Yongbing Cao

RTA2 is involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in Candida albicans

Abstract.The calcineurin pathway has been reported to be essential for the development of azole resistance in Candida albicans. The depletion or ectopic over-expression of RTA2 increased or decreased susceptibility of C. albicans to azoles, respectively. CaCl2- induced activation of the calcineurin pathway in wildtype C. albicans promoted resistance to azoles, while the Ca2+ chelator (EGTA), calcineurin inhibitors (FK506 and cyclosporin A) and the deletion of RTA2 blocked the resistance-promoting effects of CaCl2. Furthermore, we found that RTA2 was up-regulated in a calcineurin-dependent manner. The depletion of RTA2 also made the cell membrane of C. albicans liable to be destroyed by azoles and RTA2 over-expression attenuated the destroying effects. Finally, the disruption of RTA2 caused an increased accumulation of dihydrosphingosine (DHS), one of the two sphingolipid long-chain bases, by decreasing release of DHS. In conclusion, our findings suggest that RTA2 is involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans.

Triazole derivatives with improved in vitro antifungal activity over azole drugs

A series of triazole antifungal agents with piperidine side chains was designed and synthesized. The results of antifungal tests against eight human pathogenic fungi in vitro showed that all the compounds exhibited moderate-to-excellent activities. Molecular docking between 8d and the active site of Candida albicans CYP51 was provided based on the computational docking results. The triazole interacts with the iron of the heme group. The difluorophenyl group is located in the S3 subsite and its fluorine atom (2-F) can form H-bonds with Gly307. The side chain is oriented into the S4 subsite and formed hydrophobic and van der Waals interactions with the amino residues. Moreover, the phenyl group in the side chain interacts with the phenol group of Phe380 through the formation of π–π face-to-edge interactions.

Altered protein profile of lymphocytes in an antigen-specific model of colitis: a comparative proteomic study.

Abstract.Objective:Lymphocytes are deeply involved in the initiation and perpetuation of inflammatory response in inflammatory bowel disease (IBD) and lymphocyte-derived proteins are associated with the pathogenesis of the disease. The aim of this study was to identify the altered protein profiles of lymphocytes from rats with colitis.Methods:Colitis models were induced by colonic administration of trinitrobenzene sulfonic acid (TNBS) in 50% ethanol in male SD rats. Seven days after administration of TNBS/ethanol, lymphocytes were harvested from mesenteric lymph nodes (MLNs) and proteins were extracted. Two-dimensional polyacrylamide gel electrophoresis and PDQuest 2D-image-analysis software were used to display and analyze the protein spots. The differentially-expressed proteins were identified by tryptic in-gel digestion and mass spectrometry. Real-time RT-PCR was used for selected transcripts to validate the findings of the proteomics analysis.Results:A total of 1,100 protein spots including 26 proteins with at least a two-fold difference in abundance between colitis and control groups were identified. Among all the detected spots, 17 were up-regulated and 9 were down-regulated. It was found that the altered proteins included the regulators of the cell cycle and cell proliferation, signal transduction factors, inflammatory factors, apoptosis-related proteins and metabolic enzymes.Conclusions:In lymphocytes of rats with TNBS-induced colitis, 26 altered proteins were identified. They involve inflammation, apoptosis, metabolism, and regulation of the cell cycle, cell proliferation, and signal transduction.

Design, synthesis, and anticancer activity of novel berberine derivatives prepared via CuAAC “click” chemistry as potential anticancer agents

A series of novel derivatives of phenyl-substituted berberine triazolyls has been designed and synthesized via copper-catalyzed azide-alkyne cycloaddition click chemistry in an attempt to develop antitumor agents. All of the compounds were evaluated for anticancer activity against a panel of three human cancer cell lines, including MCF-7 (breast), SW-1990 (pancreatic), and SMMC-7721 (liver) and the noncancerous human umbilical vein endothelial cell (HUVEC) cell lines. The results indicated that most of the compounds displayed notable anticancer activities against the MCF-7 cells compared with berberine. Among these derivatives, compound 16 showed the most potent inhibitory activity against the SW-1990 and SMMC-7721 cell lines, with half-maximal inhibitory concentration (IC50) values of 8.54±1.97 μM and 11.87±1.83 μM, respectively. Compound 36 exhibited the most potent inhibitory activity against the MCF-7 cell line, with an IC50 value of 12.57±1.96 μM. Compound 16 and compound 36 exhibited low cytotoxicity in the HUVEC cell line, with IC50 values of 25.49±3.24 μM and 30.47±3.47 μM. Furthermore, compounds 14, 15, 16, 17, 18, 32, and 36 exhibited much better selectivity than berberine toward the normal cell line HUVEC.

The structure and retrotransposition mechanism of LTR-retrotransposons in the asexual yeast Candida albicans.

Retrotransposons constitute a major part of the genome in a number of eukaryotes. Long-terminal repeat (LTR) retrotransposons are one type of the retrotransposons. Candida albicans have 34 distinct LTR-retrotransposon families. They respectively belong to the Ty1/copia and Ty3/gypsy groups which have been extensively studied in the model yeast Saccharomyces cerevisiae. LTR-retrotransposons carry two LTRs flanking a long internal protein-coding domain, open reading frames. LTR-retrotransposons use RNA as intermediate to synthesize double-stranded DNA copies. In this article, we describe the structure feature, retrotransposition mechanism and the influence on organism diversity of LTR retrotransposons in C. albicans. We also discuss the relationship between pathogenicity and LTR retrotransposons in C. albicans.

Tolerance to Caspofungin in Candida albicans Is Associated with at Least Three Distinctive Mechanisms That Govern Expression of FKS Genes and Cell Wall Remodeling

ABSTRACT Expanding echinocandin use to prevent or treat invasive fungal infections has led to an increase in the number of breakthrough infections due to resistant Candida species. Although it is uncommon, echinocandin resistance is well documented for Candida albicans, which is among the most prevalent bloodstream organisms. A better understanding is needed to assess the cellular factors that promote tolerance and predispose infecting cells to clinical breakthrough. We previously showed that some mutants that were adapted to growth in the presence of toxic sorbose due to loss of one chromosome 5 (Ch5) also became more tolerant to caspofungin. We found here, following direct selection of mutants on caspofungin, that tolerance can be conferred by at least three mechanisms: (i) monosomy of Ch5, (ii) combined monosomy of the left arm and trisomy of the right arm of Ch5, and (iii) an aneuploidy-independent mechanism. Tolerant mutants possessed cell walls with elevated chitin and showed downregulation of genes involved in cell wall biosynthesis, namely, FKS, located outside Ch5, and CHT2, located on Ch5, irrespective of Ch5 ploidy. Also irrespective of Ch5 ploidy, the CNB1 and MID1 genes on Ch5, which are involved in the calcineurin signaling pathway, were expressed at the diploid level. Thus, multiple mechanisms can affect the relative expression of the aforementioned genes, controlling them in similar ways. Although breakthrough mutations in two specific regions of FKS1 have previously been associated with caspofungin resistance, we found mechanisms of caspofungin tolerance that are independent of FKS1 and thus represent an earlier event in resistance development.

Adaptation of Candida albicans to growth on sorbose via monosomy of chromosome 5 accompanied by duplication of another chromosome carrying a gene responsible for sorbose utilization

Candida albicans, a fungus that normally inhabits the digestive tract and other mucosal surfaces, can become a pathogen in immunocompromised individuals, causing severe or even fatal infection. Mechanisms by which C.xa0albicans can evade commonly used antifungal agents are not fully understood. We are studying a model system involving growth of C.xa0albicans on toxic sugar sorbose, which represses synthesis of cell wall glucan and, as a result, kills fungi in a manner similar to drugs from the echinocandins class. Adaptation to sorbose occurs predominantly due to reversible loss of one homolog of chromosome 5 (Ch5), which results in upregulation of the metabolic gene SOU1 (SOrbose Utilization) on Ch4. Here, we show that growth on sorbose due to Ch5 monosomy can involve a facultative trisomy of a hybrid Ch4/7 that serves to increase copy number of the SOU1 gene. This shows that control of expression of SOU1 can involve multiple mechanisms; in this case, negative regulation and increase in gene copy number operating simultaneously in cell.

Structural features and mechanism of translocation of non-LTR retrotransposons in Candida albicans

A number of abundant mobile genetic elements called retrotransposons reverse transcribe RNA to generate DNA for insertion into eukaryotic genomes. Non-long-terminal repeat (non-LTR) retrotransposons represent a major class of retrotransposons, and transposons that move by target-primed reverse transcription lack LTRs characteristic of retroviruses and retroviral-like transposons. Yeast model systems in Candida albicans and Saccharomyces cerevisiae have been developed for the study of non-LTR retrotransposons. Non-LTR retrotransposons are divided into LINEs (long interspersed nuclear elements), SINEs (short interspersed nuclear elements), and SVA (SINE, VNTR, and Alu). LINE-1 elements have been described in fungi, and several families called Zorro elements have been detected from C. albicans. They are all members of L1 clades. Through a mechanism named target-primed reverse transcription (TPRT), LINEs translocate the new copy into the target site to initiate DNA synthesis primed by the 3′ OH of the broken strand. In this article, we describe some advances in the research on structural features and origin of non-LTR retrotransposons in C. albicans, and discuss mechanisms underlying their reverse transcription and integration of the donor copy into the target site.