Leonel João Pais Pereira

Synthesis, characterization and antifungal activity of chemically and fungal‐produced silver nanoparticles against Trichophyton rubrum

To characterize and explore the potential in extracellular biosynthesis of silver nanoparticles (AgNPs) by Penicillium chrysogenum and Aspergillus oryzae and to investigate the antifungal effect of chemically vs biologically synthesized AgNPs comparing with conventional antifungal drugs against Trichophyton rubrum.

Effect of progesterone on candida albicans vaginal pathogenicity

Candida albicans is responsible for the majority of cases of vulvovaginal candidiasis (VVC), an infection which occurs mainly during the luteal phase of the menstrual cycle or during the pregnancy, when levels of progesterone are elevated. One of the most important candidal virulence factors is the ability to adhere to host surfaces and form biofilms. The aim of this study was to determine the influence of progesterone on C. albicans virulence, namely biofilm formation and colonisation/invasion of a reconstituted human vaginal epithelium (RHVE). Biofilm formation on the RHVE was evaluated by enumeration of culturable cells, total mass quantification and scanning electron microscopy. The capacity of C. albicans strains to invade and colonise the tissue was examined by fluorescence microscopy using species-specific peptide nucleic acid (PNA) probe hybridisation, and quantitatively evaluated by RT-PCR Candida quantification methodology. Furthermore, gene (BCR1 and HWP1) expression of biofilm and RHVE-colonising cells was evaluated by quantitative RT-PCR. Results confirmed that progesterone reduced the capacity of C. albicans strains to form biofilms and to colonise and invade RHVE. Additionally, it was demonstrated that progesterone decreased expression of BCR1 and HWP1, which are important virulence determinants of C. albicans. In conclusion, it was evident that progesterone can have a major influence on C. albicans pathogenicity on vaginal epithelial cells and may partly explain susceptibility of women to VVC at different stages of the menstrual cycle.

Polyphasic identification and preservation of fungal diversity : concepts and applications

Fungi are a diverse group of unique eukaryotic organisms currently accepted as the Eumycota kingdom. The (under) estimated number of fungal species is 1.5 × 106 of which only a small number have been identified (ca. 8–10%). They are ubiquitous in nature with an extraordinary ability to decompose plant wastes while also causing much spoilage of food and other relevant commodities. Certain species are used directly as food and others in the manufacture of foodstuffs, antibiotics, enzymes, organic acids and alcohol. Still others can infect humans, animals and crops. Information about each microorganism (e.g. morphological and molecular descriptions, including modern spectral data – MALDI-TOF MS, physiological and biochemical features, ecological roles, and societal risks or benefits) is the key element in fungal identification. In order to attain a sound fungal identification a polyphasic approach is required. It is achieved through the integration of all biological traits data. Fungal service culture collections have well established management systems and preservation techniques that are of elemental importance and guarantee the proper identification and characterisation of environmental fungal isolates. They also assure the continuity of taxonomic and comparative studies and fungal availability for biotechnological exploitation. To foster bio-economy and sustain the biotechnological developments new demands for quality control of fungal holdings preserved in culture collections are in course. The quality control system is associated with new guidelines for the culture collections to operate at global level and to adapt the traditional fungal repositories into the new OECD concept of Biological Resource Centres (BRCs).

The CgHaa1-Regulon Mediates Response and Tolerance to Acetic Acid Stress in the Human Pathogen Candida glabrata

To thrive in the acidic vaginal tract, Candida glabrata has to cope with high concentrations of acetic acid. The mechanisms underlying C. glabrata tolerance to acetic acid at low pH remain largely uncharacterized. In this work, the essential role of the CgHaa1 transcription factor (encoded by ORF CAGL0L09339g) in the response and tolerance of C. glabrata to acetic acid is demonstrated. Transcriptomic analysis showed that CgHaa1 regulates, directly or indirectly, the expression of about 75% of the genes activated under acetic acid stress. CgHaa1-activated targets are involved in multiple physiological functions including membrane transport, metabolism of carbohydrates and amino acids, regulation of the activity of the plasma membrane H+-ATPase, and adhesion. Under acetic acid stress, CgHaa1 increased the activity and the expression of the CgPma1 proton pump and contributed to increased colonization of vaginal epithelial cells by C. glabrata. CgHAA1, and two identified CgHaa1-activated targets, CgTPO3 and CgHSP30, are herein demonstrated to be determinants of C. glabrata tolerance to acetic acid. The protective effect of CgTpo3 and of CgHaa1 was linked to a role of these proteins in reducing the accumulation of acetic acid inside C. glabrata cells. In response to acetic acid stress, marked differences were found in the regulons controlled by CgHaa1 and by its S. cerevisiae ScHaa1 ortholog, demonstrating a clear divergent evolution of the two regulatory networks. The results gathered in this study significantly advance the understanding of the molecular mechanisms underlying the success of C. glabrata as a vaginal colonizer.

The use of MALDI-TOF ICMS as an alternative tool for Trichophyton rubrum identification and typing

AIMS In this study, the potential of matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry (MALDI-TOF ICMS) was investigated for the identification of clinical isolates. The isolates were analyzed at the species and strain level. METHODS Spectral identification by MALDI-TOF ICMS was performed for all strains, and compared with the results of sequencing of the internal transcribed spacers (ITS1 and ITS2), and the 5.8S rDNA region. PCR fingerprinting analysis using primers M13, (GACA)4, and (AC)10 was performed in order to assess the intra-specific variability of Trichophyton rubrum strains. RESULTS The identification of strains at species level by MALDI-TOF ICMS was in agreement with the previously performed morphological and biochemical analysis. Sequence data confirmed spectral mass identification at species level. Intra-specific variability was assessed. Within the T. rubrum cluster, strains were distributed into smaller highly related sub-groups with a similarity values above 85%. CONCLUSIONS MALDI-TOF ICMS was shown to be a rapid, low-cost and accurate alternative tool for the identification and strain typing of T. rubrum.