riboflavin  (Valiant Co Ltd)

Journal: The Journal of Biological Chemistry

Article Title: Crosstalk between iron and flavins in the opportunistic fungal pathogen Candida albicans

doi: 10.1016/j.jbc.2025.110396

Figure Lengend Snippet: Riboflavin as the sole flavin secreted by Fe-starved C. albicans . WT strain SC5314 ( A – C , E – H ) or the isogenic sef1Δ/Δ mutant ( A – C ) was grown under Fe-replete or Fe-starvation conditions and either analyzed for total accumulation of metals ( A – C ) or for flavin secretion ( E – H ). In part A – C , whole cell accumulation of Fe or of Mn and Cu was carried out by the BPS or AAS assays, respectively, as described in . D – F , HPLC chromatograms of flavin fluorescence at 525 nm were obtained as described in from ( D ) a mixture of 2.0 nmols FAD, FMN and riboflavin standards, or spent media from Fe-starved ( E ) or Fe-replete ( F ) WT cells. Dotted lines indicate individual times of elution for FAD, FMN and riboflavin based on standards run in parallel. Note: The decreased intensity of the FAD standard reflects it lower fluorescence constant at 525 nm (see main text for details). G , HPLC chromatograph of media alone (“no cells”) versus an equal amount of spent media from Fe-replete cultures shows the dependence on cells for the riboflavin fluorescence detected. H , The area under the curve (AUC) for the riboflavin peak was used to quantify riboflavin secreted from Fe replete (+Fe) versus Fe-starved (−Fe) cells as described in . For all bar graphs, each value represents an independent fungal culture. Bars are mean and standard deviation, and significance determined by one way ANOVA with post hoc Tukey test ( A – C ) or unpaired t test ( H ); ∗∗∗∗ p < 0.0001; ∗∗∗ p = 0.003; ∗ p = 0.021 (part C sef1 +Fe versus -Fe) and 0.022 (part C WT -Fe vs sef1 -Fe).

Article Snippet: 20 μM standards of riboflavin, FAD, and FMN (Acros Organics 132350250, MP Biomedicals 0215112780, Sigma-Aldrich F2253, respectively) were prepared in the aforementioned 70:30 mix of ammonium acetate: methanol.

Techniques: Mutagenesis, Fluorescence, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Crosstalk between iron and flavins in the opportunistic fungal pathogen Candida albicans

doi: 10.1016/j.jbc.2025.110396

Figure Lengend Snippet: The effects of Fe-starvation on intracellular flavins. A , metabolites were extracted from Fe-replete or Fe-starved cells as described in Experimental Procedures and subjected to HPLC analysis of flavins in comparison to standards as in . B – D , Flavins were quantified as in E . B , the sum of FAD, FMN and riboflavin peaks was used to derive total intracellular flavins, and ( C , D ) the percentage contribution was calculated for individual flavins in each culture. Each value represents an independent culture. Bars are mean and standard deviation, and significance determined by unpaired t-tests; ∗∗∗∗ p < 0.0001; ns, p > 0.05. Strain utilized: SC5314.

Article Snippet: 20 μM standards of riboflavin, FAD, and FMN (Acros Organics 132350250, MP Biomedicals 0215112780, Sigma-Aldrich F2253, respectively) were prepared in the aforementioned 70:30 mix of ammonium acetate: methanol.

Techniques: Comparison, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Crosstalk between iron and flavins in the opportunistic fungal pathogen Candida albicans

doi: 10.1016/j.jbc.2025.110396

Figure Lengend Snippet: The dependence on SEF1 for flavin secretion during Fe-starvation. A , picture of WT and sef1Δ/Δ cultures grown under Fe-starvation (−Fe) or Fe-replete (+Fe) conditions, showing the intense yellow color of secreted flavins from WT Fe-starved cells. B , HPLC chromatograms of flavin standards and the spent media of sef1Δ/Δ mutants were obtained as in A . C , extracellular riboflavin was quantified as in E . Each value represents an independent fungal culture. Bars are mean and standard deviation, and significance determined by unpaired t test; ∗∗∗∗ p < 0.0001. Strains utilized: WT, SC5314 ( A ) or the sef1Δ/Δ derivative of SC5314 (“ sef1” in A , and B , C ).

Article Snippet: 20 μM standards of riboflavin, FAD, and FMN (Acros Organics 132350250, MP Biomedicals 0215112780, Sigma-Aldrich F2253, respectively) were prepared in the aforementioned 70:30 mix of ammonium acetate: methanol.

Techniques: Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Crosstalk between iron and flavins in the opportunistic fungal pathogen Candida albicans

doi: 10.1016/j.jbc.2025.110396

Figure Lengend Snippet: Effects of sef1Δ/Δ mutations on intracellular flavins during Fe-starvation. A , HPLC chromatograms of flavin standards ( top ) and of intracellular flavins extracted from sef1Δ/Δ Fe-replete ( middle ) and Fe-starved ( bottom ) cells as obtained in A . B , total flavins were quantified from three individual pairs of Fe-starved and Fe-replete sef1Δ/Δ cultures as in B . C and D , quantification of individual flavins from WT versus sef1Δ/Δ cultures was carried out as in and E . E , for each pair of Fe-replete and Fe-starved cultures grown in parallel, the log2 fold change (LFC) in riboflavin from Fe-replete vs Fe-starved cells was calculated for intracellular (“InC”) and extracellular (“E x C”) riboflavin. Results show opposing effects of Fe-starvation on intracellular versus extracellular riboflavin of sef1Δ/Δ mutants. Significance was determined by one-way Anova with post hoc Tukey test and also by an unpaired t test in the case of sef1Δ/Δ FMN (part C ); ∗∗∗∗ p < 0.0001; ∗∗∗ p = 0.0003; ∗∗ p = 0.0035; ∗ p = 0.013 (WT FMN -Fe vs + Fe) or 0.0395 ( sef1 FMN -Fe vs + Fe) or 0.039 (part E); ns p > 0.05. Strains utilized: SC5314 ( C – E ) or the sef1Δ/Δ derivative ( A – E ).

Article Snippet: 20 μM standards of riboflavin, FAD, and FMN (Acros Organics 132350250, MP Biomedicals 0215112780, Sigma-Aldrich F2253, respectively) were prepared in the aforementioned 70:30 mix of ammonium acetate: methanol.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Crosstalk between iron and flavins in the opportunistic fungal pathogen Candida albicans

doi: 10.1016/j.jbc.2025.110396

Figure Lengend Snippet: Expression of riboflavin biosynthesis and flavin conversion genes. A , shown is the pathway for riboflavin biosynthesis from GTP and ribulose sugars, and the steps involving C. albicans RIB1, RIB3 and RIB4 genes. Riboflavin is converted to FMN and FAD by the Fmn1 flavin kinase and FAD synthase Fad1, respectively. See the main text for details. B , qRT-PCR analysis of the designated riboflavin biosynthesis and flavin conversion genes was carried out with the indicated strains grown in Fe-starvation or Fe-replete conditions. The results shown are compared to TUB2 expression. Each value represents an independent fungal culture. Significance was determined by one-way Anova with post hoc Tukey test and also by an unpaired t test in the case of sef1Δ/Δ FMN1 . ∗∗∗∗ p < 0.0001; ∗∗ p = 0.0015 (part B ) and 0.0034 (part C ); ∗ p = 0.048 (part C , FMN1) and 0.012 (part C , FAD1); ns p > 0.05. Strains utilized: SC5314 and the sef1Δ/Δ derivative.

Article Snippet: 20 μM standards of riboflavin, FAD, and FMN (Acros Organics 132350250, MP Biomedicals 0215112780, Sigma-Aldrich F2253, respectively) were prepared in the aforementioned 70:30 mix of ammonium acetate: methanol.

Techniques: Expressing, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Crosstalk between iron and flavins in the opportunistic fungal pathogen Candida albicans

doi: 10.1016/j.jbc.2025.110396

Figure Lengend Snippet: Model for the crosstalk between Fe and flavins in pathogenic C. albicans . Under Fe-replete conditions, FAD and FMN are the predominant intracellular flavins of C. albicans , with trace riboflavin (RF) and very little riboflavin is secreted. (Font size of FAD, FMN, and RF reflects relative abundance.) When Fe-starved, WT cells upregulate RIB genes for riboflavin synthesis (depicted in blue ) but not flavin conversion genes ( purple ). Riboflavin levels rise inside the cell with Fe-starvation, but this is not accompanied by an increase in FMN and FAD. Rather, the excess riboflavin is exported from the cell in Fe-starved WT cells. Sef1 is required to induce riboflavin biosynthesis as well as riboflavin export, and mutants of sef1Δ/Δ show an increase in intracellular RF, but a repression in RF export during Fe-starvation. The riboflavin exporter regulated by Fe and Sef1 ( red arrow ) is still unknown. The cartoon also depicts possible roles for secreted riboflavin in Fe-starved C. albicans , including increased Fe uptake from serum as documented in our studies, and activities as an immune suppressant and antioxidant (see main text for details).

Article Snippet: 20 μM standards of riboflavin, FAD, and FMN (Acros Organics 132350250, MP Biomedicals 0215112780, Sigma-Aldrich F2253, respectively) were prepared in the aforementioned 70:30 mix of ammonium acetate: methanol.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Crosstalk between iron and flavins in the opportunistic fungal pathogen Candida albicans

doi: 10.1016/j.jbc.2025.110396

Figure Lengend Snippet: Effects of riboflavin on Fe uptake in C. albicans . The WT strain SC5314 ( A – E , G ) and fre10Δ/Δ mutant ( F ) were assayed for either Fe-accumulation ( A – C , E – G ) or for cell morphology by dark field microscopy ( D ) as described in . The Fe uptake studies used the following sources of Fe: ( A ) 35 μM FeCl 3 added to a yeast nitrogen base (YNB) media under aerobic (+O 2 ) or anaerobic (−O 2 ) conditions in unbuffered media or media "buffered" to pH 5.8 as indicated; ( B , C , F ) 50% fetal calf serum in YNB media; ( E ) 35 μM FeCl 3 added to IMDM; ( G ) 5 μM diferric-transferrin added to IMDM at the indicated pH. With the exception of ( A ), which was conducted at 30 °C, all experiments were carried out at 37 °C under anaerobic conditions. Accumulation of Fe ( A – C and E – G ) or of Mn and Cu ( B ) was monitored in cells cultured in the absence or presence of 10 μM riboflavin (+RF). Individual values represent independent fungal cultures. In ( C ), values were plotted relative to the no-riboflavin mean for each experimental trial. Significance was determined by one-way Anova with post hoc Tukey test ( A ) or unpaired t-tests ( B and C , E – G ). ∗∗∗∗ p < 0.0001; ∗∗∗ p = 0.0007 (part G -RF pH 6.0 vs pH 5.5) and 0.0002 (part G -RF pH 5.5 vs pH 5.0); ∗∗ p = 0.0064 (part B ) and 0.0084 (part F ); ∗ p = 0.031, ns p > 0.05. ( D ) Shown are images of cell morphology from the various Fe uptake studies. In experiments with 35 μM FeCl 3 in YNB, cells are in yeast form. Serum induces the formation of short hyphae/germ tubes, as does IMDM. The addition of riboflavin (RF) does not alter overall cell morphology.

Article Snippet: 20 μM standards of riboflavin, FAD, and FMN (Acros Organics 132350250, MP Biomedicals 0215112780, Sigma-Aldrich F2253, respectively) were prepared in the aforementioned 70:30 mix of ammonium acetate: methanol.

Techniques: Mutagenesis, Microscopy, Cell Culture