaptation to the upper sections of the gut. Genome‐wide location analyses revealed that Crz2p directly modulates the expression of many mannosyltransferase‐ and cell‐wall protein‐encoding genes, suggesting a link with cell‐wall function. We show that CRZ2 overexpression alters cell‐wall phosphomannan abundance and increases sensitivity to tunicamycin, suggesting a role in protein glycosylation. Our study reflects the powerful use of gene overexpression as a complementary approach to gene deletion to identify relevant biological pathways involved in C. albicans interaction with the host environment.

Highlights

Candida albicans is part of the human microbiota
Among the PTET‐CRZ2 upregulated genes, we found a high proportion of those encoding cell‐surface proteins (e.g., PGA6, ECM331, PLB1, PLB4.5, KRE1, and SCW11), mannosyltransferases (e.g., MNN1, MNN24, MNN22, and BMT5), proteins involved in methionine/cysteine metabolism (e.g., MET15, MET3, MET10, and ECM17), and small molecule/amino‐acid transporters (e.g., orf19.4690, SSU1, FRP3, FIGURE 4 Transcriptomic analysis of CRZ2 overexpression strains. (a) Heat maps of the 40 highest transcriptionally modulated genes in PTET‐CRZ2 transcript profiling data at time points 2 and 4 hr post‐induction with 40 μg ml−1 doxycycline
As this phenotype could be a consequence of tunicamycin‐induced endoplasmic reticulum (ER) stress, we examined the susceptibility of the PTDH3‐CRZ2 strain to another potent inducer of ER stress, FIGURE 9 CRZ2 is required for adaptation to acidic pH and bile salts under hypoxia at 37°C. pH 3‐ and bile salt‐ susceptibility phenotypes of two independent crz2Δ/crz2Δ (#1 and #2) and the corresponding wild‐type control (CRZ2/CRZ2) strains were analysed together with two independent PTDH3‐driven CRZ2 overexpressers and the matched control strain (PTDH3‐GTW) by spot assay on SD plates supplemented with 150 mM HEPES at pH 3 and 0.1% bile salts, respectively

Summary

| INTRODUCTION

Candida albicans is part of the human microbiota. As a commensal, C. albicans is present within the gastrointestinal (GI) and genital tracts of healthy humans. The same approaches demonstrated that RTG1, RTG3, TYE7, and LYS144 mediate GI tract colonisation by controlling the expression of genes involved in the acquisition and metabolism of specific nutrients, reflecting the importance of nutrient sensing/uptake during C. albicans commensalism (Perez & Johnson, 2014) Such systems biology‐driven strategies are cornerstone for mapping biological networks operating during C. albicans interaction with the host. We established a new library carrying 572 signature‐tagged strains with potent, pNIMX‐driven, conditional overexpression (Chauvel et al, 2012) We used it in a mouse model of GI tract colonisation to propose a new role for the TF Crz2p: the regulation of processes controlling the ability of C. albicans to efficiently proliferate within the host

| RESULTS

| DISCUSSION

| EXPERIMENTAL PROCEDURES