Relevance of Nutrient-Sensing in the Pathogenesis of Trichophyton rubrum and Trichophyton interdigitale.

The growth and development of organisms depend on nutrient availability. Dermatophytes must sense nutrient levels and adapt to the host environment to colonize human and animal keratinized tissues. Owing to the clinical importance of the Trichophyton genus, this study compared the expression profile of genes involved in metabolism, cell cycle control, and proteases in two Trichophyton species, Trichophyton rubrum, and Trichophyton interdigitale, in response to nutrients and environmental pH. In addition, we evaluated the activity of enzymes in the tricarboxylic acid, glyoxylate, and methylcitrate cycles. Moreover, the effects of interruption of the transcription factor pacC on T. interdigitale in the same conditions as for the wild-type strain were determined. Our analyses revealed specific responses in each species to the nutritional and pH variation. An improved adaptation of T. interdigitale to keratin was observed, compared with that of T. rubrum. T. rubrum growth in buffered keratin media indicated pH 8.0 as an optimal pH condition for metabolic activity, which differed from that for T. interdigitale. Tricarboxylic acid components in T. rubrum showed increased enzymatic activity and transcript accumulation. In T. interdigitale, a higher activity of enzymes in glyoxylate and methylcitrate cycles was observed, with no direct correlation to the transcriptional profile. T. interdigitale fungal metabolism suggests the requirement of anaplerotic pathways in the late cultivation period. The identified differences between T. rubrum and T. interdigitale may represent determinants for adaptation to the host and the incidence of infection with each species.

STE20/PAKA Protein Kinase Gene Releases an Autoinhibitory Domain through Pre-mRNA Alternative Splicing in the Dermatophyte Trichophyton rubrum.

Signaling pathways are highly diverse in filamentous fungi, allowing the cells to receive and process ambient information. Interaction of components from different pathways results in signaling networks. The mitogen-activated protein kinase (MAPK) pathway is dependent on phosphorylation that is accomplished by kinase proteins. Thus, the STE/PAK protein kinase family plays essential roles in MAPK signal transduction, regulating several cellular functions. The STE/PAK protein displays an autoinhibitory (Cdc42/Rac interactive binding-CRIB) domain on its N-terminal portion, which interacts with the C-terminal catalytic kinase domain. Based on current knowledge, for the STE/PAK kinase to be activated, molecular signals (e.g., interaction with the activated form of Rac1 and Cdc42 proteins) or proteolytic cleavage by caspase 3 is necessary. Both mechanisms release the kinase domain from the CRIB interaction. Here, we hypothesize a novel molecular mechanism for the activation of STE20/PAKA kinase in Trichophyton rubrum based on an alternative pre-mRNA splicing process. Our data suggest that, because of the retention of intron 1 of this gene, it is theoretically possible that the translation of STE20/PAKA kinase will be free of its autoinhibitory CRIB domain. These findings indicate a rapid response system to environmental changes. Furthermore, STE20/PAKA may be a potential T. rubrum virulence factor and an interesting target for new drugs against dermatophytes.

Transcriptome-wide survey of gene expression changes and alternative splicing in Trichophyton rubrum in response to undecanoic acid.

While fatty acids are known to be toxic to dermatophytes, key physiological aspects of the Trichophyton rubrum response to undecanoic acid (UDA), a medium chain saturated fatty acid (C11:0), are not well understood. Thus, we analysed RNA-seq data from T. rubrum exposed to sub-lethal doses of UDA for 3 and 12 h. Three putative pathways were primarily involved in UDA detoxification: lipid metabolism and cellular membrane composition, oxidative stress, and pathogenesis. Biochemical assays showed cell membrane impairment, reductions in ergosterol content, and an increase in keratinolytic activity following UDA exposure. Moreover, we assessed differential exon usage and intron retention following UDA exposure. A key enzyme supplying guanine nucleotides to cells, inosine monophosphate dehydrogenase (IMPDH), showed high levels of intron 2 retention. Additionally, phosphoglucomutase (PGM), which is involved in the glycogen synthesis and degradation as well as cell wall biosynthesis, exhibited a significant difference in exon 4 usage following UDA exposure. Owing to the roles of these enzymes in fungal cells, both have emerged as promising antifungal targets. We showed that intron 2 retention in impdh and exon 4 skipping in pgm might be related to an adaptive strategy to combat fatty acid toxicity. Thus, the general effect of UDA fungal toxicity involves changes to fungal metabolism and mechanisms for regulating pre-mRNA processing events.

Pre-mRNA splicing is modulated by antifungal drugs in the filamentous fungus Neurospora crassa.

For this study, we sought to identify pre-mRNA processing events modulated by changes in extracellular pH, inorganic phosphate, and antifungal drugs. We examined genes with at least four putative introns whose transcriptional level responded to these effectors. We showed that the intron retention levels of genes encoding asparagine synthetase 2, C6-zinc finger regulator (fluffy), and a farnesyltransferase respond to amphotericin B, ketoconazole, and other effectors. In general, the assayed antifungals promoted the disruption of the structural domains of these proteins probably leading to their inactivation, which emphasize the complexity of the metabolic modulation exerted by antifungal signaling.

Ambient pH sensing in filamentous fungi: pitfalls in elucidating regulatory hierarchical signaling networks.

In this article, the experiments used to construct the ambient pH-signaling network involved in the secretion of enzymes by filamentous fungi have been reviewed, focusing on the phosphate-repressible phosphatases in Aspergillus nidulans. Classic and molecular genetics have been used to demonstrate that proteolysis of the transcription factor PacC at alkaline ambient pH is imperative for its action, implying that the full-length version is not an active molecular form of PacC. It has been hypothesized that the transcriptional regulator PacC may be functional at both acidic and alkaline ambient pH, in either the full-length or the proteolyzed form, if it carries a pal-dependent molecular tag. The products of the pal genes are involved in a metabolic pathway that led to the synthesis of effector molecules that tag the pacC product, perhaps facilitating its proteolysis.

Transcription of N- and O-linked mannosyltransferase genes is modulated by the pacC gene in the human dermatophyte Trichophyton rubrum.

In fungi, ambient pH sensing involves the activation of the Pal/PacC signalling pathway. In the dermatophyte Trichophyton rubrum, pH-dependent secretion of keratinases, which are major virulence determinants, is affected by disruption of the pacC gene. Here, the transcription profiling of the genes coding for N- and O-linked mannosyltransferases, enzymes involved in protein glycosylation, was evaluated in T. rubrum in response to disruption of the pacC gene and growth in keratin, glucose, and glucose plus glycine. We show that transcription of these mannosyltransferase genes is affected by nutrients at acidic pH and by PacC.