Herein we describe the changes in the gene expression profile of Candida parapsilosis associated with the acquisition of experimentally induced resistance to azole antifungal drugs. Three resistant strains of C. parapsilosis were obtained following prolonged in vitro exposure of a susceptible clinical isolate to constant concentrations of fluconazole, voriconazole, or posaconazole. We found that after incubation with fluconazole or voriconazole, strains became resistant to both azoles but not to posaconazole, although susceptibility to this azole decreased, whereas the strain incubated with posaconazole displayed resistance to the three azoles. The resistant strains obtained after exposure to fluconazole and to voriconazole have increased expression of the transcription factor MRR1, the major facilitator transporter MDR1, and several reductases and oxidoreductases. Interestingly, and similarly to what has been described in C. albicans, upregulation of MRR1 and MDR1 is correlated with point mutations in MRR1 in the resistant strains. The resistant strain obtained after exposure to posaconazole shows upregulation of two transcription factors (UPC2 and NDT80) and increased expression of 13 genes involved in ergosterol biosynthesis. This is the first study addressing global molecular mechanisms underlying azole resistance in C. parapsilosis; the results suggest that similarly to C. albicans, tolerance to azoles involves the activation of efflux pumps and/or increased ergosterol synthesis.Candida parapsilosis is the second most common Candida species isolated from patients with bloodstream infections in Latin America and Asia (46, 60), and it is also commonly found in European surveys (4,17,43,67). It is responsible for a broad variety of clinical manifestations that generally occur in individuals with impaired immune systems, in neutropenic or burn patients, as well as in patients admitted to medical or surgical intensive care units (43), especially pediatric units (26,48).Azoles are the most commonly used drugs for the treatment of Candida infections (13). They target lanosterol 14␣-demethylase, a member of the cytochrome P450 enzymes, which is required for the synthesis of ergosterol (1, 76). Ergosterol is a major and essential lipid constituent of the fungal cell membrane (1). The acquisition of azole resistance, particularly after prolonged exposure, as happens with prophylactic overuse, is a well-known phenomenon in fungi (5,6,29). The widespread use of azole antifungals, especially fluconazole (FLC), resulted in a growing incidence of Candida species in which resistance is easily induced, such as Candida glabrata (75), or species that show intrinsic resistance, such as C. krusei (74). Previous studies with C. albicans (38), C. dubliniensis (59), and C. tropicalis (9) demonstrated that resistance to fluconazole can be promoted following repeated in vitro exposure to the drug. The ability of a drug to induce in vitro resistance suggests that similar mechanisms may also occur in vivo, which may thus became proble...

The risk factors for coronavirus disease 2019 (COVID-19) severity are still poorly understood. Considering the pivotal role of the gut microbiota on host immune and inflammatory functions, we investigated the association between changes in the gut microbiota composition and the clinical severity of COVID-19. We conducted a multicenter cross-sectional study prospectively enrolling 115 COVID-19 patients categorized according to: (1) the WHO Clinical Progression Scale—mild, 19 (16.5%); moderate, 37 (32.2%); or severe, 59 (51.3%), and (2) the location of recovery from COVID-19—ambulatory, 14 (household isolation, 12.2%); hospitalized in ward, 40 (34.8%); or hospitalized in the intensive care unit, 61 (53.0%). Gut microbiota analysis was performed through 16S rRNA gene sequencing, and the data obtained were further related to the clinical parameters of COVID-19 patients. The risk factors for COVID-19 severity were identified by univariate and multivariable logistic regression models. In comparison to mild COVID-19 patients, the gut microbiota of moderate and severe patients have: (a) lower Firmicutes/Bacteroidetes ratio; (b) higher abundance of Proteobacteria; and (c) lower abundance of beneficial butyrate-producing bacteria such as the genera Roseburia and Lachnospira. Multivariable regression analysis showed that the Shannon diversity index [odds ratio (OR) = 2.85, 95% CI = 1.09–7.41, p = 0.032) and C-reactive protein (OR = 3.45, 95% CI = 1.33–8.91, p = 0.011) are risk factors for severe COVID-19 (a score of 6 or higher in the WHO Clinical Progression Scale). In conclusion, our results demonstrated that hospitalized patients with moderate and severe COVID-19 have microbial signatures of gut dysbiosis; for the first time, the gut microbiota diversity is pointed out as a prognostic biomarker of COVID-19 severity.

In a restricted group of opportunistic fungal pathogens the universal leucine CUG codon is translated both as serine (97%) and leucine (3%), challenging the concept that translational ambiguity has a negative impact in living organisms. To elucidate the molecular mechanisms underlying the in vivo tolerance to a nonconserved genetic code alteration, we have undertaken an extensive structural analysis of proteins containing CUG-encoded residues and solved the crystal structures of the two natural isoforms of Candida albicans seryl-tRNA synthetase. We show that codon reassignment resulted in a nonrandom genome-wide CUG redistribution tailored to minimize protein misfolding events induced by the large-scale leucine-to-serine replacement within the CTG clade. Leucine or serine incorporation at the CUG position in C. albicans seryl-tRNA synthetase induces only local structural changes and, although both isoforms display tRNA serylation activity, the leucine-containing isoform is more active. Similarly, codon ambiguity is predicted to shape the function of C. albicans proteins containing CUGencoded residues in functionally relevant positions, some of which have a key role in signaling cascades associated with morphological changes and pathogenesis. This study provides a first detailed analysis on natural reassignment of codon identity, unveiling a highly dynamic evolutionary pattern of thousands of fungal CUG codons to confer an optimized balance between protein structural robustness and functional plasticity.aminoacyl-tRNA synthetase | morphogenesis | mitogen-activated protein kinase pathway | Ras1 | X-ray crystallography G enetic code alterations and ambiguity are widespread in nature even though it is not yet clear how their negative impact is overcome (1). Expansion of the genetic code to selenocysteine (Sec) and pyrrolysine (Pyl) provides, however, a glimpse of advantages that may explain the evolution of codon reassignments under negative selective pressure (2). Sec is inserted into the genetic code of bacteria and eukaryotes by a specific selenocysteyl-tRNA Sec , which places selenocysteine in the catalytic site of selenoproteins increasing their chemical reaction rate relative to cysteine-containing homologues (3). Similarly, Pyl is cotranslationally introduced into the active center of methyltransferases of Methanosarcineace spp. and of Desulfitobacterium hafniense, a symbiont of the gutless worm Olavius algarvensis, where it plays a fundamental role in methane biosynthesis (2, 4). Another interesting case involves the mammalian methionyl-tRNA synthetase (MetRS), which is modified under environmental stress and misacylates noncognate tRNAs with reactive oxygen species (ROS)-scavenging methionine (5), therefore protecting proteins from oxidative damage. A similar adaptive mechanism apparently drove mitochondrial reassignment of Ile AUA codons to methionine (6).In Candida albicans and in most other CTG clade species a mutant serine tRNA (tRNA CAG Ser ) has the peculiarity of decoding leucine CUG codons both as s...