scholarly journals Strains and Strategies for Large-Scale Gene Deletion Studies of the Diploid Human Fungal Pathogen Candida albicans

2005 ◽  
Vol 4 (2) ◽  
pp. 298-309 ◽  
Author(s):  
Suzanne M. Noble ◽  
Alexander D. Johnson
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Large Scale ◽  
Gene Deletion ◽  
Selectable Marker ◽  
Wild Type ◽  
Position Effects ◽  
Human Fungal Pathogen ◽  
Chromosome Position ◽  
Knockout Mutants

ABSTRACT Candida albicans is the most common human fungal pathogen and causes significant morbidity and mortality worldwide. Nevertheless, the basic principles of C. albicans pathogenesis remain poorly understood. Of central importance to the study of this organism is the ability to generate homozygous knockout mutants and to analyze them in a mammalian model of pathogenesis. C. albicans is diploid, and current strategies for gene deletion typically involve repeated use of the URA3 selectable marker. These procedures are often time-consuming and inefficient. Moreover, URA3 expression levels—which are susceptible to chromosome position effects—can themselves affect virulence, thereby complicating analysis of strains constructed with URA3 as a selectable marker. Here, we describe a set of newly developed reference strains (leu2Δ/leu2Δ, his1Δ/his1Δ; arg4Δ/arg4Δ, his1Δ/his1Δ; and arg4Δ/arg4Δ, leu2Δ/leu2Δ, his1Δ/his1Δ) that exhibit wild-type or nearly wild-type virulence in a mouse model. We also describe new disruption marker cassettes and a fusion PCR protocol that permit rapid and highly efficient generation of homozygous knockout mutations in the new C. albicans strains. We demonstrate these procedures for two well-studied genes, TUP1 and EFG1, as well as a novel gene, RBD1. These tools should permit large-scale genetic analysis of this important human pathogen.

scholarly journals Pmt-Mediated O Mannosylation Stabilizes an Essential Component of the Secretory Apparatus, Sec20p, in Candida albicans

2004 ◽  
Vol 3 (5) ◽  
pp. 1164-1168 ◽  
Author(s):  
Yvonne Weber ◽  
Stephan K.-H. Prill ◽  
Joachim F. Ernst
Keyword(s):  
Endoplasmic Reticulum ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Wild Type ◽  
Rapid Degradation ◽  
Vesicle Traffic ◽  
Human Fungal Pathogen ◽  
Low Levels ◽  
Lumenal Domain ◽  
Secretory Apparatus

ABSTRACT Sec20p is an essential endoplasmic reticulum (ER) membrane protein in yeasts, functioning as a tSNARE component in retrograde vesicle traffic. We show that Sec20p in the human fungal pathogen Candida albicans is extensively O mannosylated by protein mannosyltransferases (Pmt proteins). Surprisingly, Sec20p occurs at wild-type levels in a pmt6 mutant but at very low levels in pmt1 and pmt4 mutants and also after replacement of specific Ser/Thr residues in the lumenal domain of Sec20p. Pulse-chase experiments revealed rapid degradation of unmodified Sec20p (38.6 kDa) following its biosynthesis, while the stable O-glycosylated form (50 kDa) was not formed in a pmt1 mutant. These results suggest a novel function of O mannosylation in eukaryotes, in that modification by specific Pmt proteins will prevent degradation of ER-resident membrane proteins via ER-associated degradation or a proteasome-independent pathway.

scholarly journals Altered Expression of Selectable Marker URA3 in Gene-Disrupted Candida albicans Strains Complicates Interpretation of Virulence Studies

1998 ◽  
Vol 66 (11) ◽  
pp. 5301-5306 ◽  
Author(s):  
Jennifer Lay ◽  
L. Keith Henry ◽  
Julie Clifford ◽  
Yigal Koltin ◽  
Christine E. Bulawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Fungal Pathogen ◽  
Selectable Marker ◽  
Activity Levels ◽  
Wild Type ◽  
Altered Expression ◽  
Genes Encoding

ABSTRACT The ura-blaster technique for the disruption of Candida albicans genes has been employed in a number of studies to identify possible genes encoding virulence factors of this fungal pathogen. In this study, the URA3-encoded orotidine 5′-monophosphate (OMP) decarboxylase enzyme activities of C. albicans strains with ura-blaster-mediated genetic disruptions were measured. All strains harboring genetic lesions via the ura-blaster construct showed reduced OMP decarboxylase activities compared to that of the wild type when assayed. The activity levels in different gene disruptions varied, suggesting a positional effect on the level of gene expression. Because the URA3 gene ofC. albicans has previously been identified as a virulence factor for this microorganism, our results suggest that decreased virulence observed in strains constructed with the ura-blaster cassette cannot accurately be attributed, in all cases, to the targeted genetic disruption. Although revised methods for validating aURA3-disrupted gene as a target for antifungal drug development could be devised, it is clearly desirable to replaceURA3 with a different selectable marker that does not influence virulence.

scholarly journals SpRY Cas9 Can Utilize a Variety of Protospacer Adjacent Motif Site Sequences To Edit the Candida albicans Genome

mSphere ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Ben A. Evans ◽  
Douglas A. Bernstein
Keyword(s):  
Candida Albicans ◽  
Genome Editing ◽  
Fungal Pathogen ◽  
Wild Type ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Protospacer Adjacent Motif ◽  
Motif Site ◽  
Life Threatening ◽  
Genomic Locations

ABSTRACT Candida albicans is a human fungal pathogen capable of causing life-threatening infections. The ability to edit the C. albicans genome using CRISPR/Cas9 is an important tool investigators can leverage in their search for novel therapeutic targets. However, wild-type Cas9 requires an NGG protospacer adjacent motif (PAM), leaving many AT-rich regions of DNA inaccessible. A recently described near-PAMless CRISPR system that utilizes the SpRY Cas9 variant can target non-NGG PAM sequences. Using this system as a model, we developed C. albicans CRISPR/SpRY. We tested our system by mutating C. albicans ADE2 and show that CRISPR/SpRY can utilize non-NGG PAM sequences in C. albicans. Our CRISPR/SpRY system will allow researchers to efficiently modify C. albicans DNA that lacks NGG PAM sequences. IMPORTANCE Genetic modification of the human fungal pathogen Candida albicans allows us to better understand how fungi differ from humans at the molecular level and play essential roles in the development of therapeutics. CRISPR/Cas9-mediated genome editing systems can be used to introduce site-specific mutations to C. albicans. However, wild-type Cas9 is limited by the requirement of an NGG PAM site. CRISPR/SpRY targets a variety of different PAM sequences. We modified the C. albicans CRISPR/Cas9 system using the CRISPR/SpRY as a guide. We tested CRISPR/SpRY on C. albicans ADE2 and show that our SpRY system can facilitate genome editing independent of an NGG PAM sequence, thus allowing the investigator to target AT-rich sequences. Our system will potentially enable mutation of the 125 C. albicans genes which have been previously untargetable with CRISPR/Cas9. Additionally, our system will allow for precise targeting of many genomic locations that lack NGG PAM sites.

scholarly journals Genetic analysis of sirtuin deacetylases in hyphal growth of Candida albicans

2021 ◽  
Vol 3 (12) ◽  
Author(s):  
Guolei Zhao ◽  
Laura Rusche
Keyword(s):  
Candida Albicans ◽  
Nuclear Localization ◽  
Fungal Pathogen ◽  
Environmental Changes ◽  
Hyphal Growth ◽  
Morphological Plasticity ◽  
Environmental Cues ◽  
Wild Type ◽  
Human Fungal Pathogen ◽  
Redundant Role

Candida albicans is a major human fungal pathogen that encounters varied host environments during infection. In response to environmental cues, C. albicans switches between ovoid yeast and elongated hyphal growth forms, and this morphological plasticity contributes to virulence. Environmental changes that alter the cell’s metabolic state could be sensed by sirtuins, which are NAD+-dependent deacetylases. Here we studied the roles of three sirtuin deacetylases, Sir2, Hst1, and Hst2, in hyphal growth of C. albicans. We made single, double, and triple sirtuin knockout strains and tested their ability to switch from yeast to hyphae. We found that true hyphae formation was significantly reduced by the deletion of SIR2 but not HST1 or HST2. Moreover, the expression of hyphal-specific genes HWP1, ALS3, and ECE1 decreased in the sir2Δ/Δ mutant compared to wild-type. This regulation of hyphae formation was dependent on the deacetylase activity of Sir2, as a point mutant lacking deacetylase activity had a similar defect in hyphae formation as the sir2Δ/Δ mutant. Finally, we found that Sir2 and Hst1 were localized to the nucleus, with Sir2 specifically focused in the nucleolus. This nuclear localization suggests a role for Sir2 and Hst1 in regulating gene expression. In contrast, Hst2 was localized to the cytoplasm. In conclusion, our results suggest that Sir2 plays a critical and non-redundant role in hyphal growth of C. albicans.

scholarly journals pH-Dependant Antifungal Activity of Valproic Acid against the Human Fungal Pathogen Candida albicans

2017 ◽  
Vol 8 ◽  
Author(s):  
Julien Chaillot ◽  
Faiza Tebbji ◽  
Carlos García ◽  
Hugo Wurtele ◽  
René Pelletier ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Candida Albicans ◽  
Antifungal Activity ◽  
Fungal Pathogen ◽  
Human Fungal Pathogen

scholarly journals The 5’ untranslated region of theEFG1transcript promotes its translation to regulate hyphal morphogenesis inCandida albicans

2018 ◽  
Author(s):  
Prashant R. Desai ◽  
Klaus Lengeler ◽  
Mario Kapitan ◽  
Silas Matthias Janßen ◽  
Paula Alepuz ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Regulatory Mechanisms ◽  
Untranslated Regions ◽  
Regulatory Sequence ◽  
Transcriptional Level ◽  
Human Fungal Pathogen ◽  
Hyphal Morphogenesis ◽  
Cellular Morphogenesis ◽  
Incandida Albicans

ABSTRACTExtensive 5’ untranslated regions (UTR) are a hallmark of transcripts determining hyphal morphogenesis inCandida albicans.The major transcripts of theEFG1gene, which are responsible for cellular morphogenesis and metabolism, contain a 5’ UTR of up to 1170 nt. Deletion analyses of the 5’ UTR revealed a 218 nt sequence that is required for production of the Efg1 protein and its functions in filamentation, without lowering the level and integrity of theEFG1transcript. Polysomal analyses revealed that the 218 nt 5’ UTR sequence is required for efficient translation of the Efg1 protein. Replacement of theEFG1ORF by the heterologous reporter geneCaCBGlucconfirmed the positive regulatory importance of the identified 5’ UTR sequence. In contrast to other reported transcripts containing extensive 5’ UTR sequences, these results indicate the positive translational function of the 5’ UTR sequence in theEFG1transcript, which is observed in context of the nativeEFG1promoter. The results suggest that the 5’ UTR recruits regulatory factors, possibly during emergence of the native transcript, which aid in translation of theEFG1transcript.IMPORTANCEMany of the virulence traits that makeCandida albicansan important human fungal pathogen are regulated on a transcriptional level. Here we report an important regulatory contribution of translation, which is exerted by the extensive 5’ untranslated regulatory sequence (5’ UTR) of the transcript for the protein Efg1, which determines growth, metabolism and filamentation in the fungus. Presence of the 5’ UTR is required for efficient translation of Efg1, to promote filamentation. Because transcripts for many relevant regulators contain extensive 5’ UTR sequences, it appears that virulence ofC. albicansdepends on the combination of transcriptional and translation regulatory mechanisms.

scholarly journals Sub-MIC levels of purpurin inhibit membrane ATPase-mediated proton efflux activity in the human fungal pathogen Candida albicans

2014 ◽  
Vol 67 (4) ◽  
pp. 349-350 ◽  
Author(s):  
Paul Wai-Kei Tsang ◽  
Alan Pak-Kin Wong ◽  
Han-Sung Jung ◽  
Wing-Ping Fong
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Proton Efflux ◽  
Human Fungal Pathogen ◽  
Membrane Atpase

scholarly journals Restructured Lactococcus lactis strains with emergent properties constructed by a novel highly efficient screening system

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Fulu Liu ◽  
Yating Zhang ◽  
Wanjin Qiao ◽  
Duolong Zhu ◽  
Haijin Xu ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Large Scale ◽  
Gene Deletion ◽  
Selectable Marker ◽  
Genome Reduction ◽  
Emergent Properties ◽  
Metabolic Capacity ◽  
Efficient System ◽  
Cell Factories ◽  
Efficient Screening

Abstract Background After 2.83% genome reduction in Lactococcus lactis NZ9000, a good candidate host for proteins production was obtained in our previous work. However, the gene deletion process was time consuming and laborious. Here, we proposed a convenient gene deletion method suitable for large-scale genome reduction in L. lactis NZ9000. Results Plasmid pNZ5417 containing a visually selectable marker PnisZ-lacZ was constructed, which allowed more efficient and convenient screening of gene deletion mutants. Using this plasmid, two large nonessential DNA regions, L-4A and L-5A, accounting for 1.25% of the chromosome were deleted stepwise in L. lactis 9k-3. When compared with the parent strain, the mutant L. lactis 9k-5A showed better growth characteristics, transformability, carbon metabolic capacity, and amino acids biosynthesis. Conclusions Thus, this study provides a convenient and efficient system for large-scale genome deletion in L. lactis through application of visually selectable marker, which could be helpful for rapid genome streamlining and generation of restructured L. lactis strains that can be used as cell factories.

CSU57 encodes a novel repressor of sorbose utilization in opportunistic human fungal pathogen Candida albicans

Yeast ◽  
2020 ◽  
Author(s):  
Praveen Kumar Reddy ◽  
Dileep Pullepu ◽  
Darshan Dhabalia ◽  
Sagunthala Murugesan Udaya Prakash ◽  
Mohammad Anaul Kabir
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Human Fungal Pathogen
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