Development of a Shuttle Vector That Transforms at High Frequency for the Emerging Human Fungal Pathogen: Candida...

Area of Science:

• Fungal molecular biology
• Medical mycology
• Genetics

Background:

• Molecular manipulation of *Candida auris* is hindered by the lack of suitable plasmids.
• Existing transformation methods in *C. auris* lack efficient cloning or shuttle vectors.
• Centromeric sequences in *C. auris* are characterized but not utilized as molecular tools.

Purpose of the Study:

• To identify *C. auris* centromeric sequences capable of forming stable, transformable plasmids.
• To develop a novel shuttle vector for *C. auris* molecular research.
• To assess the utility of centromere-derived sequences as plasmid maintenance elements.

Main Methods:

• Cloning of all seven *C. auris* centromeric sequences.
• Testing of cloned sequences for transformation frequency and plasmid stability.
• Construction and characterization of a shuttle vector using a high-frequency transforming subclone.
• Plasmid rescue and analysis in *E. coli*.
• Transformation efficiency assessment across *C. auris* clades.

Main Results:

• Transformation frequency varied significantly among tested centromeric sequences.
• A 1.7 Kb subclone demonstrated high transformation frequency and ARS-like properties in a plasmid.
• The developed shuttle vector was stable under selection (~1 copy per cell) but easily lost without selection.
• The plasmid remained intact in *C. auris* even with large inserts and transformed all four *C. auris* clades.

Conclusions:

• *C. auris* centromeric sequences can be exploited to create functional plasmids.
• The developed plasmid serves as a valuable and versatile tool for *C. auris* research.
• This new reagent will significantly advance the investigation of *Candida auris* as a human fungal pathogen.