Accessibility and contribution to glucan masking of natural and genetically tagged versions of yeast wall protein 1 of Candida albicans

Yeast wall protein 1 (Ywp1) is an abundant glycoprotein of the cell wall of the yeast form of Candida albicans, the most prevalent fungal pathogen of humans. Antibodies that bind to the polypeptide backbone of isolated Ywp1 show little binding to intact yeast cells, presumably because the Ywp1 epitopes are masked by the polysaccharides of the mannoproteins that form the outer layer of the cell wall. Rare cells do exhibit much greater anti-Ywp1 binding, however, and one of these was isolated and characterized. No differences were seen in its Ywp1, but it exhibited greater adhesiveness, sensitivity to wall perturbing agents, and exposure of its underlying β-1,3-glucan layer to external antibodies. The molecular basis for this greater epitope accessibility has not been determined, but has facilitated exploration of how these properties change as a function of cell growth and morphology. In addition, previously engineered strains with reduced quantities of Ywp1 in their cell walls were also found to have greater β-1,3-glucan exposure, indicating that Ywp1 itself contributes to the masking of wall epitopes, which may be important for understanding the anti-adhesive effect of Ywp1. Ectopic production of Ywp1 by hyphae, which reduces the adhesivity of these filamentous forms of C. albicans, was similarly found to reduce exposure of the β-1,3-glucan in their walls. To monitor Ywp1 in the cell wall irrespective of its accessibility, green fluorescent protein (Gfp) was genetically inserted into wall-anchored Ywp1 using a bifunctional cassette that also allowed production from a single transfection of a soluble, anchor-free version. The wall-anchored Ywp1-Gfp-Ywp1 accumulated in the wall of the yeast forms but not hyphae, and appeared to have properties similar to native Ywp1, including its adhesion-inhibiting effect. Some pseudohyphal walls also detectably accumulated this probe. Strains of C. albicans with tandem hemagglutinin (HA) epitopes inserted into wall-anchored Ywp1 were previously created by others, and were further explored here. As above, rare cells with much greater accessibility of the HA epitopes were isolated, and also found to exhibit greater exposure of Ywp1 and β-1,3-glucan. The placement of the HA cassette inhibited the normal N-glycosylation and propeptide cleavage of Ywp1, but the wall-anchored Ywp1-HA-Ywp1 still accumulated in the cell wall of yeast forms. Bifunctional transformation cassettes were used to additionally tag these molecules with Gfp, generating soluble Ywp1-HA-Gfp and wall-anchored Ywp1-HA-Gfp-Ywp1 molecules. The former revealed unexpected electrophoretic properties caused by the HA insertion, while the latter further highlighted differences between the presence of a tagged Ywp1 molecule (as revealed by Gfp fluorescence) and its accessibility in the cell wall to externally applied antibodies specific for HA, Gfp and Ywp1, with accessibility being greatest in the rapidly expanding walls of budding daughter cells. These strains and results increase our understanding of cell wall properties and how C. albicans masks itself from recognition by the human immune system.

The 199 bp partial sequence of GFP (nucleotides 2176-2374, as numbered above) omits the last 5 codons and stop codon of GFP, so that the final Gfp that is inserted internally into the endogenous protein lacks the C-terminal 5 amino acids of Gfp (i.e., -DELYK.) This segment is unnecessary for creation of a stable, fluorescent Gfp [3].

Instructions for Use
A PCR amplicon encompassing the GFP-URA3-GFP segment serves as the transfecting DNA for ura3 strains of yeast (which require uracil or uridine for growth because their URA3 is defective or missing). Transformants grow in the absence of exogenous uracil or uridine upon stable integration of the GFP-URA3-GFP cassette into their genome by homologous recombination. PCR primers are designed to target the insertion to the point of interest (within the coding sequence of a gene). Subsequent growth of transformants in the presence of 5-FOA selects for rare individual cells that have lost their inserted URA3 through recombination of the homologous flanking GFP sequences (which share 199 bp of identical sequence); this fuses the upstream GFP coding sequence with the downstream coding sequence, resulting in a protein with an internal Gfp insertion.
PCR primer design: 80-nucleotide DNA primers have been used routinely and successfully for these transformations. The 5' 60 or so nucleotides should match the genomic insertion target, and the 3' 20 or so nucleotides should match the ends of the GFP-URA3-GFP cassette; the amplicon will therefore have about 60 base pairs of target sequence at each end, with any length of sequence between those two targets in the genomic DNA (even none, if a lossless insertion is desired rather than a replacement). The upstream primer sequence is an in-frame fusion of the target coding sequence and the GFP coding sequence (possibly bypassing the first codon or two of GFP, if desired). The downstream primer sequence is an in-frame fusion of the complement of the target sequence and the complement of the end of the GFP gene (with or without part or all of the optional linker segment, which encodes SSASPSGS).
Success has routinely been achieved using primers from IDT that have simply been desalted, not gelpurified.
If your PCR polymerase is likely to add an untemplated "A" to the 3' ends of the amplicons, make sure this addition will match the target sequence (i.e., the target should have a "T" immediately upstream from the first [5'] base of your primers).
For the most efficient amplification of the GFP-URA3-GFP cassette from the pGFP-URA3-GFP plasmid, pre-digest the plasmid with Hind III and Apa I. The liberated 2.33 kbp insert can also be gel purified for amplification if you anticipate multiple uses of this cassette.
This system has been used successfully in each attempt with six different strains of Candida albicans (all derivatives of strains CaI4 and BWP17, which are both derivatives of strain SC5314); it has not yet been attempted in other strains, species or genera. So far, it has been tried only for a GPI-anchored cell wall protein (Ywp1); the initial transformants secreted the Ywp1-Gfp fusion protein into the culture medium, as the chimera lacked the C-terminal anchor of Ywp1; the 5-FOA survivors incorporated Ywp1-Gfp-Ywp1 into the cell wall, as no amino acids were altered or lost from Ywp1 in this assembly and the inserted Gfp did not prevent transport to that destination.

Alternatives
A similar plasmid template (pMG2082 = pGUG = pGFP-URA3-GFP = pGF-URA3-FP) has been described by Gerami-Nejad et al. [4]. That plasmid consists of URA3 flanked by partial GFP sequences, so that fluorescent Gfp is observed only after recombinative excision of the URA3. In contrast, the current pGEM-GFP-URA3-GFP plasmid has a full upstream GFP that allows initial transformants to be confirmed visually or spectroscopically prior to 5-FOA selection, and creates a fusion protein (with Gfp comprising the C-terminal moiety) that may also be useful for study; the PCR amplicon used for transfection is also more streamlined, being almost 1 kbp shorter than the one generated from pMG2082.
Gerami-Nejad et al. also constructed a plasmid template similar to pMG2082, but with URA3 replaced by NAT1, which confers resistance to nourseothricin [5]; this allows positive selection of transformants, and thus does not rely on ura3 auxotrophs as hosts. Correct insertion verification requires PCR analysis of the initial transformants; loss of NAT1 and generation of Gfp fluorescence then requires visual or flowcytometric screening for identification.