The invasive multidrug resistant fungus Candida auris has been emerging globally; between 2009 and 2015 it was reported in at least a dozen countries and four continents. The isolates are difficult to identify using traditional biochemical lab techniques and the mortality rates are very high. Candida auris has been associated with healthcare outbreaks and some isolates are resistant to all three classes of antifungal drugs. It can infect wounds, infiltrate the bloodstream and take root in the urinary tract.
Preliminary data indicates that multi-drug resistant Candida auris express Candida albicans Als3p homolog on its surface. Human serum and PBMC from NDV-3A vaccinated subjects may have C. auris cross-reactive antibodies and T cells. The goal of this study was to clone and express Als3p homolog from C. auris in E. coli. The homolog can be used to generate data in support of the NDV-3A vaccine.
Als3p homolog and primer design
The Als3p homolog on Candida auris was identified by Als3p amino acid sequence homology search using NCBI BLAST. The PCR primer was designed for the N-terminal region of the Als3p homolog and introduced AsisI and Pmel restriction cutting sites.
We used pFN18 HaloTag® T7 Flexi® Vector for the cloning of the Als3p homolog. The plasmid vector contains AsisI and PmeI restriction cutting sites.
PCR amplification and cloning
The C. auris genomic DNA, purified from overnight grown culture, was used to amplify the N-terminal region (1322 bp) of ALS3 homolog by PCR. The amplified ALS3 gene and plasmid DNA were digested at AsisI and PmeI restriction cutting sites, and ligated together
Protein expression and purification
Recombinant plasmid vector was transferred to ClearColi® BL21(DE3) competent cells. The cloning was confirmed by the restriction digestion analysis and sequencing of the plasmid extracted from the E. coli. After confirmation and incubation, the cells were pelleted and lysed by sonication and freeze thawing, and protein was purified.
The lysis supernatant and purified protein fractions were ran on SDS-PAGE. The protein bands were also transferred on nitrocellulose membrane and Als3p homolog band was detected by anti-Als3p mouse serum as primary antibody and peroxidase conjugated anti-mouse IgG as secondary antibodies.
- Als3p homolog was successfully amplified and cloned in pFN18 HaloTag® T7 Flexi® Vector.
- We were able to express the Als3p homolog in ClearColi® E. coli, which was confirmed by Western blotting.
- The cloned Als3p homolog will be used in future research to evaluate the quality and magnitude of cross-reactive immune responses in the vaccinated patients recruited in phase I/II clinical trial of NDV-3A vaccine.