An integrated transcriptomic and metabolomic approach to investigate the heterogeneous Candida albicans biofilm phenotype

Biofilm · 8 authors, 5 centres

AI SUMMARY

FIDELITY 92%

POPULATIONClinical Candida albicans bloodstream isolates (n=60), stratified into high biofilm-forming (HBF) and low biofilm-forming (LBF) groups

INTERVENTIONExposure to fetal calf serum (FCS) or arachidonic acid (AA), with and without the COX pathway inhibitor salicylic acid

COMPARISONGrowth in RPMI-1640 medium without serum vs. with serum; HBF vs. LBF isolates; AA treatment vs. AA + salicylic acid

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TL;DR

This study investigated why different clinical isolates of Candida albicans form biofilms with varying strength. The researchers found that low biofilm-forming strains can be induced to form robust biofilms when exposed to serum, and this induction is driven by arachidonic acid metabolism via the COX pathway. These findings reveal a potential new drug target for treating C. albicans bloodstream infections.

Full summary

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**Background:** Candida albicans is the most prevalent fungal pathogen in bloodstream infections and is classified as a critical priority pathogen by the WHO. Biofilm formation is a key virulence trait that varies widely among clinical isolates and is associated with increased mortality. While much is known about constitutive high biofilm formers, the mechanisms by which low biofilm-forming strains adapt to form biofilms under host-relevant conditions remain poorly understood. Serum exposure is known to induce filamentation and biofilm formation, but the metabolic and transcriptional pathways driving this adaptive phenotype in heterogeneous clinical isolates have not been fully characterized.

**Methods:** The study used 60 clinical C. albicans bloodstream isolates previously characterized as either high biofilm-forming (HBF) or low biofilm-forming (LBF). Biofilm formation was assessed using crystal violet assays, and antifungal tolerance was evaluated via population analysis profiling (PAP) for fluconazole, caspofungin, and amphotericin B. For induction experiments, biofilms were grown in RPMI-1640 with or without 25% fetal calf serum (FCS) or dialysed serum. Hyphal length was measured microscopically. RNA-Seq was performed on one HBF and one LBF isolate at 90 min, 4 h, and 24 h, yielding an average of 24.4 million reads per sample with 95% average alignment to the reference genome. Differential expression analysis used DESeq2 with cutoffs of log2FC > 1.5 and FDR-adjusted p < 0.05. Metabolomics was performed on 3 HBF and 3 LBF isolates using LC-MS, identifying 49 compounds against authentic standards. Integrated analysis combined PAPi pathway activity profiling with transcriptomic data. Biofilm induction by arachidonic acid (5 mM) with and without salicylic acid (5 mM) was tested on 5 HBF and 5 LBF isolates.

**Key Results:** Fluconazole tolerance (measured as AUC) showed a significant positive correlation with biofilm formation across 60 isolates (R = 0.34, p < 0.05), while no significant correlation was found for caspofungin or amphotericin B. Serum exposure induced a 5- to 7.4-fold increase in biofilm biomass in LBF isolates (p < 0.01) and increased germ tube length by 3.5-fold in LBF and 1.8-fold in HBF (p < 0.01). Dialysed serum did not induce biofilm formation (p > 0.05), indicating small molecules are critical. Transcriptomic analysis revealed 316 genes uniquely upregulated in HBF, 96 shared, and 164 unique to LBF in response to serum. Only LBF showed significantly enriched KEGG pathways, including fatty acid metabolism genes (FOX2, FOX3, ECI1, CAT2, PEX11, ANT1) and acetate transport genes (ATO1, ATO10, ATO9). Drug transporter analysis showed CDR1, CDR2, CDR3, and CDR11 were upregulated in HBF at 24 h. Metabolomics identified arachidonic acid (AA) metabolism as the most significantly regulated pathway (p = 4.37e-13), with AA detected only in serum-containing cultures. Exogenous AA (5 mM) induced an approximately 3-fold increase in biofilm biomass in LBF isolates (p < 0.0001), and this effect was reversed by the COX pathway inhibitor salicylic acid. HBF isolates showed a non-significant increase with AA but a significant reduction with salicylic acid.

**Clinical Implications:** This study demonstrates that C. albicans clinical isolates exhibit substantial heterogeneity in biofilm formation and antifungal tolerance, with low biofilm formers showing remarkable metabolic plasticity. The finding that LBF strains can utilize host-derived arachidonic acid via the COX pathway to induce biofilm formation identifies a potential therapeutic target. The arachidonic acid cascade, which is not endogenously produced by C. albicans but is scavenged from the host, represents a vulnerability that could be exploited for drug discovery. The study also highlights the importance of using diverse clinical isolates rather than standard laboratory strains to uncover clinically relevant pathogenic mechanisms.

PICO

PPOPULATION

Clinical Candida albicans bloodstream isolates (n=60), stratified into high biofilm-forming (HBF) and low biofilm-forming (LBF) groups

IINTERVENTION

Exposure to fetal calf serum (FCS) or arachidonic acid (AA), with and without the COX pathway inhibitor salicylic acid

OOUTCOME

Biofilm biomass (crystal violet absorbance at 570 nm), hyphal length, fluconazole tolerance (AUC), transcriptional changes (RNA-Seq), and metabolic changes (LC-MS)

STUDY TYPE

other

SPECIALTY

infectious_disease

SUMMARISED BY

AI pipeline

FIDELITY CHECK

92% · A

ORIGINAL PAPER

PMC10034394

An integrated transcriptomic and metabolomic approach to investigate the heterogeneous Candida albicans biofilm phenotype

Biofilm

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ABSTRACT

Candida albicans is the most prevalent and notorious of the Candida species involved in bloodstream infections, which is characterised by its capacity to form robust biofilms. Biofilm formation is an important clinical entity shown to be highly variable among clinical isolates. There are various environmental and physiological factors, including nutrient availability which influence the phenotype of Candida species. However, mechanisms underpinning adaptive biofilm heterogeneity have not yet been fully explored. Within this study we have profiled previously characterised and phenotypically distinct C. albicans bloodstream isolates. We assessed the dynamic susceptibility of these differing populations to antifungal treatments using population analysis profiling in addition to assessing biofilm formation and morphological changes. High throughput methodologies of RNA-Seq and LC-MS were employed to map and integrate the transcriptional and metabolic reprogramming undertaken by heterogenous C. albicans isolates in response to biofilm and hyphal inducing serum. We found a significant relationship between biofilm heterogeneity and azole resistance (P < 0.05). In addition, we observed that in response to serum our low biofilm forming (LBF) C. albicans exhibited a significant increase in biofilm formation and hyphal elongation. The transcriptional reprogramming of LBF strains compared to high biofilm forming (HBF) was distinct, indicating a high level of plasticity and variation in stress responses by heterogenous strains. The metabolic responses, although variable between LBF and HBF, shared many of the same responses to serum. Notably, a high upregulation of the arachidonic acid cascade, part of the COX pathway, was observed and this pathway was found to induce biofilm formation in LBF 3-fold. C. albicans is a highly heterogenous bloodstream pathogen with clinical isolates varying in antifungal tolerance and biofilm formation. In addition to this, C. albicans is capable of highly complex and variable regulation of transcription and metabolic pathways and heterogeneity across isolates further increases the complexity of these pathways. Here we have shown with a dual and integrated approach, the importance of studying a diverse panel of C. albicans isolates, which has the potential to reveal distinct pathways that can harnessed for drug discovery.

CITATION EXPORT

Christopher Delaney, Bryn Short, Ranjith Rajendran et al.. An integrated transcriptomic and metabolomic approach to investigate the heterogeneous Candida albicans biofilm phenotype. Biofilm. (2023). https://doi.org/10.1016/j.bioflm.2023.100112

AUTHORS

8 · 5 CENTRES

Christopher Delaney, Bryn Short, Ranjith Rajendran, Ryan Kean, Karl Burgess, Craig Williams, Carol A. Munro, Gordon Ramage

aSchool of Medicine, College of Medical, Veterinary and Life Sciences (MVLS), University of Glasgow, UK
bDepartment of Biological and Biomedical Sciences, School of Health and Life Sciences, Glasgow Caledonian University, Glasgow, UK
cInstitute of Quantitative Biology, Biochemistry and Biotechnology, University of Edinburgh, Max Born Crescent, Edinburgh, UK
dMicrobiology Department, Lancaster Royal Infirmary, University of Lancaster, Lancaster, UK
eSchool of Medicine, Medical Sciences and Nutrition, Institute of Medical Sciences, University of Aberdeen, UK