Verkenning van het Potentieel van Riboflavine Biosynthese in Candida albicans: Van Veelbelovende Antischimmeldoelwit tot Waardevolle Vitaminebron

Fungal infections have increased in prevalence over recent decades, yet treatment options remain limited, with only three major classes of antifungal drugs available. The efficacy of these drugs is further constrained by host toxicity, narrow activity spectra, poor pharmacokinetics, and both intrinsic and acquired resistance. To address this, novel antifungal targets with distinct modes of action are urgently needed. One promising avenue is the riboflavin biosynthesis pathway, which is absent in humans but essential for fungi. Our research demonstrates that all enzymes in the riboflavin biosynthesis pathway are essential for Candida albicans, Candida. glabrata, and Saccharomyces cerevisiae. Riboflavin auxotrophs, mimicking drug-inhibited strains, showed rapid mortality in a physiologically relevant medium. Furthermore, Rib1 is critical for fungal virulence in C. albicans and C. glabrata, as RIB1 deletion significantly reduced fungal burdens in infected mice, particularly in the kidneys and brain. Despite this, C. albicans and C. glabrata auxotrophs can absorb external riboflavin when available. We identified Orf19.4337 as the riboflavin importer in C. albicans and named it Rut1. We found that Rut1 only facilitates growth at external riboflavin concentrations that exceed the physiological concentrations in the human body, suggesting that riboflavin uptake is unlikely to provide resistance to biosynthesis-targeting drugs. In contrast, S. cerevisiae displayed more efficient RF uptake, allowing it to outcompete C. albicans under low RF conditions. An auxotrophic S. cerevisiae strain survives in blood simulative medium, indicating that a compound targeting the riboflavin biosynthetic pathway could selectively inhibit C. albicans and C. glabrata while potentially sparing S. cerevisiae. This could offer potential therapeutic applications, especially considering S. cerevisiae's probiotic properties that inhibit C. albicans in certain niches. As a flavinogenic yeast, Candida albicans secretes riboflavin in quantities that exceed its metabolic needs, though the physiological drivers behind this remain unclear. Notably, we observed substantial variation in riboflavin production across yeast species and among clinical isolates of the same species. We explored the environmental factors influencing RF production and secretion in C. albicans, finding that factors like medium composition, temperature, oxygen, carbon source, and pH significantly affect secretion levels. Additionally, riboflavin secretion increased under oxidative, nitrosative, and osmotic stress, though it decreased under cell wall stress. Despite these insights, overexpression or partial deletion of RIB1 did not alter stress tolerance, suggesting a complex relationship between riboflavin production and stress responses. Given the association between riboflavin secretion and iron levels, we hypothesized that riboflavin might enhance iron bioavailability by reducing ferric iron to its more soluble ferrous form. However, our experimental results did not support this hypothesis. It should be further investigated if riboflavin can indeed facilitate iron uptake. Additionally, we found no link between riboflavin secretion and virulence traits like biofilm formation or host cytotoxicity. Next, we shifted the focus to a host perspective, investigating riboflavin secretion by C. albicans within the human gut and its potential impact on host health. We demonstrated that C. albicans does secrete riboflavin in the murine gut, depending on the strain tested, it can even prevent ariboflavinosis caused by a riboflavin deficient diet. Riboflavin secretion by C. albicans might offer health benefits that could explain, from an evolutionary standpoint, why this opportunistic pathogen persists as a commensal in humans. Beyond preventing riboflavin deficiency, the riboflavin produced by C. albicans may enhance gut health by maintaining the integrity of the GI epithelial lining, modulating the immune system, and potentially offering protection against gut pathogens. One exciting possiblity is that C. albicans could activate mucosal-associated invariant T cells in the gut, which recognize derivatives of riboflavin biosynthesis. This activation may help prevent the fungus from transitioning from a commensal to a pathogenic form. Lastly, to address the urgent need for new antifungal agents, we conducted a high-throughput screening of 20,000 compounds, identifying five structurally distinct and promising candidates. The compounds were characterized and chemogenomic profiling was employed to uncover their modes of action. For one compound, we identified a clear profile suggesting that it targets the sphingolipid biosynthesis pathway. Further investigation revealed that the compound inhibits the first and essential step in this pathway, specifically serine palmitoyltransferase. However, its mode of action differs from other known inhibitors of serine palmitoyltransferase such as myriocin.