PDR16-mediated azole resistance in Candida albicans

Many Candida albicans azole-resistant (AR) clinical isolates overexpress the CDR1 and CDR2 genes encoding homologous multidrug transporters of the ATP-binding cassette family. We show here that these strains also overexpress the PDR16 gene, the orthologue of Saccharomyces cerevisiae PDR16 encoding a phosphatidylinositol transfer protein of the Sec14p family. It has been reported that S. cerevisiae pdr16Delta mutants are hypersusceptible to azoles, suggesting that C. albicans PDR16 may contribute to azole resistance in these isolates. To address this question, we deleted both alleles of PDR16 in an AR clinical strain overexpressing the three genes, using the mycophenolic acid resistance flipper strategy. Our results show that the homozygous pdr16Delta/pdr16Delta mutant is approximately twofold less resistant to azoles than the parental strain whereas reintroducing a copy of PDR16 in the mutant restored azole resistance, demonstrating that this gene contributes to the AR phenotype of the cells. In addition, overexpression of PDR16 in azole-susceptible (AS) C. albicans and S. cerevisiae strains increased azole resistance by about twofold, indicating that an increased dosage of Pdr16p can confer low levels of azole resistance in the absence of additional molecular alterations. Taken together, these results demonstrate that PDR16 plays a role in C. albicans azole resistance.     

Responses of pathogenic and nonpathogenic yeast species to steroids reveal the functioning and evolution of multidrug resistance transcriptional networks

Steroids are known to induce pleiotropic drug resistance states in hemiascomycetes, with tremendous potential consequences for human fungal infections. Our analysis of gene expression in Saccharomyces cerevisiae and Candida albicans cells subjected to three different concentrations of progesterone revealed that their pleiotropic drug resistance (PDR) networks were strikingly sensitive to steroids. In S. cerevisiae, 20 of the Pdr1p/Pdr3p target genes, including PDR3 itself, were rapidly induced by progesterone, which mimics the effects of PDR1 gain-of-function alleles. This unique property allowed us to decipher the respective roles of Pdr1p and Pdr3p in PDR induction and to define functional modules among their target genes. Although the expression profiles of the major PDR transporters encoding genes ScPDR5 and CaCDR1 were similar, the S. cerevisiae global PDR response to progesterone was only partly conserved in C. albicans. In particular, the role of Tac1p, the main C. albicans PDR regulator, in the progesterone response was apparently restricted to five genes. These results suggest that the C. albicans and S. cerevisiae PDR networks, although sharing a conserved core regarding the regulation of membrane properties, have different structures and properties. Additionally, our data indicate that other as yet undiscovered regulators may second Tac1p in the C. albicans drug response.     

NCI60 cancer cell line panel data and RNAi analysis help identify EAF2 as a modulator of simvastatin and lovastatin response in HCT-116 cells

Simvastatin and lovastatin are statins traditionally used for lowering serum cholesterol levels. However, there exists evidence indicating their potential chemotherapeutic characteristics in cancer. In this study, we used bioinformatic analysis of publicly available data in order to systematically identify the genes involved in resistance to cytotoxic effects of these two drugs in the NCI60 cell line panel. We used the pharmacological data available for all the NCI60 cell lines to classify simvastatin or lovastatin resistant and sensitive cell lines, respectively. Next, we performed whole-genome single marker case-control association tests for the lovastatin and simvastatin resistant and sensitive cells using their publicly available Affymetrix 125K SNP genomic data. The results were then evaluated using RNAi methodology. After correction of the p-values for multiple testing using False Discovery Rate, our results identified three genes (NRP1, COL13A1, MRPS31) and six genes (EAF2, ANK2, AKAP7, STEAP2, LPIN2, PARVB) associated with resistance to simvastatin and lovastatin, respectively. Functional validation using RNAi confirmed that silencing of EAF2 expression modulated the response of HCT-116 colon cancer cells to both statins. In summary, we have successfully utilized the publicly available data on the NCI60 cell lines to perform whole-genome association studies for simvastatin and lovastatin. Our results indicated genes involved in the cellular response to these statins and siRNA studies confirmed the role of the EAF2 in response to these drugs in HCT-116 colon cancer cells.     

Applying the PDR principle to AIDS

The principle of pathogen-derived resistance (the PDR principle) has been put forward as a broadly-applicable conceptual tool for use in designing genes which will confer resistance to pathogens. This paper reveals an example of how the PDR principle may be applied in the field of human medicine. Specifically it is shown how the PDR principle can be employed in designing a series of genes which should be capable of protecting human blood cells from the retrovirus causing the AIDS disease. Prospects are discussed for using such genes in gene therapy treatment of people infected with this virus.     

植物抗病毒病育种策略

为了得到抗病毒的寄主植物,植物育种学家进行了许多有益研究,形成了许多行之有效的抗病毒病育种策略。利用植物本身对病毒侵染所具有的一些免疫功能及其本身的一些抗性基因来获得抗性;利用来源于病毒自身基因的一些抗病性策略(PDR),如利用病毒外壳蛋白基因,病毒复制酶基因,病毒移动蛋白基因,病毒卫星RNA和反义RNA等,植物也可以获得抗性。近年来对由转录后RNA沉默引起的由RNA介导的病毒抗性策略(RMVR)也进行了深入地研究。除了PDR和RMVR以外,还有一些导致植物抗病毒的策略,包括利用美国商陆的病毒抗性蛋白(PAP),2',5’-寡腺苷酸合成酶,“植物抗体”以及病毒蛋白多肽来获得病毒抗性等。     

Engineered bakers yeast as a sensitive bioassay indicator organism for the trichothecene toxin deoxynivalenol

The aim of this study was to increase the sensitivity of Saccharomyces cerevisiae towards trichothecene toxins, in particular to deoxynivalenol (DON), in order to improve the utility of this yeast as a bioassay indicator organism. We report the construction of a strain with inactivated genes (PDR5, PDR10, PDR15) encoding ABC transporter proteins with specificity for the trichothecene deoxynivalenol, with inactivated AYT1 (encoding a trichothecene-3-O-acetyltransferase), and inactivated UBI4 and UBP6 genes. Inactivation of the stress inducible polyubiquitin gene UBI4 or the ubiquitin protease UBP6 increased DON sensitivity, the inactivation of both genes had a synergistic effect. The resulting pdr5 pdr10 pdr15 ayt1 ubp6 ubi4 mutant strain showed 50% growth inhibition at a DON concentration of 5 mg/l under optimal conditions. The development of a simple two step assay for microbial DON degradation in 96 well microtiter format and its testing with the DON detoxifying bacterium BBSH 797 is reported.     

Regulation of osteoclast differentiation by statins

HMG-CoA reductase inhibitor (statin) treatment is frontline therapy for lowering plasma cholesterol levels in patients with hyperlipidemia. In a few case studies, analysis of clinical data has revealed a decreased risk of fracture in patients on statin therapy. However, this reduction in the incidence of fracture is not always observed nor is it supported by an increase in bone density, which further complicates our understanding of the role of statins in bone metabolism. Thus, the precise role of statins in bone metabolism remains poorly understood. In this study, we examined the effect of statin treatment on osteoclastogenesis. Treatment with lovastatin resulted in a significant, dose-dependent decrease in the numbers of differentiated osteoclasts and decreased cholesterol biosynthesis activity with an EC(50) similar to that observed in freshly isolated rat or cultured human liver cells. Studies assessing the role of mevalonate metabolites in the development of the osteoclasts demonstrated that geranylgeraniol, but not squalene or farnesol was important for the development and differentiation of osteoclasts, implicating protein geranylgeranylation rather than protein farnesylation as a key factor in the osteoclast differentiation process. In conclusion, our data indicate that lovastatin inhibits osteoclast development through inhibition of geranylgeranylation of key prenylated proteins and that the bone effects of statins are at least partially due to their effects on osteoclast numbers.     

Cross-resistance to strobilurin fungicides in mitochondrial and nuclear mutants of<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In yeast the resistance to kresoxim-methyl and azoxystrobin, like the resistance to strobilurin A (mucidin) is under the control of both mitochondrial cob gene and the PDR network of nuclear genes involved in multidrug resistance. The mucidin-resistant mucl (G137R) and muc2 (L275S) mutants of Saccharomyces cerevisiae containing point mutations in mtDNA were found to be cross-resistant to kresoxim-methyl and azoxystrobin. Cross-resistance to all three strobilurin fungicides was also observed in yeast transformants containing gain-of-function mutations in the nuclear PDR3 gene. On the other hand, nuclear mutants containing disrupted chromosomal copies of the PDR1 and PDR3 genes or the PDR5 gene alone were hypersensitive to kresoxim-methyl, azoxystrobin and strobilurin A. The frequencies of spontaneous mutants selected for resistance either to kresoxim-methyl, azoxystrobin or strobilurin A were similar and resulted from mutations both in mitochondrial and nuclear genes. The results indicate that resistance to strobilurin fungicides, differing in chemical structure and specific activity, can be caused by the same molecular mechanism involving changes in the structure of apocytochrome b and/or increased efflux of strobilurins from fungal cells.