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Previous reports have demonstrated that the peptide derived from LfcinB, R-1-R, exhibits anti-Candida activity, which is enhanced when combined with an extract from the Bidens pilosa plant. However, the mechanism of action remains unexplored. In this research, a proteomic study was carried out, followed by a bioinformatic analysis and biological assays in both the SC5314 strain and a fluconazole-resistant isolate of Candida albicans after incubation with R-1-R. The proteomic data revealed that treatment with R-1-R led to the up-regulation of most differentially expressed proteins compared to the controls in both strains. These proteins are primarily involved in membrane and cell wall biosynthesis, membrane transport, oxidative stress response, the mitochondrial respiratory chain, and DNA damage response. Additionally, proteomic analysis of the C. albicans parental strain SC5314 treated with R-1-R combined with an ethanolic extract of B. pilosa was performed. The differentially expressed proteins following this combined treatment were involved in similar functional processes as those treated with the R-1-R peptide alone but were mostly down-regulated (data are available through ProteomeXchange with identifier PXD053558). Biological assays validated the proteomic results, evidencing cell surface damage, reactive oxygen species generation, and decreased mitochondrial membrane potential. These findings provide insights into the complex antifungal mechanisms of the R-1-R peptide and its combination with the B. pilosa extract, potentially informing future studies on natural product derivatives. [ABSTRACT FROM AUTHOR]

Candida albicans Prn1 is a protein with an unknown function similar to mammalian Pirin. It also has orthologues in other pathogenic fungi, but not in Saccharomyces cerevisiae. Prn1 highly increases its abundance in response to H2O2 treatment; thus, to study its involvement in the oxidative stress response, a C. albicans prn1∆ mutant and the corresponding wild-type strain SN250 have been studied. Under H2O2 treatment, Prn1 absence led to a higher level of reactive oxygen species (ROS) and a lower survival rate, with a higher percentage of death by apoptosis, confirming its relevant role in oxidative detoxication. The quantitative differential proteomics studies of both strains in the presence and absence of H2O2 indicated a lower increase in proteins with oxidoreductase activity after the treatment in the prn1∆ strain, as well as an increase in proteasome-activating proteins, corroborated by in vivo measurements of proteasome activity, with respect to the wild type. In addition, remarkable differences in the abundance of some transcription factors were observed between mutant and wild-type strains, e.g., Mnl1 or Nrg1, an Mnl1 antagonist. orf19.4850, a protein orthologue to S. cerevisiae Cub1, has shown its involvement in the response to H2O2 and in proteasome function when Prn1 is highly expressed in the wild type. [ABSTRACT FROM AUTHOR]

Pst1p was previously identified as a protein secreted by yeast regenerating protoplasts, which suggests a role in cell wall construction. ECM33 encodes a protein homologous to Pst1p, and both of them display typical features of GPI-anchored proteins and a characteristic receptor L-domain. Pst1p and Ecm33p are both localized to the cell surface, Pst1p being at the cell membrane and possibly also in the periplasmic space. Here, the characterization of pst1[DELTA], ecm33[DELTA] and pst1[DELTA] ecm33[DELTA] mutants is described. Deletion of ECM33 leads to a weakened cell wall, and this defect is further aggravated by simultaneous deletion of PST1. As a result, the ecm33[DELTA] mutant displays increased levels of activated Slt2p, the MAP kinase of the cell integrity pathway, and relies on a functional Slt2-mediated cell integrity pathway to ensure viability. Analyses of model glycosylated proteins show glycosylation defects in the ecm33[DELTA] mutant. Ecm33p is also important for proper cell wall ultrastructure organization and, furthermore, for the correct assembly of the mannoprotein outer layer of the cell wall. Pst1p seems to act in the compensatory mechanism activated upon cell wall damage and, in these conditions, may partially substitute for Ecm33p.

Ecm33p is a widely distributed fungal protein with functional relevance, clearly demonstrated by ecm33[DELTA] mutant phenotypes, mainly related to the cell wall. Homology searches with Saccharomyces cerevisiae genes identified Candida albicans Ecm33p, as well as the two other proteins of its family: Pst1p and the product of YCL048w. C. albicans Ecm33p is a 423 aa protein which has the typical features of cell-surface GPI proteins and is able to complement S. cerevisiae ecm33[DELTA] cell wall defects. Heterozygous (RML1) and homozygous (RML2) mutants of CaECM33 were obtained, as well as a single and a double reintegrant (RML3 and RML4, respectively). Caecm33 mutant strains displayed an aberrant morphology, being more rounded and bigger than the wild-type, suggesting morphogenetic defects. They also exhibited cell wall defects, with enhanced sensitivity to different compounds that interfere in polymerization of cell wall components (Calcofluor white, Congo red and hygromycin B) and a marked tendency to flocculate extensively, in addition, CaEcm33p is required for normal C. albicans yeast-to-hyphae transition in vitro. In liquid medium (5% serum), the transition was delayed in Caecm33 mutants, and after 24 h the culture contained very abnormal large and rounded cells. On solid medium (10% serum, Spider or SLADH) RML2 failed to produce hyphae and media invasiveness. CaECM33 showed a gene dosage effect, demonstrated by the intermediate phenotype of the heterozygous mutants RML1 and confirmed by Northern blot analysis. Furthermore, CaEcm33p is also involved in C. albicans virulence. In a murine systemic model of infection, 100% mouse survival and no kidney or brain colonization were obtained 30 days after infection with [10.sup.6) Candida cells of any homozygous or heterozygous Caecm33[DELTA] mutant tested. In contrast, all mice infected with parental or RML4 (two CaECM33 copies reintegrated) strains died in a few days, showing that, in these conditions, two CaECM33 copies were required for virulence.

Background: Echinocandin resistance represents a great concern, as these drugs are recommended as first-line therapy for invasive candidiasis. Echinocandin resistance is conferred by mutations in FKS genes. Nevertheless, pathways are crucial for enabling tolerance, evolution, and maintenance of resistance. Therefore, understanding the biological processes and proteins involved in the response to caspofungin may provide clues indicating new therapeutic targets.Objectives: We determined the resistance mechanism and assessed the proteome response to caspofungin exposure. We then evaluated the phenotypic impact of calcineurin inhibition by FK506 and cephalosporine A (CsA) on caspofungin-resistant Candida glabrata isolates.Methods: Twenty-five genes associated with caspofungin resistance were analysed by NGS, followed by studies of the quantitative proteomic response to caspofungin exposure. Then, susceptibility testing of caspofungin in presence of FK506 and CsA was performed. The effects of calcineurin inhibitor/caspofungin combinations on heat stress (40°C), oxidative stress (0.2 and 0.4 mM menadione) and on biofilm formation (polyurethane catheter) were analysed. Finally, a Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of the combinations on larval survival.Results: F659-del was found in the FKS2 gene of resistant strains. Proteomics data showed some up-regulated proteins are involved in cell-wall biosynthesis, response to stress and pathogenesis, some of them being members of calmodulin-calcineurin pathway. Therefore, the impact of calmodulin inhibition was explored. Calmodulin inhibition restored caspofungin susceptibility, decreased capacity to respond to stress conditions, and reduced biofilm formation and in vivo pathogenicity.Conclusions: Our findings confirm that calmodulin-calcineurin-Crz1 could provide a relevant target in life-threatening invasive candidiasis. [ABSTRACT FROM AUTHOR]