Expression vectors for C-terminal fusions with fluorescent proteins and epitope tags in Candida glabrata

Yáñez-Carrillo, Patricia; Orta-Zavalza, Emmanuel; Gutiérrez-Escobedo, Guadalupe; Patrón‐Soberano, Araceli; Peñas, Alejandro De Las; Castaño, Irene

doi:10.1016/j.fgb.2015.04.020

Cited by 21 publications

(12 citation statements)

References 34 publications

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“…Complemented strains were generated by reintegrating VHR1 and VHT1 into the TRP1 locus. This locus was chosen as tryptophan auxotrophy ( trp1 Δ) had no negative impact on C. glabrata in vivo fitness and in vitro mutant growth phenotypes (Jacobsen et al, ; Yanez‐Carrillo et al, ). In detail, the TRP1 gene and its 5' upstream region (−984 bp to +750 bp) was PCR‐amplified with primers introducing flanking SphI restriction sites and ligated into the SphI‐linearized pUC19 vector (In‐Fusion HD Cloning Kit) to generate pUC19‐ TRP1 .…”

Section: Methodsmentioning

confidence: 99%

Fungal biotin homeostasis is essential for immune evasion after macrophage phagocytosis and virulence

Sprenger

Hartung

Allert

et al. 2020

Cellular Microbiology

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Biotin is an important cofactor for multiple enzymes in central metabolic processes.While many bacteria and most fungi are able to synthesise biotin de novo, Candida spp. are auxotrophic for this vitamin and thus require efficient uptake systems to facilitate biotin acquisition during infection. Here we show that Candida glabrata and Candida albicans use a largely conserved system for biotin uptake and regulation, consisting of the high-affinity biotin transporter Vht1 and the transcription factor Vhr1. Both species induce expression of biotin-metabolic genes upon in vitro biotin depletion and following phagocytosis by macrophages, indicating low biotin levels in the Candida-containing phagosome. In line with this, we observed reduced intracellular proliferation of both Candida cells pre-starved of biotin and deletion mutants lacking VHR1 or VHT1 genes. VHT1 was essential for the full virulence of C. albicans during systemic mouse infections, and the lack of VHT1 led to reduced fungal burden in C. glabrata-infected brains and C. albicans-infected brains and kidneys. Together, our data suggest a critical role of Vht1-mediated biotin acquisition for C. glabrata and C. albicans during intracellular growth in macrophages and systemic infections. K E Y W O R D S biotin, Candida albicans, Candida glabrata, macrophage, metabolism, virulence

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Section: Methodsmentioning

confidence: 99%

Fungal biotin homeostasis is essential for immune evasion after macrophage phagocytosis and virulence

Sprenger

Hartung

Allert

et al. 2020

Cellular Microbiology

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“…Plasmid and C. glabrata Strain Construction C. glabrata mCherry tagging was done via C-terminal fusion of TDH3 (CAGL0G09383 g) with mCherry based on a previously published strategy (Yá ñ ez-Carrillo et al, 2015) using the pYC56 plasmid (Addgene) as a mCherry donor. Briefly, the last 485 bp of the TDH3 coding sequence (CDS; excluding the stop codon) and 118 bp downstream (DR) of the open reading frame were PCR amplified and cloned into pYC56 using SacI, BamHI and KpnI, XhoI (all Thermo Fisher Scientific), respectively, yielding the final plasmid pYC56-TDH3urdr.…”

Section: Methods Detailsmentioning

confidence: 99%

Type I Interferon Response Dysregulates Host Iron Homeostasis and Enhances Candida glabrata Infection

Riedelberger¹,

Penninger²,

Tscherner³

et al. 2020

Cell Host & Microbe

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“…The NLS-RFP construct was sub-cloned from plasmid pML85 (gift of Michael Lisby) into C. glabrata plasmid pMJ22 82 (obtained from Addgene) using XhoI and NotI restriction sites. Slides for time-lapse microscopy were prepared by pipetting warm YPD containing 1% low melting agarose (with or without 0.03% MMS) onto glass slides and letting it solidify, forming YPD-agarose pads.…”

Section: Methodsmentioning

confidence: 99%

A non-canonical DNA damage checkpoint response in a major fungal pathogen

Shor

Garcia-Rubio

DeGregorio

et al. 2020

Preprint

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To protect genome integrity, eukaryotic cells respond to DNA damage by triggering highly conserved checkpoint mechanisms involving the phosphorylation of Rad53/CHK2 kinase. Budding yeast Candida glabrata, closely related to model eukaryote Saccharomyces cerevisiae, is an opportunistic pathogen characterized by high genetic diversity and rapid emergence of drug resistant mutants. However, the mechanisms enabling this genetic variability are unclear. Here we show that C. glabrata cells exposed to DNA damage neither induce CgRad53 phosphorylation nor accumulate in S phase, and exhibit higher lethality than S. cerevisiae. Furthermore, time-lapse microscopy showed C. glabrata cells continuing to divide in the presence of DNA damage, resulting in mitotic errors and cell death. Finally, RNAseq analysis revealed transcriptional rewiring of the DNA damage response in C. glabrata and identified several key protectors of genome stability upregulated by DNA damage in S. cerevisiae but downregulated in C. glabrata, including PCNA. Together, our results reveal a non-canonical fungal DNA damage response, which may contribute to rapidly generating genetic change and drug resistance.

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Expression vectors for C-terminal fusions with fluorescent proteins and epitope tags in Candida glabrata

Cited by 21 publications

References 34 publications

Fungal biotin homeostasis is essential for immune evasion after macrophage phagocytosis and virulence

Fungal biotin homeostasis is essential for immune evasion after macrophage phagocytosis and virulence

Type I Interferon Response Dysregulates Host Iron Homeostasis and Enhances Candida glabrata Infection

A non-canonical DNA damage checkpoint response in a major fungal pathogen

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