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The antifungal activity of the laccase inhibitor DMSA against Cryptococcus neoformans is linked to the perturbation of histidine biosynthesis

ABSTRACT: As a globally distributed opportunistic fungus, Cryptococcus neoformans poses a significant threat to human health by causing fatal meningitis, which is associated with high mortality and morbidity rates. Current antifungals—polyenes, flucytosine, and azoles—are limited by toxicity and resistance, underscoring the need for new agents. This study evaluated dimercaptosuccinic acid (DMSA), beginning with its effect on the virulence factor laccase. DMSA showed broad-spectrum activity against standard (H99, B3501) and clinical strains, with MIC values of 32–64 μg/mL. It inhibited laccase (IC₅₀ = 43.98 μM; Km = 0.547 mM with L-dopa) and impaired multiple virulence factors, including capsule formation (43.2% reduction in H99), urease activity (68.6% inhibition in H99 and 53.4% in B3501), as well as biofilm formation and its metabolic activity. In a Galleria mellonella infection model, although survival was not significantly improved, DMSA reduced fungal burden by 30% (P < 0.05), supported by histology and CFU counts. Further transcriptomic and nutrient supplementation assays indicated that the antifungal action of DMSA is mediated through interference with histidine biosynthesis, suggesting a mechanism beyond laccase inhibition.

ORGANISM(S): Cryptococcus neoformans

PROVIDER: GSE307105 | GEO | 2026/08/30

REPOSITORIES: GEO

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Project description:A family of APSES transcription factor is known to be fungal-specific transcriptional regulators and play important roles in governing growth, differentiation, and virulence of diverse fungal pathogens. Yet none of APSES-like transcription factors have been identified and investigated in a basidiomycetous fungal pathogen, Cryptococcus neoformans. In the present study we discovered an APSES-like transcription factor, Msa1 (Mbp1/Swi4-like APSES protein 1), as one of novel flucytosine-responsive genes (total 194 genes) identified through comparative transcriptome analysis of C. neoformans hybrid sensor kinase mutants, tco1 and tco2 mutants, which displayed differential flucytosine-susceptibility. Supporting the microarray data, Northern blot and quantitative RT-PCR analysis confirmed that expression of MSA1 is rapidly induced in response to flucytosine in the wild-type strain, but not in the tco1 and tco2 mutants. Furthermore, C. neoformans with deletion of the MSA1 gene exhibited increased susceptibility to flucytosine. Intriguingly, Msa1 plays pleiotropic roles in diverse cellular process of C. neoformans. Msa1 positively regulates ergosterol biosynthesis and thereby its inhibition confers increased susceptibility and resistance to amphotericin B and azole drugs, respectively. Msa1 is also involved in DNA damage repair counteracting genotoxic stresses. During sexual differentiation Msa1 represses pheromone production, but promotes cell-cell fusion. Furthermore Msa1 is required for production of antioxidant melanin pigment and full virulence of C. neoformans. Finally we also performed DNA microarrray analysis to identify Msa1-regulated genes in C. neoformans. A majority of them were found to be involved in cell cycle regulation and DNA repair. Therefore, this study provides a novel antifungal therapeutic method for treatment of cryptococcosis. There are more than 95% of genome homology between JEC21 and H99. Therefore 24 slides of JEC21 (cryptococcus neoformans var. neoformans serotype D) 70-mer oligo are used in this analysis, 3 biological replicate experiments are performed, total RNAs are extracted under 2 conditions (with or without treatment of Flucytosine) with 4 strains from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), tco1Δ , tco2Δ , skn7Δ), We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye.

Project description:A family of APSES transcription factor is known to be fungal-specific transcriptional regulators and play important roles in governing growth, differentiation, and virulence of diverse fungal pathogens. Yet none of APSES-like transcription factors have been identified and investigated in a basidiomycetous fungal pathogen, Cryptococcus neoformans. In the present study we discovered an APSES-like transcription factor, mbs1 (Mbp1/Swi4-like APSES protein 1), as one of novel flucytosine-responsive genes (total 194 genes) identified through comparative transcriptome analysis of C. neoformans hybrid sensor kinase mutants, tco1 and tco2 mutants, which displayed differential flucytosine-susceptibility. Supporting the microarray data, Northern blot and quantitative RT-PCR analysis confirmed that expression of mbs1 is rapidly induced in response to flucytosine in the wild-type strain, but not in the tco1 and tco2 mutants. Furthermore, C. neoformans with deletion of the mbs1 gene exhibited increased susceptibility to flucytosine. Intriguingly, mbs1 plays pleiotropic roles in diverse cellular process of C. neoformans. mbs1 positively regulates ergosterol biosynthesis and thereby its inhibition confers increased susceptibility and resistance to amphotericin B and azole drugs, respectively. mbs1 is also involved in DNA damage repair counteracting genotoxic stresses. During sexual differentiation mbs1 represses pheromone production, but promotes cell-cell fusion. Furthermore mbs1 is required for production of antioxidant melanin pigment and full virulence of C. neoformans. Finally we also performed DNA microarrray analysis to identify mbs1-regulated genes in C. neoformans. A majority of them were found to be involved in cell cycle regulation and DNA repair. Therefore, this study provides a novel antifungal therapeutic method for treatment of cryptococcosis. total RNAs are extracted 2 strains from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), mbs1Δ), We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye.

Project description:Secreted laccases are important enzymes on an ecological scale for their role in mediating plant-fungal interactions, but their function in fungal pathogenesis has yet to be elucidated. Ascomycete laccases have been primarily associagted with cell wall melanin deposition, and laccase mutants in ascomycete species often demonstrate reduced pigmentation. In this study, a putatively secreted laccase, Sslac2, was characterized from the broad host-range plant pathogen Sclerotinia sclerotiorum, which is largely unpigmented and is not dependent on melanogenesis for plant infection. Of the seven putative laccases in the S. sclerotiorum genome, Sslac2 was the only one found to be highly upregulated during pathogenesis of soybeans and was additionally found to be induced during growth on solid surfaces. Gene knockoues ot Sslac2 demonstrate wide ranging developmental defects, including abolished sclerotial formation, and are functionally non-pathogenic on unwounded tissue. While these mutants demonstrated a clear radial growth defect, enhanced growth was observed in liquid culture, likely due to altered hydrophobicity and thigmotropic responsiveness. Sslac2 mutants were also unable to respond to a host environment, and accordingly unable to differentiate penetration structures, respond appropriately to chemical stress, or produce the key virulence factor oxalic acid. Transmission and scanning electron microscopy of WT and mutant strains show apparent differences in extracellular matrix structure that may explain the inability of the mutant to respond to environmental cues. Targeting Sslac2 using host-induced gene silencing significantly improved resistance to S. sclerotiorum, suggesting that fungal laccases could be a valuable target of disease control. Collectively, we identified a laccase critical to the development and virulence of the broad host-range pathogen S. sclerotiorum and propose a potentially novel role for funal laccases in modulating environmental sensing.

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The antifungal activity of the laccase inhibitor DMSA against Cryptococcus neoformans is linked to the perturbation of histidine biosynthesis

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