Gene Expression Response of<i>Trichophyton rubrum</i>during Coculture on Keratinocytes Exposed to Antifungal Agents

Komoto, Tatiana Takahasi; Bitencourt, Tamires Aparecida; Silva, Gabriel; Beleboni, Renê Oliveira; Marins, Mozart; Fachin, Ana Lúcia

doi:10.1155/2015/180535

Cited by 28 publications

(27 citation statements)

References 42 publications

(46 reference statements)

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“…RNA extraction and cDNA synthesis were performed according to Komoto et al, 22 using the Illustrate RNAspin Mini RNA Isolation kit (GE Healthcare). Complementary DNA was synthesised from 2 μg of total RNA in a 20‐μL reaction volume using the RevertAID H Minus First Strand cDNA synthesis kit (Fermentas ® ).…”

Section: Methodsmentioning

confidence: 99%

Expression of genes containing tandem repeat patterns involved in the fungal‐host interaction and in the response to antifungals inTrichophyton rubrum

Abreu

Bitencourt²,

Franco

et al. 2020

Mycoses

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Background: Trichophyton rubrum is the most common aetiological agent of human dermatophytoses. These infections mainly occur in keratinised layers such as skin, hair and nails because the fungus uses keratin as a nutrient source. Fluconazole and amphotericin are antifungal agents most commonly used to treat dermatophytoses and acts on cell membrane ergosterol. Despite the clinical importance of T rubrum, the mechanisms underlying the fungal-host relationship have not yet been clarified. Tandem repeats (TRs) are short DNA sequences that are involved in a variety of adaptive functions, including the process of fungal infection. It is known that the larger the number of TRs in the genome, the greater the capacity of cell-cell junction and surface adhesion, especially when these repeats are present in regions encoding cell surface proteins. Objectives: To identify in silico T rubrum genes containing TR patterns and to analyse the modulation of these genes in culture medium containing keratin (a model simulating skin infection) and antifungal drugs. Methods: The Dermatophyte Tandem Repeats Database (DTRDB) and the FaaPred tool were used to identify four T rubrum genes containing TR patterns. Quantitative real-time (RT) PCR was used to evaluate the gene expression during the growth of T rubrum on keratin and in the presence of fluconazole, amphotericin B and Congo red (acts in the cell wall).Results: The expression of these genes was found to be induced in culture medium containing keratin. In addition, these genes were induced in the presence of antifungal agents, especially fluconazole, indicating an adaptive response to the stress caused by this drug. Conclusion:The results suggest an important role of genes containing TRs in the fungal-host interaction and in the susceptibility to inhibitory compounds, indicating these sequences as new potential targets for the development of antifungal agents. K E Y W O R D S adhesin, cell wall proteins, fluconazole, fungal infection, gene expression | 611 de ABReU et Al.

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Section: Methodsmentioning

confidence: 99%

Expression of genes containing tandem repeat patterns involved in the fungal‐host interaction and in the response to antifungals inTrichophyton rubrum

Abreu

Bitencourt²,

Franco

et al. 2020

Mycoses

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“…The plates were incubated at 28 • C for 72 h, and fungal growth was assessed by light microscopy (Leica DMI3000B). The human keratinocytes (HaCaT) cell line was cultured, as previously described (Komoto et al, 2015). The coculture assay was performed using 2 × 10 6 conidia/mL and 2.5 10 5 keratinocyte cells/mL.…”

Section: Coculture and Nail Infectionmentioning

confidence: 99%

HacA Governs Virulence Traits and Adaptive Stress Responses in Trichophyton rubrum

et al. 2020

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The ability of fungi to sense environmental stressors and appropriately respond is linked to secretory system functions. The dermatophyte infection process depends on an orchestrated signaling regulation that triggers the transcription of genes responsible for adherence and penetration of the pathogen into host-tissue. A high secretion system is activated to support the host-pathogen interaction and assures maintenance of the dermatophyte infection. The gateway of secretion machinery is the endoplasmic reticulum (ER), which is the primary site for protein folding and transport. Current studies have shown that ER stress that affects adaptive responses is primarily regulated by UPR and supports fungal pathogenicity; this has been assessed for yeasts and Aspergillus fumigatus, in regard to how these fungi cope with host environmental stressors. Fungal UPR consists of a transmembrane kinase sensor (Ire1/IreA) and a downstream target Hac1/HacA. The active form of Hac is achieved via non-spliceosomal intron removal promoted by endonuclease activity of Ire1/IreA. Here, we assessed features of HacA and its involvement in virulence and susceptibility in Trichophyton rubrum. Our results showed that exposure to antifungals and ER-stressing agents initiated the activation of HacA from T. rubrum. Interestingly, the activation occurs when a 20 nt fragment is removed from part of the exon-2 and part of intron-2, which in turn promotes the arisen of the DNA binding site motif and a dimer interface domain. Further, we found changes in the cell wall and cellular membrane composition in the hacA mutant as well as an increase in susceptibility toward azole and cell wall disturbing agents. Moreover, the hacA mutant presented significant defects in important virulence traits like thermotolerance and growth on keratin substrates. For instance, the development of the hacA mutant was impaired in co-culture with keratinocytes or human nail fragments. Changes in the pro-inflammatory cytokine release were verified for the hacA mutant during the co-culture assay, which might be related to differences in pathogen-associated molecular patterns (PAMPs) in the cell wall. Together, these results suggested that HacA is an integral part of T. rubrum physiology and virulence, implying that it is an important molecular target for antidermatophytic therapy.

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“…Trichophyton rubrum, the most prevalent dermatophyte, is attributed to 80% of dermatomycoses affecting keratinized tissues of humans, and so they are anthropophilic. It uses keratin as a nutrient during scalp, skin, and nail infections (Komoto et al, 2015). In cutaneous lesions, the pathogen adheres and may penetrate the host tissue to scavenge nutrient macromolecules as carbon, nitrogen, phosphorus, and sulfur sources.…”

Section: Introductionmentioning

confidence: 99%

Preparation, and Assessment of Antidermatophyte Activity of Miconazole–Urea Water-Soluble Film

2020

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Cutaneous mycoses, particularly tinea pedis caused by Trichophyton rubrum, are commonly known infections in humans. They are still considered as a major public health problem worldwide affecting the quality of life due to prolonged period of treatment and development of drug resistance, which leads to recurrence of infections. The objective of our study was to assess the effectiveness of miconazole in the presence and absence of urea, as a penetration enhancer, against T. rubrum and to formulate both of them in a water-soluble film to be applied topically for the purpose of treating tinea pedis caused by this fungus. Drug combination revealed synergism where miconazole minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) (0.5 and 1 mg/L) were considerably declined to 0.001 and 0.004 mg/L, respectively, when combined with 20% urea. This enhanced drug interaction activity against the test strain was explained by the alterations raised on the morphology and ultrastructures observed microscopically. Minimal fungicidal dose of miconazole/urea combination displayed plasmolysis and shrink cytoplasm; however, necrotic cells with punctured walls and degraded cytoplasmic content were observed at high fungicidal dose. Watersoluble films, prepared using increasing values of miconazole MFC and urea, were transparent, smooth, uniform, and flexible. Their physicochemical characters showed homogeneity in weight, thickness, drug content, and folding endurances with normal surface pH values, indicating the reproducibility of the preparation method. The novel simulation model for the film mechanism of action supported the idea and the suggested application method of the new dosage form. Evaluation of these films was carried in vitro using disk diffusion assay as well as in vivo using guinea pig dermatophytosis model. The in vitro assessment revealed an increase in the inhibition zone diameters in a concentration-dependent manner upon using 10 or 20% of urea combined with miconazole. In vivo test showed that combination of 0.004 mg/L miconazole with 20% urea (M + U20) showed the highest efficacy percentage (95.83%), which was statistically superior to the infected untreated control (p < 0.001) in fungal burden reduction as well as improvement in clinical scores (p < 0.001). This work supports the hypothesis and suggests a new promising dosage form for the treatment of T. rubrum infections.

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Gene Expression Response ofTrichophyton rubrumduring Coculture on Keratinocytes Exposed to Antifungal Agents

Cited by 28 publications

References 42 publications

Expression of genes containing tandem repeat patterns involved in the fungal‐host interaction and in the response to antifungals inTrichophyton rubrum

Expression of genes containing tandem repeat patterns involved in the fungal‐host interaction and in the response to antifungals inTrichophyton rubrum

HacA Governs Virulence Traits and Adaptive Stress Responses in Trichophyton rubrum

Preparation, and Assessment of Antidermatophyte Activity of Miconazole–Urea Water-Soluble Film

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