scholarly journals ACandida albicansCRISPR system permits genetic engineering of essential genes and gene families

2015 ◽  
Vol 1 (3) ◽  
pp. e1500248 ◽  
Author(s):  
Valmik K. Vyas ◽  
M. Inmaculada Barrasa ◽  
Gerald R. Fink
Keyword(s):  
Candida Albicans ◽  
Genetic Engineering ◽  
High Frequency ◽  
Target Genes ◽  
Gene Families ◽  
Induced Mutations ◽  
Pathogenic Yeast ◽  
Crispr System ◽  
Gene Knockouts ◽  
Major Impediment

Candida albicansis a pathogenic yeast that causes mucosal and systematic infections with high mortality. The absence of facile molecular genetics has been a major impediment to analysis of pathogenesis. The lack of meiosis coupled with the absence of plasmids makes genetic engineering cumbersome, especially for essential functions and gene families. We describe aC. albicansCRISPR system that overcomes many of the obstacles to genetic engineering in this organism. The high frequency with which CRISPR-induced mutations can be directed to target genes enables easy isolation of homozygous gene knockouts, even without selection. Moreover, the system permits the creation of strains with mutations in multiple genes, gene families, and genes that encode essential functions. This CRISPR system is also effective in a fresh clinical isolate of undetermined ploidy. Our method transforms the ability to manipulate the genome ofCandidaand provides a new window into the biology of this pathogen.

scholarly journals Induction of Candida albicans Drug Resistance Genes by Hybrid Zinc Cluster Transcription Factors

2014 ◽  
Vol 59 (1) ◽  
pp. 558-569 ◽  
Author(s):  
Sabrina Schneider ◽  
Joachim Morschhäuser
Keyword(s):  
Candida Albicans ◽  
Transcription Factors ◽  
Dna Binding ◽  
Target Genes ◽  
Efflux Pumps ◽  
Pathogenic Yeast ◽  
Fluconazole Resistance ◽  
Dna Binding Domains ◽  
Zinc Cluster ◽  
Binding Domains

ABSTRACTThe pathogenic yeastCandida albicanscan develop resistance to azole antifungal drugs by overexpressingERG11, which encodes the drug target, or the multidrug efflux pumpsMDR1andCDR1/CDR2. The constitutive upregulation of these genes is usually caused by gain-of-function mutations in the zinc cluster transcription factors Upc2, Mrr1, and Tac1, respectively. These transcription factors are also required for the induction of their target genes in drug-susceptible strains in the presence of specific stimuli. By swapping the DNA-binding domains of Mrr1, Tac1, and Upc2 we investigated if the hybrid transcription factors could activate their new target genes in response to the same signals. When Tac1 was targeted to theMDR1andERG11promoters, the expression of these genes became inducible by fluphenazine. Similarly,MDR1andCDR2were strongly upregulated by fluconazole when Upc2 was fused to the DNA-binding domains of Mrr1 and Tac1, respectively. In contrast, Mrr1 was unable to promote gene expression in response to benomyl when it was targeted to theCDR2andERG11promoters instead of theMDR1promoter. These results suggest that Tac1 and Upc2 themselves are activated by the inducers fluphenazine and fluconazole, respectively, whereas benomyl does not activate Mrr1 itself but a coregulatory factor that is present at the promoters of Mrr1 target genes. Strains in which the expression levels of Mrr1 and Tac1 target genes were controlled by Upc2 exhibited increased fluconazole resistance, demonstrating that the ability to efficiently upregulate the expression of efflux pumps in the presence of the drug results in enhanced intrinsic fluconazole resistance.

scholarly journals CRISPR-Cas9-mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus polymyxa

2021 ◽  
Author(s):  
Meliawati Meliawati ◽  
Christa Teckentrup ◽  
Jochen Schmid
Keyword(s):  
Genetic Engineering ◽  
Genome Editing ◽  
Related Species ◽  
Paenibacillus Polymyxa ◽  
Genomic Region ◽  
Large Cluster ◽  
Future Research ◽  
Engineering Research ◽  
Crispr System ◽  
Gene Knockouts

Clustered regularly interspaced short palindromic repeats (CRISPR) system has rapidly advanced genetic engineering research. The system has been applied for different genetic engineering purposes in multiple organisms including the quite rarely explored Paenibacillus polymyxa. Only limited studies on CRISPR-based system have been described for this highly interesting and versatile bacterium. Here, we demonstrated the utilization of a Cas9-based system to realize 32.8 kb deletion of genomic region by using a single targeting sgRNA. Large cluster deletion was successfully performed with remarkable efficiency of 97 %. Furthermore, we also exploited the system for multiplexing by editing of two distantly located genes at once. We investigated double gene knockouts as well as simultaneous gene integrations and reached editing efficiencies of 78 % and 50 %, respectively. The findings reported in this study are anticipated to accelerate future research in P. polymyxa and related species.

scholarly journals Roles of Different Peptide Transporters in Nutrient Acquisition in Candida albicans

2013 ◽  
Vol 12 (4) ◽  
pp. 520-528 ◽  
Author(s):  
Nico Dunkel ◽  
Tobias Hertlein ◽  
Renate Franz ◽  
Oliver Reuß ◽  
Christoph Sasse ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Nitrogen Sources ◽  
Gene Families ◽  
Peptide Transport ◽  
Transport Systems ◽  
Pathogenic Yeast ◽  
Content Type ◽  
Peptide Transporters ◽  
Gastrointestinal Colonization

ABSTRACT Fungi possess two distinct proton-coupled peptide transport systems, the dipeptide/tripeptide transporters (PTR) and the oligopeptide transporters (OPT), which enable them to utilize peptides as nutrients. In the pathogenic yeast Candida albicans , peptide transporters are encoded by gene families consisting of two PTR genes and eight OPT genes. To gain insight into the functions and importance of specific peptide transporters, we generated mutants lacking the two dipeptide/tripeptide transporters Ptr2 and Ptr22, as well as the five major oligopeptide transporters Opt1 to Opt5. These mutants were unable to grow in media containing peptides as the sole nitrogen source. Forced expression of individual peptide transporters in the septuple mutants showed that Ptr2 and Ptr22 could utilize all tested dipeptides as substrates but differed in their abilities to transport specific tripeptides. Interestingly, several oligopeptide transporters, which are thought to transport peptides consisting of more than three amino acids, also mediated the uptake of tripeptides. Opt1 especially turned out to be a highly flexible transporter that enabled growth on all tripeptides tested and could even utilize a dipeptide, a function that has never been ascribed to this family of peptide transporters. Despite their inability to grow on proteins or peptides, the opt1 Δ opt2 Δ opt3 Δ opt4 Δ opt5 Δ ptr2 Δ ptr22 Δ septuple mutants had no in vivo fitness defect in a mouse model of gastrointestinal colonization. Therefore, the nutritional versatility of C. albicans enables it to utilize alternative nitrogen sources in this host niche, which probably contributes to its success as a commensal and pathogen in mammalian hosts.

Ultrastructural Changes of Candida albicans Species Induced by the Presence of Sodium Diclofenac

2017 ◽  
Vol 68 (11) ◽  
pp. 2566-2569 ◽  
Author(s):  
Elena Rusu ◽  
Ionela Sarbu ◽  
Magdalena Mitache ◽  
Horatiu Moldovan ◽  
Carmen Ioana Biris ◽  
...  
Keyword(s):  
Candida Albicans ◽  
High Frequency ◽  
Fungal Infections ◽  
Diagnostic Methods ◽  
Ultrastructural Changes ◽  
Frequency Of Occurrence ◽  
Medical Treatments ◽  
Sodium Diclofenac ◽  
Cell Wall Disruption ◽  
Candidiasis Treatment

The high frequency of occurrence of candidiasis as well as high mortality of patients with immunosuppression cause a tendency toward better understanding of Candida albicans species virulence factors and developing sensitive and specific diagnostic methods, and appropriate strategies of candidiasis treatment. In recent decades the incidence of fungal infections has alarming increases because of advanced medical treatments. In this study was analyzed possible ultrastructural changes of the species C. albicans cells following treatment with sodium diclofenac at various concentrations. Following treatment of C. albicans cells with sodium diclofenac 1 mM and 2 mM changes in the plasmalemma can be noticed, changes in the density of cell wall, disruption and necrotic appearance of the cytoplasm.

scholarly journals TUP1, CPH1 and EFG1 Make Independent Contributions to Filamentation in Candida albicans

Genetics ◽  
2000 ◽  
Vol 155 (1) ◽  
pp. 57-67 ◽  
Author(s):  
Burkhard R Braun ◽  
Alexander D Johnson
Keyword(s):  
Candida Albicans ◽  
Environmental Conditions ◽  
Target Genes ◽  
Single Cells ◽  
Filamentous Growth ◽  
Surface Proteins ◽  
Pathway Expression ◽  
Separate Pathway ◽  
The Common ◽  
Growth Pathway

Abstract The common fungal pathogen, Candida albicans, can grow either as single cells or as filaments (hyphae), depending on environmental conditions. Several transcriptional regulators have been identified as having key roles in controlling filamentous growth, including the products of the TUP1, CPH1, and EFG1 genes. We show, through a set of single, double, and triple mutants, that these genes act in an additive fashion to control filamentous growth, suggesting that each gene represents a separate pathway of control. We also show that environmentally induced filamentous growth can occur even in the absence of all three of these genes, providing evidence for a fourth regulatory pathway. Expression of a collection of structural genes associated with filamentous growth, including HYR1, ECE1, HWP1, ALS1, and CHS2, was monitored in strains lacking each combination of TUP1, EFG1, and CPH1. Different patterns of expression were observed among these target genes, supporting the hypothesis that these three regulatory proteins engage in a network of individual connections to downstream genes and arguing against a model whereby the target genes are regulated through a central filamentous growth pathway. The results suggest the existence of several distinct types of filamentous forms of C. albicans, each dependent on a particular set of environmental conditions and each expressing a unique set of surface proteins.

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