Antifungal effects of a 1,3,4-thiadiazole derivative determined by cytochemical and vibrational spectroscopic studies.

Compounds belonging to the group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diols exhibit a broad spectrum of biological activity, including antibacterial, antifungal, and anticancer properties. The mechanism of the antifungal activity of compounds from this group has not been described to date. Among the large group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diol derivatives, the compound 4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol, abbreviated as C1, was revealed to be one of the most active agents against pathogenic fungi, simultaneously with the lowest toxicity to human cells. The C1 compound is a potent antifungal agent against different Candida species, including isolates resistant to azoles, and molds, with MIC100 values ranging from 8 to 96 µg/ml. The antifungal activity of the C1 compound involves disruption of the cell wall biogenesis, as evidenced by the inability of cells treated with C1 to maintain their characteristic cell shape, increase in size, form giant cells and flocculate. C1-treated cells were also unable to withstand internal turgor pressure causing protoplast material to leak out, exhibited reduced osmotic resistance and formed buds that were not covered with chitin. Disturbances in the chitin septum in the neck region of budding cells was observed, as well as an uneven distribution of chitin and ß(1→3) glucan, and increased sensitivity to substances interacting with wall polymerization. The ATR-FTIR spectral shifts in cell walls extracted from C. albicans cells treated with the C1 compound suggested weakened interactions between the molecules of ß(1→3) glucans and ß(1→6) glucans, which may be the cause of impaired cell wall integrity. Significant spectral changes in the C1-treated cells were also observed in bands characteristic for chitin. The C1 compound did not affect the ergosterol content in Candida cells. Given the low cytotoxicity of the C1 compound to normal human dermal fibroblasts (NHDF), it is possible to use this compound as a therapeutic agent in the treatment of surface and gastrointestinal tract mycoses.

Triintsin, a human pathogenic fungus-derived defensin with broad-spectrum antimicrobial activity.

Since there is a symbiotic and competitive relationship between microorganisms in the same ecological niche, fungal defensins have been found to be important resources for antimicrobial peptides. Here, a fungal defensin, triintsin, was characterized in a clinical isolate of Trichophyton interdigitale from a patient with onychomycosis. The comparison of its genomic and mRNA sequences showed the gene organization and structure of three coding exons separated by two introns. The precursor peptide of triintsin contained 85 amino acid residues, which were composed of three parts including an N-terminal signal domain of 21 residues, a pro-peptide of 47 residues that ended at lysine-arginine and a mature peptide of 38 residues at the C-terminus. The 3D-structure established by homology modeling revealed that triintsin presented a representative typical cysteine-stabilized α-helical and ß-sheet fold. The reductive linear peptide of triintsin was obtained by chemical synthesis. After cyclization to form three pairs of disulfide bonds, the oxidative-type peptide displayed broad-spectrum antimicrobial activity against both gram-positive and gram-negative bacteria but also showed anti-fungal activity. Moreover, triintsin can effectively inhibit the growth of clinical strains. Altogether, the peptide is a human pathogenic fungus-derived defensin with broad-spectrum antimicrobial activity.

Nail Raman spectroscopy: A promising method for the diagnosis of onychomycosis. An ex vivo pilot study.

Trichophyton rubrum and Candida species comprise the majority of onychomycosis pathogens. The aim of this study was to evaluate Raman spectroscopy for the differentiation between healthy and either T. rubrum or Candida infected nails. Raman measurements were performed on clippings (N = 52) infected either by T. rubrum (N = 12) or Candida species (N = 14; C. parapsilosis (sensu lato): N = 11, C. glabrata: N = 1, C. albicans: N = 2) with healthy nails (N = 26) used as controls. Systematic spectral differences were observed in the 500-520 cm-1 band region, attributable to a diverting imprint of the disulfide stretching of cystine and cysteine residues among samples. Particularly, Candida infected nails demonstrated a shoulder at 519 cm-1, corresponding to the signal of the less stable gauche-gauche-trans conformation of the disulfide bond. Two additional bands at 619 and 648 cm-1, corresponding to the C-S stretching vibration, were more evident in the T. rubrum infected nails. Finally, a Raman band at 1550 cm-1, attributable to amide II and tryptophan (Trp) content, was undetectable in Candida infected nails. Using principal component analysis (PCA), efficient differentiation of healthy, T. rubrum and Candida species infected nails was achieved. Soft independent modeling of class analogy (SIMCA) and partial least squares-discriminant analysis (PLS-DA) were further applied to generate diagnostic algorithms for the classification of Raman spectra. Both techniques succeeded in modeling clinical nail samples in three groups according to their mycological categories. Raman spectroscopy is a promising method for the differentiation of healthy vs. diseased nails, including efficient differentiation between onychomycosis caused by T. rubrum and Candida species.

Proteogenomic Analysis of Trichophyton rubrum Aided by RNA Sequencing.

Infections caused by dermatophytes, Trichophyton rubrum in particular, are among the most common diseases in humans. In this study, we present a proteogenomic analysis of T. rubrum based on whole-genome proteomics and RNA-Seq studies. We confirmed 4291 expressed proteins in T. rubrum and validated their annotated gene structures based on 35 874 supporting peptides. In addition, we identified 323 novel peptides (not present in the current annotated protein database of T. rubrum) that can be used to enhance current T. rubrum annotations. A total of 104 predicted genes supported by novel peptides were identified, and 127 gene models suggested by the novel peptides that conflicted with existing annotations were manually assigned based on transcriptomic evidence. RNA-Seq confirmed the validity of 95% of the total peptides. Our study provides evidence that confirms and improves the genome annotation of T. rubrum and represents the first survey of T. rubrum genome annotations based on experimental evidence. Additionally, our integrated proteomics and multisourced transcriptomics approach provides stronger evidence for annotation refinement than proteomic data alone, which helps to address the dilemma of one-hit wonders (uncertainties supported by only one peptide).