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Huangqin Decoction Exerts Antifungal Effects by Interfering with Energy Metabolism of Trichophyton rubrum with Glyoxylate Cycle as a Potential Target

ABSTRACT: Background: The dermatophyte Trichophyton rubrum (T. rubrum) is a highly specialized filamentous fungus that infects keratinized tissues and is the most common pathogen isolated from human dermatophytosis. The clinical treatment of the infections is challenging due to the prolonged treatment duration, limited efficacy, antifungal resistance and adverse effects of available drugs. Huangqin decoction (HQD) is a classical traditional Chinese medicine (TCM) formula, whose inhibitory efficiency against T. rubrum was disclosed in our previous study. However, the responsible mechanisms are not well understood. Aim: This study aimed to elucidate the mode of action of Huangqin decoction (HQD) against T. rubrum as a whole by using transcriptome sequencing combined with verification of qRT-PCR and enzymatic activity assays. Methods: By treating T. rubrum with HQD at minimum inhibitory concentration (MIC), the differentially expressed genes (DEGs) of T. rubrum in response to HQD based on transcriptome profiles were recognized. Gene function analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed functions and signaling pathways modulated by the DEGs. The transcriptome results were validated by quantitative real-time PCR (qRT-PCR). The potential targets were further explored using enzymatic activity assays. Results: At MIC, a total of 338 differentially expressed genes (DEGs) were detected in T. rubrum after HQD exposure compared with the control group (q-value < 0.05), of which 199 genes were significantly up-regulated and 139 genes were down-regulated. Gene function analysis indicated that COG functions of DEGs mainly covered “carbohydrate transport and metabolism” and “energy production and conversion”. The most frequently found GO terms were “metabolic process” and “catalytic activity”. KEGG enrichment analysis revealed that DEGs were predominately enriched in 6 pathways of “valine, leucine and isoleucine degradation”, “indole alkaloid biosynthesis”, “ABC transporters”, “synthesis and degradation of ketone bodies”, “starch and sucrose metabolism”, and “glyoxylate and dicarboxylate metabolism” (q-value < 0.05). Three KEGG pathways were closely related to energy metabolism, which was consistent with the COG and GO results. A subset of eight DEGs by the involvement of the biosynthesis and degradation of cell walls and energy metabolism including amino acid metabolism and the glyoxylate cycle were further confirmed by qRT-PCR and the results verified the transcriptome data. Enzymatic activity assays showed that compared to the control group, activities of citrate synthase (CS), aconitate hydratase (ACO) and isocitrate lyase (ICL) in the glyoxylate cycle were affected by HQD. Conclusion: The overall evidence suggested that the interference of energy metabolism might contribute to the mode of action of HQD against dermatophyte pathogen T. rubrum, and the glyoxylate cycle may be the potential antifungal target of HQD against the fungi. Our study provides a theoretical basis for the mechanisms of HQD upon T. rubrum infections and offers an important insight regarding the glyoxylate cycle especially enzyme isocitrate lyase as indispensable targets of the herbal formulations.

ORGANISM(S): Trichophyton rubrum

PROVIDER: GSE300994 | GEO | 2025/06/28

REPOSITORIES: GEO

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Project description:Trichophyton mentagrophytes (T. mentagrophytes) is a prevalent pathogen that causes human and animal dermatophytosis. The clinical treatment of these infections is challenging due to the prolonged treatment duration, limited efficacy, antifungal resistance and side effects of existing drugs. It has been reported that the classic Traditional Chinese medicine (TCM) prescription Huangqin decoction (HQD) along with its principal ingredients could exhibit antifungal properties. Given the valued advantages of TCM such as broad-spectrum antifungal activity, low incidence of drug resistance and low toxicity, this study investigated the antifungal activity of HQD against T. mentagrophytes and explored the potential inhibitory mechanism, aimed to provide new clues for the treatment of dermatophytosis. By detecting minimal inhibitory concentration (MIC) using the broth microdilution method, the result showed that HQD could significantly inhibit the growth of T. mentagrophytes, with a MIC of 3.13 mg/mL. The transcriptome sequencing and quantitative real-time PCR (qRT-PCR) technology were combined to shed light on the complicated adaptive responses of T. mentagrophytes upon HQD. The results demonstrated that a total of 730 differentially expressed genes (DEGs) were detected in T. mentagrophytes after HQD exposure, of which 547 were up-regulated and 183 were down-regulated. These DEGs were abundant in “single-organism metabolic process”, “catalytic activity” and “oxidoreductase activity”, and were significantly enriched in seven signaling pathways including glutathione metabolism, DNA replication, glyoxylate and dicarboxylate metabolism, taurine and hypotaurine metabolism, carotenoid biosynthesis, ubiquitin mediated proteolysis, and cyanoamino acid metabolism. The results of transcriptome profiling were verified using qRT-PCR for a subset of 10 DEGs. The overall evidence indicated that HQD had a significant anti-dermatophyte activity through the modulation of glutathione metabolism by regulating GST, γ-GT genes, the inhibition of DNA replication pathway by downgrading MCM3, MCM5, RNaseH1 genes, and the repressed expression of PSD gene related to phospholipid synthesis in T. mentagrophytes.

Project description:Although dermatophytes are the most common agents of superficial mycoses in humans and animals, the molecular basis of the pathogenicity of these fungi is largely unknown. In vitro digestion of keratin by dermatophytes is associated with the secretion of multiple proteases, which are assumed to be responsible for their particular specialization to colonize and degrade keratinized host structures during infection. To address this hypothesis a guinea pig infection model was established for the zoophilic dermatophyte Arthroderma benhamiae which causes highly inflammatory cutaneous infections in humans and rodents. Microarray analysis revealed a distinct in vivo protease gene expression profile in the fungal cells, which is surprisingly different from the pattern elicited during in vitro growth on keratin. Instead of the major in vitro expressed proteases others were activated specifically during infection. These enzymes are therefore suggested to fulfill important functions that are not exclusively associated with the degradation of keratin. As the most upregulated in vivo specific A. benhamiae sequence we discovered the gene encoding the serine protease subtilisin 6, which is a known major allergen in the related dermatophyte Trichophyton rubrum and putatively linked to host inflammation. In addition, our approach identified other candidate pathogenicity related factors in A. benhamiae, such as genes encoding key enzymes of the glyoxylate cycle and an opsin-related protein. This first broad transcriptional profiling approach during dermatophyte infection gives new molecular insights into pathogenicity associated mechanisms that make these microorganisms the most successful etiologic agents of superficial mycoses. Keywords: Two-condition experiment, strong proteolytic activity in the supernatant versus no proteolytic activity or infected tissue versus no proteolytic activity Three independently prepared A. benhamiae replicates grown in each of the three media, Sabouraud, soy and keratin-soy medium (designated SabA/B/C, soyA/B/C and keratin-soyA/B/C) were used. ARN from skin samples and fungus together of Guinea Pig infected with A. benhamieae were prepared. Pairwise transcriptional comparisons, i.e. soy versus Sabouraud, keratin-soy versus Sabouraud and Guinea Pig infected versus Sabouraud were done. The total number of slides in this study was 18.

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Huangqin Decoction Exerts Antifungal Effects by Interfering with Energy Metabolism of Trichophyton rubrum with Glyoxylate Cycle as a Potential Target

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