RESUMEN
Dermatophytes are fungal pathogens that cause cutaneous infections such as onychomycosis and athlete's foot in both healthy and immunocompromised patients.Trichophyton rubrum is the most prevalent dermatophyte causing human nail and skin infections worldwide, and because of its anthropophilic nature, animal infection models are limited. The purpose of this work was to compare the expression profile of T. rubrum genes encoding putative virulence factors during growth in ex vivo and in vitro infection models. The efficiency of the ex vivo skin infection model was confirmed by scanning electron microscopy (SEM), which showed that the conidia had produced hyphae that penetrated into the epidermis. Quantitative RT-PCR (qRT-PCR) analysis showed that the expression of some genes is modulated in response to the infection model used, as compared to that observed in cells grown in glucose-containing media. We concluded that ex vivo infection models help assess the molecular aspects of the interaction of T. rubrum with the host milieu, and thus provide insights into the modulation of genes during infection.
Asunto(s)
Interacciones Huésped-Patógeno , Modelos Biológicos , Onicomicosis/microbiología , Trichophyton , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Humanos , Uñas/microbiología , Trichophyton/genética , Trichophyton/patogenicidad , Factores de VirulenciaRESUMEN
BACKGROUND: The dermatophyte Trichophyton rubrum is an anthropophilic filamentous fungus that infects keratinized tissues and is the most common etiologic agent isolated in human dermatophytoses. The clinical treatment of these infections is challenging because only few antifungal drugs are commercially available. To understand the mode of action of cytotoxic drugs against fungi, we evaluated the time-dependent effects of acriflavine on T. rubrum transcriptome using high-throughput RNA-sequencing (RNA-seq) technology. RESULTS: RNA-seq analysis generated approximately 200 million short reads that were mapped to the Broad Institute's Dermatophyte Comparative Database before differential gene expression analysis was performed. By employing a stringent cut-off threshold of -1.5 and 1.5 log2-fold changes in gene expression, a subset of 490 unique genes were found to be modulated in T. rubrum in response to acriflavine exposure. Among the selected genes, 69 genes were modulated at all exposure time points. Functional categorization indicated the putative involvement of these genes in various cellular processes such as oxidation-reduction reaction, transmembrane transport, and metal ion binding. Interestingly, genes putatively involved in the pathogenicity of dermatophytoses were down-regulated suggesting that this drug interferes with the virulence of T. rubrum. Moreover, we identified 159 novel putative transcripts in intergenic regions and two transcripts in intron regions of T. rubrum genome. CONCLUSION: The results provide insights into the molecular events underlying the stress responses of T. rubrum to acriflavine, revealing that this drug interfered with important molecular events involved in the establishment and maintenance of fungal infection in the host. In addition, the identification of novel transcripts will further enable the improvement of gene annotation and open reading frame prediction of T. rubrum and other dermatophyte genomes.
Asunto(s)
Acriflavina/farmacología , Antifúngicos/farmacología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Trichophyton/efectos de los fármacos , Trichophyton/genética , ARN de Hongos/efectos de los fármacos , Análisis de Secuencia de ARN , Trichophyton/patogenicidadRESUMEN
Genomic sequencing of several dermatophyte species has revealed that they show small differences in genetic content and genome organization, although each fungus has adapted to specific niches. Thus, it seemed relevant to compare gene expression between species. Here, we examined the transcription modulation of three ATP-binding cassette (ABC) transporter genes (pdr1, mdr2 and mdr4), which code for membrane transporter proteins in four species of Trichophyton ; T. interdigitale, T. rubrum, T. tonsurans and T. equinum . These fungal species were challenged with sub-lethal doses of griseofulvin, itraconazole, terbinafine and amphotericin B. A mutant strain of T. interdigitale, Δmdr2, was also analysed for the modulation of pdr1 and mdr4 genes to evaluate the possible functional interaction among these three genes. Disruption of the mdr2 gene resulted in the accumulation of high levels of mdr4 transcripts when challenged with griseofulvin, suggesting that the mdr4 gene is compensating for the inactivation of mdr2 by providing resistance to this antifungal. Although the three ABC transporter genes have high homology between the four dermatophytes analysed, it is likely that they have specific functions, suggesting that the action of each drug is dependent on other factors inherent to each species. Our data suggest that these ABC transporter genes act synergistically in dermatophytes, and they may compensate for one another when challenged with antifungal drugs. This may be an important cause of therapeutic failure when treating fungal infections.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Antifúngicos/farmacología , Farmacorresistencia Fúngica Múltiple , Regulación Fúngica de la Expresión Génica , Genes MDR , Trichophyton/efectos de los fármacos , Trichophyton/genética , Transportadoras de Casetes de Unión a ATP/genética , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Transcripción GenéticaRESUMEN
The type IV secretion system (T4SS) is used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Xanthomonas citri subsp. citri contains two copies of the T4SS, one in the chromosome and the other is plasmid-encoded. To understand the conditions that induce expression of the T4SS in Xcc, we analyzed, in vitro and in planta, the expression of 18 ORFs from the T4SS and 7 hypothetical flanking genes by RT-qPCR. As a positive control, we also evaluated the expression of 29 ORFs from the type III secretion system (T3SS), since these genes are known to be expressed during plant infection condition, but not necessarily in standard culture medium. From the 29 T3SS genes analyzed by qPCR, only hrpA was downregulated at 72 h after inoculation. All genes associated with the T4SS were downregulated on Citrus leaves 72 h after inoculation. Our results showed that unlike the T3SS, the T4SS is not induced during the infection process.