Vitamin B2 Kills Cryptococcal Fungal Infections by Destroying Membranes and Spiking Oxidative Stress
Riboflavin (vitamin B2) inhibits Cryptococcus neoformans growth, dismantles biofilms, and reduces organ fungal burden in mouse models of meningitis and pneumonia.
Summary
Researchers at Southwest Medical University tested riboflavin (vitamin B2) against Cryptococcus neoformans, the fungus responsible for life-threatening meningitis and pneumonia, particularly in immunocompromised patients. In laboratory experiments, riboflavin inhibited 90% of fungal growth at 0.4 mg/mL, disrupted cell membranes and walls, triggered toxic accumulation of reactive oxygen species (ROS), and blocked biofilm formation. It also suppressed key virulence factors including the protective polysaccharide capsule, melanin production, and urease secretion. In mouse models of both lung and brain infection, riboflavin significantly reduced fungal counts in organs, lessened tissue damage, and lowered inflammatory cytokines. These findings suggest riboflavin — already FDA-approved and inexpensive — could be repurposed as an antifungal agent, offering a promising low-cost addition to the limited treatment arsenal for cryptococcal disease.
Detailed Summary
Cryptococcus neoformans causes an estimated 152,000 cases of meningitis annually worldwide, resulting in approximately 112,000 deaths, with HIV-associated cryptococcal meningitis carrying a mortality rate near 70% in low-income African settings. Current treatment relies on liposomal amphotericin B plus flucytosine followed by fluconazole, but up to 60% of patients relapse due to fluconazole resistance, and drug costs remain prohibitive in high-burden regions. This study investigated riboflavin (RF), an essential water-soluble B vitamin already widely approved and inexpensive, as a repurposed antifungal candidate against the reference strain C. neoformans H99.
Using broth microdilution per CLSI M27-A4 standards, researchers determined the MIC90 of riboflavin against H99 to be 0.4 mg/mL. The minimum fungicidal concentration (MFC) was 0.8 mg/mL, yielding an MFC/MIC ratio of 2, which classifies riboflavin as fungicidal rather than merely fungistatic. Growth curve and spot dilution assays confirmed dose-dependent suppression across concentrations of 0.1–0.8 mg/mL, with effects comparable to the fluconazole positive control at 32 µg/mL. Biofilm inhibition assays using XTT reduction showed significant dose-dependent reductions in metabolic activity of both newly forming and pre-established biofilms, with amphotericin B (0.5 µg/mL) used as a positive comparator.
Mechanistic studies demonstrated multi-target effects. Transmission electron microscopy revealed membrane blebbing, cytoplasmic vacuolization, and cell wall thickening in RF-treated cells. Fluorescent staining with FITC-WGA and propidium iodide confirmed cell wall compromise and membrane permeabilization. DCFH-DA flow cytometry showed excessive intracellular ROS accumulation, and eosin Y uptake further supported membrane disruption. RT-qPCR analysis revealed that RF up-regulated cell wall biosynthesis genes CHS3, CDA1, and FKS1, as well as stress-response pathway genes Pkc1 and Mpk1, consistent with a cellular attempt to repair ongoing wall damage. Virulence factor genes CAP59 and Lac1/Lac2 (capsule and melanin pathways) were up-regulated under RF stress, yet functional assays showed significantly reduced capsule thickness and melanin production, suggesting the transcriptional response was insufficient to overcome riboflavin's inhibitory effect. Urease gene Ure1 was transcriptionally down-regulated, correlating with reduced urease activity in phenol-red broth assays.
In vivo efficacy was evaluated in two mouse models: intranasal infection simulating pulmonary cryptococcosis, and intravenous infection simulating disseminated meningitis. Riboflavin treatment significantly reduced colony-forming unit (CFU) counts in lungs, brain, spleen, and kidneys compared to untreated infected controls. Histopathological analysis of lung and brain sections showed markedly reduced inflammatory infiltrates, granuloma burden, and tissue destruction in RF-treated animals. Plasma cytokine profiling at early infection time points revealed significant decreases in IFN-γ, TNF-α, and IL-4 levels in riboflavin-treated mice, suggesting concurrent attenuation of the damaging hyperinflammatory response.
This study provides the first comprehensive evidence that riboflavin exerts fungicidal activity against C. neoformans through membrane disruption, ROS-mediated oxidative stress, and virulence suppression, with translatable in vivo efficacy. As an already-approved, low-cost, well-tolerated micronutrient, riboflavin presents an attractive candidate for drug repurposing in resource-limited settings. However, the work is preclinical and pharmacokinetic/pharmacodynamic studies, dose optimization, and combination therapy trials will be required before clinical application can be considered.
Key Findings
- MIC90 of riboflavin against C. neoformans H99 was 0.4 mg/mL; MFC was 0.8 mg/mL (MFC/MIC ratio = 2), classifying it as fungicidal
- Riboflavin significantly inhibited biofilm formation and dispersed pre-formed biofilms in a dose-dependent manner (0.1–0.8 mg/mL), assessed by XTT metabolic activity
- Flow cytometry with DCFH-DA confirmed excessive intracellular ROS accumulation in RF-treated cells; propidium iodide and eosin Y staining confirmed membrane permeabilization
- RT-qPCR showed up-regulation of cell wall stress genes CHS3, CDA1, FKS1, Pkc1, and Mpk1, while urease gene Ure1 was significantly down-regulated after RF treatment
- Capsule thickness and melanin production were functionally reduced in RF-treated cells despite transcriptional up-regulation of CAP59 and Lac1/Lac2 virulence genes
- In mouse intranasal and intravenous infection models, RF treatment significantly reduced CFU counts in lungs, brain, spleen, and kidneys vs. untreated infected controls
- Plasma levels of IFN-γ, TNF-α, and IL-4 were significantly decreased in RF-treated infected mice at early infection time points, indicating reduced hyperinflammatory response
Methodology
In vitro studies used CLSI M27-A4 broth microdilution against C. neoformans H99 with RF concentrations from 0.025 to 0.8 mg/mL; fluconazole (32 µg/mL) and amphotericin B (0.5 µg/mL) served as positive controls. Virulence and mechanistic assays included India ink capsule staining, L-DOPA melanin plates, phenol-red urease broth, XTT biofilm assays, TEM, FITC-WGA/PI fluorescent staining, DCFH-DA ROS flow cytometry, and RT-qPCR for 8 target genes. In vivo efficacy was tested in two mouse models (intranasal and intravenous C. neoformans infection) with organ CFU counts, histopathology, and multiplex plasma cytokine measurements; all in vitro assays were performed in triplicate.
Study Limitations
The study is entirely preclinical, with no pharmacokinetic data on whether the effective in vitro concentrations (0.4–0.8 mg/mL) are achievable in human plasma or CNS tissue at safe doses. Mouse infection models do not fully recapitulate human immunocompromised states (e.g., advanced HIV/AIDS), limiting direct translational inference. The authors do not report potential conflicts of interest, and no combination therapy experiments with standard-of-care drugs were conducted to assess synergy or antagonism.
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