Disclosing azole resistance mechanisms in resistant Candida glabrata strains encoding wild-type or gain-of-function CgPDR1 alleles through comparative genomics and transcriptomics.

The pathogenic yeast Candida glabrata is intrinsically resilient to azoles and rapidly acquires resistance to these antifungals, in vitro and in vivo. In most cases azole-resistant C. glabrata clinical strains encode hyperactive CgPdr1 variants, however, resistant strains encoding wild-type CgPDR1 alleles have also been isolated, although remaining to be disclosed the underlying resistance mechanism. In this study, we scrutinized the mechanisms underlying resistance to azoles of 8 resistant clinical C. glabrata strains, identified along the course of epidemiological surveys undertaken in Portugal. Seven of the strains were found to encode CgPdr1 gain-of-function variants (I392M, E555K, G558C, and I803T) with the substitutions I392M and I803T being herein characterized as hyper-activating mutations for the first time. While cells expressing the wild-type CgPDR1 allele required the mediator subunit Gal11A to enhance tolerance to fluconazole, this was dispensable for cells expressing the I803T variant indicating that the CgPdr1 interactome is shaped by different gain-of-function substitutions. Genomic and transcriptomic profiling of the sole azole-resistant C. glabrata isolate encoding a wild-type CgPDR1 allele (ISTB218) revealed that under fluconazole stress this strain over-expresses various genes described to provide protection against this antifungal, while also showing reduced expression of genes described to increase sensitivity to these drugs. The overall role in driving the azole-resistance phenotype of the ISTB218 C. glabrata isolate played by these changes in the transcriptome and genome of the ISTB218 isolate are discussed shedding light into mechanisms of resistance that go beyond the CgPdr1-signalling pathway and that may alone, or in combination, pave the way for the acquisition of resistance to azoles in vivo.

An Overview on Conventional and Non-Conventional Therapeutic Approaches for the Treatment of Candidiasis and Underlying Resistance Mechanisms in Clinical Strains.

Fungal infections and, in particular, those caused by species of the Candida genus, are growing at an alarming rate and have high associated rates of mortality and morbidity. These infections, generally referred as candidiasis, range from common superficial rushes caused by an overgrowth of the yeasts in mucosal surfaces to life-threatening disseminated mycoses. The success of currently used antifungal drugs to treat candidiasis is being endangered by the continuous emergence of resistant strains, specially among non-albicans Candida species. In this review article, the mechanisms of action of currently used antifungals, with emphasis on the mechanisms of resistance reported in clinical isolates, are reviewed. Novel approaches being taken to successfully inhibit growth of pathogenic Candida species, in particular those based on the exploration of natural or synthetic chemicals or on the activity of live probiotics, are also reviewed. It is expected that these novel approaches, either used alone or in combination with traditional antifungals, may contribute to foster the identification of novel anti-Candida therapies.

Mechanistic Insights Underlying Tolerance to Acetic Acid Stress in Vaginal Candida glabrata Clinical Isolates.

During colonization of the vaginal tract Candida glabrata cells are challenged with the presence of acetic acid at a low pH, specially when dysbiosis occurs. To avoid exclusion from this niche C. glabrata cells are expected to evolve efficient adaptive responses to cope with this stress; however, these responses remain largely uncharacterized, especially in vaginal strains. In this work a cohort of 18 vaginal strains and 2 laboratory strains (CBS138 and KUE100) were phenotyped for their tolerance against inhibitory concentrations of acetic acid at pH 4. Despite some heterogeneity has been observed among the vaginal strains tested, in general these strains were considerably more tolerant to acetic acid than the laboratory strains. To tackle the mechanistic insights behind this differential level of tolerance observed, a set of vaginal strains differently tolerant to acetic acid (VG281∼VG49 < VG99 < VG216) and the highly susceptible laboratory strain KUE100 were selected for further studies. When suddenly challenged with acetic acid the more tolerant vaginal strains exhibited a higher activity of the plasma membrane proton pump CgPma1 and a reduced internal accumulation of the acid, these being two essential features to maximize tolerance. Based on the higher level of resistance exhibited by the vaginal strains against the action of a ß-1,3-glucanase, it is hypothesized that the reduced internal accumulation of acetic acid inside these strains may originate from them having a different cell wall structure resulting in a reduced porosity to undissociated acetic acid molecules. Both the vaginal and the two laboratory strains were found to consume acetic acid in the presence of glucose indicating that metabolization of the acid is used by C. glabrata species as a detoxification mechanism. The results gathered in this study advance the current knowledge on the mechanisms underlying the increased competitiveness of C. glabrata in the vaginal tract, a knowledge that can be used to guide more suitable strategies to treat infections caused by this pathogenic yeast.

Genome Sequence of the Nonconventional Wine Yeast Hanseniaspora guilliermondii UTAD222.

In this work, we disclose the genome sequence and a corresponding manually curated annotation of the non-Saccharomyces yeast Hanseniaspora guilliermondii UTAD222, a strain shown to have interesting oenological traits for the production of wines with improved aromatic properties.

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.