Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Mini-blaster-mediated targeted gene disruption and marker complementation in Candida albicans.

ABSTRACT: Several gene disruption strategies have been described in Candida albicans to create homozygous mutants. We describe here a recyclable mini-blaster cassette containing C. albicans URA3 gene and 200-bp flanking repeats that is useful for disruption of C. albicans genes. The cassette can be used to create unmarked homozygous mutants which can be complemented at the HIS1 gene locus. This strategy of creating gene disruptions and subsequent complementation can be used to study gene function.

SUBMITTER: Ganguly S

PROVIDER: S-EPMC3918946 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Mini-blaster-mediated targeted gene disruption and marker complementation in Candida albicans.

Ganguly Shantanu S Mitchell Aaron P AP

Methods in molecular biology (Clifton, N.J.) 20120101

Several gene disruption strategies have been described in Candida albicans to create homozygous mutants. We describe here a recyclable mini-blaster cassette containing C. albicans URA3 gene and 200-bp flanking repeats that is useful for disruption of C. albicans genes. The cassette can be used to create unmarked homozygous mutants which can be complemented at the HIS1 gene locus. This strategy of creating gene disruptions and subsequent complementation can be used to study gene function. ...[more]

PMID: 22328365

Similar Datasets

New Clox Systems for rapid and efficient gene disruption in Candida albicans.

Project description:Precise genome modification is essential for the molecular dissection of Candida albicans, and is yielding invaluable information about the roles of specific gene functions in this major fungal pathogen of humans. C. albicans is naturally diploid, unable to undergo meiosis, and utilizes a non-canonical genetic code. Hence, specialized tools have had to be developed for gene disruption in C. albicans that permit the deletion of both target alleles, and in some cases, the recycling of the Candida-specific selectable markers. Previously, we developed a tool based on the Cre recombinase, which recycles markers in C. albicans with 90-100% efficiency via site-specific recombination between loxP sites. Ironically, the utility of this system was hampered by the extreme efficiency of Cre, which prevented the construction in Escherichia coli of stable disruption cassettes carrying a methionine-regulatable CaMET3p-cre gene flanked by loxP sites. Therefore, we have significantly enhanced this system by engineering new Clox cassettes that carry a synthetic, intron-containing cre gene. The Clox kit facilitates efficient transformation and marker recycling, thereby simplifying and accelerating the process of gene disruption in C. albicans. Indeed, homozygous mutants can be generated and their markers resolved within two weeks. The Clox kit facilitates strategies involving single marker recycling or multi-marker gene disruption. Furthermore, it includes the dominant NAT1 marker, as well as URA3, HIS1 and ARG4 cassettes, thereby permitting the manipulation of clinical isolates as well as genetically marked strains of C. albicans. The accelerated gene disruption strategies afforded by this new Clox system are likely to have a profound impact on the speed with which C. albicans pathobiology can be dissected.

| S-EPMC4062495 | biostudies-literature

A TRP1-marker-based system for gene complementation, overexpression, reporter gene expression and gene modification in Candida glabrata.

Project description:Although less prevalent than its relative Candida albicans, the yeast Candida glabrata is a successful pathogen of humans, which causes life-threatening candidiasis. It is thus vital to understand the pathogenicity mechanisms and contributing genes in C. glabrata. However, gene complementation as a tool for restoring the function of a previously deleted gene is not standardized in C. glabrata, and it is less frequently used than in C. albicans. In this study, we established a gene complementation strategy using genomic integration at the TRP1 locus. We prove that our approach can not only be used for integration of complementation cassettes, but also for overexpression of markers like fluorescent proteins and the antigen ovalbumin, or of potential pathogenicity-related factors like the biotin transporter gene VHT1. With urea amidolyase Dur1,2 as an example, we demonstrate the application of the gene complementation approach for the expression of sequence-modified genes. With this approach, we found that a lysine-to-arginine mutation in the biotinylation motif of Dur1,2 impairs urea-dependent growth of C. glabrata and C. albicans. Taken together, the TRP1-based gene complementation approach is a valuable tool for investigating novel gene functions and for elucidating their role in the pathobiology of C. glabrata.

| S-EPMC7787354 | biostudies-literature

Cloning and sequencing of a Candida albicans catalase gene and effects of disruption of this gene.

Project description:Catalase plays a key role as an antioxidant, protecting aerobic organisms from the toxic effects of hydrogen peroxide, and in some cases has been postulated to be a virulence factor. To help elucidate the function of catalase in Candida albicans, a single C. albicans-derived catalase gene, designated CAT1, was isolated and cloned. Degenerate PCR primers based on highly conserved areas of other fungal catalase genes were used to amplify a 411-bp product from genomic DNA of C. albicans ATCC 10261. By using this product as a probe, catalase clones were isolated from genomic libraries of C. albicans. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 487 amino acid residues. Construction of a CAT1-deficient mutant was achieved by using the Ura-blaster technique for sequential disruption of multiple alleles by integrative transformation using URA3 as a selectable marker. Resulting mutants exhibited normal morphology and comparable growth rates of both yeast and mycelial forms. Enzymatic analysis revealed an abundance of catalase in the wild-type strain but decreasing catalase activity in heterozygous mutants and no detectable catalase in a homozygous null mutant. In vitro assays showed the mutant strains to be more sensitive to damage by both neutrophils and concentrations of exogenous peroxide that were sublethal for the parental strain. Compared to the parental strain, the homozygous null mutant strain was far less virulent for mice in an intravenous infection model of disseminated candidiasis. Definitive linkage of CAT1 with virulence would require restoration of activity by reintroduction of the gene into mutants. However, initial results in mice, taken together with the enhanced susceptibility of catalase-deficient hyphae to damage by human neutrophils, suggest that catalase may enhance the pathogenicity of C. albicans.

| S-EPMC108149 | biostudies-literature

Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

Project description:The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

| S-EPMC4592247 | biostudies-literature

Isolation of the MIG1 gene from Candida albicans and effects of its disruption on catabolite repression.

Project description:We have cloned a Candida albicans gene (CaMIG1) that encodes a protein homologous to the DNA-binding protein Mig1 from Saccharomyces cerevisiae (ScMig1). The C. albicans Mig1 protein (CaMig1) differs from ScMig1, in that, among other things, it lacks a putative phosphorylation site for Snf1 and presents several long stretches rich in glutamine or in asparagine, serine, and threonine and has the effector domain located at some distance (50 amino acids) from the carboxy terminus. Expression of CaMIG1 was low and was similar in glucose-, sucrose-, or ethanol-containing media. Disruption of the two CaMIG1 genomic copies had no effect in filamentation or infectivity. Levels of a glucose-repressible alpha-glucosidase, implicated in both sucrose and maltose utilization, were similar in wild-type or mig1/mig1 cells. Disruption of CaMIG1 had also no effect on the expression of the glucose-repressed gene CaGAL1. CaMIG1 was functional in S. cerevisiae, as judged by its ability to suppress the phenotypes produced by mig1 or tps1 mutations. In addition, CaMig1 formed specific complexes with the URS1 region of the S. cerevisiae FBP1 gene. The existence of a possible functional analogue of CaMIG1 in C. albicans was suggested by the results of band shift experiments.

| S-EPMC94279 | biostudies-literature

Disruption of Streptococcus mutans and Candida albicans synergy by a commensal streptococcus.

Project description:Polymicrobial interactions in dental plaque play a significant role in dysbiosis and homeostasis in the oral cavity. In early childhood caries, Streptococcus mutans and Candida albicans are often co-isolated from carious lesions and associated with increased disease severity. Studies have demonstrated that metabolic and glucan-dependent synergism between C. albicans and S. mutans contribute to enhanced pathogenesis. However, it is unclear how oral commensals influence pathogen synergy. Streptococcus parasanguinis, a hydrogen peroxide (H2O2) producing oral commensal, has antimicrobial activity against S. mutans. In this study, we utilized a three species biofilm model to understand the impact of S. parasanguinis on S. mutans and C. albicans synergy. We report that S. parasanguinis disrupts S. mutans and C. albicans biofilm synergy in a contact and H2O2-independent manner. Further, metabolomics analysis revealed a S. parasanguinis-driven alteration in sugar metabolism that restricts biofilm development by S. mutans. Moreover, S. parasanguinis inhibits S. mutans glucosyltransferase (GtfB) activity, which is important for glucan matrix development and GtfB-mediated binding to C. albicans mannan. Taken together, our study describes a new antimicrobial role for S. parasanguinis and highlights how this abundant oral commensal may be utilized to attenuate pathogen synergism.

| S-EPMC7661713 | biostudies-literature

Sequencing, disruption, and characterization of the Candida albicans sterol methyltransferase (ERG6) gene: drug susceptibility studies in erg6 mutants.

Project description:The rise in the frequency of fungal infections and the increased resistance noted to the widely employed azole antifungals make the development of new antifungals imperative for human health. The sterol biosynthetic pathway has been exploited for the development of several antifungal agents (allylamines, morpholines, azoles), but additional potential sites for antifungal agent development are yet to be fully investigated. The sterol methyltransferase gene (ERG6) catalyzes a biosynthetic step not found in humans and has been shown to result in several compromised phenotypes, most notably markedly increased permeability, when disrupted in Saccharomyces cerevisiae. The Candida albicans ERG6 gene was isolated by complementation of a S. cerevisiae erg6 mutant by using a C. albicans genomic library. Sequencing of the Candida ERG6 gene revealed high homology with the Saccharomyces version of ERG6. The first copy of the Candida ERG6 gene was disrupted by transforming with the URA3 blaster system, and the second copy was disrupted by both URA3 blaster transformation and mitotic recombination. The resulting erg6 strains were shown to be hypersusceptible to a number of sterol synthesis and metabolic inhibitors, including terbinafine, tridemorph, fenpropiomorph, fluphenazine, cycloheximide, cerulenin, and brefeldin A. No increase in susceptibility to azoles was noted. Inhibitors of the ERG6 gene product would make the cell increasingly susceptible to antifungal agents as well as to new agents which normally would be excluded and would allow for clinical treatment at lower dosages. In addition, the availability of ERG6 would allow for its use as a screen for new antifungals targeted specifically to the sterol methyltransferase.

| S-EPMC105764 | biostudies-literature

CaNAT1, a heterologous dominant selectable marker for transformation of Candida albicans and other pathogenic Candida species.

Project description:A dominant selectable marker for Candida albicans and other Candida species, which confers resistance to nourseothricin, was characterized. In a heterologous promoter system and a recyclable cassette, the marker efficiently permitted deletion and complementation of C. albicans genes. Neither growth nor filamentous development was affected in strains expressing this marker.

| S-EPMC547112 | biostudies-literature

Establishment of tetracycline-regulated bimolecular fluorescence complementation assay to detect protein-protein interactions in Candida albicans.

Project description:To visualize protein-protein interactions in Candida albicans with the bimolecular fluorescence complementation (BiFC) approach, we created a Tet-on system with the plasmids pWTN1 and pWTN2. Both plasmids bear a hygromycin B-resistant marker (CaHygB) that is compatible with the original Tet-on plasmid pNIM1, which carries a nourseothricin-resistant marker (CaSAT1). By using GFPmut2 and mCherry as reporters, we found that the two complementary Tet-on plasmids act synergistically in C. albicans with doxycycline in a dose-dependent manner and that expression of the fusion proteins, CaCdc11-GFPmut2 and mCherry-CaCdc10, derived from this system, is septum targeted. Furthermore, to allow detection of protein-protein interactions with the reassembly of a split fluorescent protein, we incorporated mCherry into our system. We generated pWTN1-RN and pNIM1-RC, which express the N-terminus (amino acids 1-159) and C-terminus (amino acids 160-237) of mCherry, respectively. To verify BiFC with mCherry, we created the pWTN1-CDC42-RN (or pWTN1-RN-CDC42) and pNIM1-RC-RDI1 plasmids. C. albicans cells containing these plasmids treated with doxycycline co-expressed the N- and C-terminal fragments of mCherry either N-terminally or C-terminally fused with CaCdc42 and CaRdi1, respectively, and the CaCdc42-CaRdi1 interaction reconstituted a functional form of mCherry. The establishment of this Tet-on-based BiFC system in C. albicans should facilitate the exploration of protein-protein interactions under a variety of conditions.

| S-EPMC7031294 | biostudies-literature

A Bimolecular Fluorescence Complementation Tool for Identification of Protein-Protein Interactions in <i>Candida albicans</i>.

Project description:Investigation of protein-protein interactions (PPI) in Candida albicans is essential for understanding the regulation of the signal transduction network that triggers its pathogenic lifestyle. Unique features of C. albicans, such as its alternative codon usage and incomplete meiosis, have enforced the optimization of standard genetic methods as well as development of novel approaches. Since the existing methods for detection of PPI are limited for direct visualization of the interacting complex in vivo, we have established a bimolecular fluorescence complementation (BiFC) assay in C. albicans, a powerful technique for studying PPI. We have developed an optimized set of plasmids that allows for N- and C-terminal tagging of proteins with split yeast-enhanced monomeric Venus fragments, so that all eight combinations of fusion orientations can be analyzed. With the use of our BiFC assay we demonstrate three interaction complexes in vivo, which were also confirmed by two-hybrid analysis. Our Candida-optimized BiFC assay represents a useful molecular tool for PPI studies and shows great promise in expanding our knowledge of molecular mechanisms of protein functions.

| S-EPMC5633398 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data