Fusarium:Molecular Diversity and Intrinsic Drug Resistance

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Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency

A leading pharmacological strategy toward HIV cure requires "shock" or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription

Phylogeny, diagnostics and antifungal susceptibility of clinically relevant Fusarium species

During the last decades, human infections by Fusarium species (Fungi) have shown global increase, both in immunocompromised and in immunocompetent patients. These infections can be classified in four classes: (1) superficial infections of skin and nails, (2) keratitis of the cornea, and (3) deep localized and (4) disseminated infections. In this PhD project we addressed some of the major problems connected to human fusarioses. (1) We first identified and delimited the species that are etiological agents of different types of infections. Our molecular and phylogenetic studies were performed by using multi-gene analysis of BT, TEF1, TOPO1, PGK, RPB2 and ITS. The results confirmed that Fusarium is monophyletic. (2) Subsequently we aimed to understand which properties are essential in virulence of these opportunists, given the fact that the great majority of their saprobic and plant-pathogenic relatives are not known to be involved in human disease. (3) Furthermore, we developed diagnostic tools for the clinical laboratory. Knowing that current culture-dependent characterization methods for deep infections are often too slow, we introduced faster DNA-based or whole cell-based identification techniques such as MALDI-TOF MS to characterize these species. MALDI-TOF MS approach can be performed with minimal amounts of sample and takes only 15−30 minutes and are cheap. AFLP fingerprinting has successfully been applied in this study with a large numbers of clinical and environmental strains. Our findings indicate that AFLP analysis and MLSA analysis provide high resolution data allowing discrimination between Fusarium species and genotypes. (4) Finally, once etiological agents have been properly recognized and identified, we tested susceptibility profiles of most of opportunistic Fusarium against a panel of anitifungal compounds. In vitro antifungal testing showed that amphotericin B and voriconazole were the antimycotics with the best overall in vitro activity, followed by posaconazole. In vitro combination activity of natamycin alone and in combination with voriconazole was judged optimal for Fusarium keratitis and showed 70% synergism

Species distinction in the Trichophyton rubrum complex

PubMedID: 31189587The Trichophyton rubrum species complex comprises commonly encountered dermatophytic fungi with a worldwide distribution. The members of the complex usually have distinct phenotypes in culture and cause different clinical symptoms, despite high genome similarity. In order to better delimit the species within the complex, molecular, phenotypic, and physiological characteristics were combined to reestablish a natural species concept. Three groups, T. rubrum, T. soudanense, and T. violaceum, could be distinguished based on the sequence of the internal transcribed spacer (ITS) ribosomal DNA barcode gene. On average, strains within each group were similar by colony appearance, microscopy, and physiology, but strains between groups showed significant differences. Trichophyton rubrum strains had higher keratinase activity, whereas T. violaceum strains tended to be more lipophilic; however, none of the phenotypic features were diagnostic. The results of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and amplified fragment length polymorphism (AFLP) were partially consistent with the ITS data but failed to distinguish the species unambiguously. Despite their close similarity, T. violaceum, T. soudanense, and T. rubrum can be regarded as independent species with distinct geographical distributions and clinical predilections. Trichophyton soudanense is pheno- and genotypically intermediate between T. rubrum and T. violaceum. For routine diagnostics, ITS sequencing is recommended. Copyright © 2019 American Society for Microbiology. All Rights Reserved