Shuttle vectors for facile gap repair cloning and integration into a neutral locus in Candida albicans

Abstract

Candida albicans is the most prevalent fungal pathogen of humans. The current techniques used to construct C. albicans strains require integration of exogenous DNA at ectopic locations, which can exert position effects on gene expression that can confound the interpretation of data from critical experiments such as virulence assays. We have identified a large intergenic region, NEUT5L, which facilitates the integration and expression of ectopic genes. To construct and integrate inserts into this novel locus, we re-engineered yeast/bacterial shuttle vectors by incorporating 550 bp of homology to NEUT5L. These vectors allow rapid, facile cloning through in vivo recombination (gap repair) in Saccharomyces cerevisiae and efficient integration of the construct into the NEUT5L locus. Other useful features of these vectors include a choice of three selectable markers (URA3, the recyclable URA3-dpl200 or NAT1), and rare restriction enzyme recognition sites for releasing the insert from the vector prior to transformation into C. albicans, thereby reducing the insert size and preventing integration of non-C. albicans DNA. Importantly, unlike the commonly used RPS1/RP10 locus, integration at NEUT5L has no negative effect on growth rates and allows native-locus expression levels, making it an ideal genomic locus for the integration of exogenous DNA in C. albicans.

Fig. 1.

The intergenic region between orf19.1961 and orf19.1963 is a neutral locus for integration and expression of exogenous DNA. (a) Region of chromosome 5 showing CLN3/orf19.1960, orf19.1961, GDS1/orf19.1963, and the 550 bp NEUT5L region (white box, which corresponds to Chromosome 5L coordinates 232782–233331); (b) Western blot showing expression of Eno1–GFP integrated at ENO1 (strain YJB11372), NEUT5L region (YJB12257), or RPS1 (YJB12780) detected with anti-GFP antibody. The wild-type strain SC5314 was used as a negative control. Anti-H4 antibody was used as a loading control. Indicated below is the mean±sd of the GFP signal intensity normalized to the loading control and to the ENO1 native locus of two independent experiments. (c) Overnight YPAD cultures of strains SC5314 (WT, dashed and dotted black line), YJB11372 (ENO1 : : GFP, continuous grey line), YJB12257 (neut5l : : ENO1 : : GFP, dashed black line), and YJB12780 (rps1 : : ENO1 : : GFP, dotted black line) were diluted 1 : 1000 in YPAD, YPAD buffered at pH 3, 6 or 9, or YPAD containing 1 M NaCl or 4 mM H₂O₂, and incubated for 24 h 25 min at 30 °C with high-speed shaking. OD₅₉₅ was measured every 15 min using a Tecan Sunrise microplate. The growth curves show the combined results of two independent experiments in which each strain was tested in triplicate. (d) Doubling times calculated from the growth curves of two independent experiments in which each strain was tested in triplicate (shown is the mean±se, total n = 6). * P-value <0.0083.

Fig. 2.

Maps of the S. cerevisiae–E. coli shuttle vectors for integration into the C. albicans NEUT5L locus. (a) pDUP shuttle vector; and (b) pDIS shuttle vector. Common features of the vectors include the Amp^R Ampicilin resistance gene for selection in E. coli, the TRP1 marker for selection on S. cerevisiae trp- strains, and a centromeric (CEN6) and autonomous replicating sequence (ARSH4) to ensure replicative stability in S. cerevisiae. The selectable markers for the resulting vectors are: pDUP3 and pDIS3, NAT1; pDUP4 and pDIS4, URA3; pDUP5 and pDIS5, URA3-dpI200. (c, d) Representation of SfiI/NgoMIV restriction enzyme digestion of inserts from pDUP and pDIS vectors and integration into NEUT5L. (e) Schematic representation of the primer design for gap repair. These primers work in both pDUP and pDIS vectors. Primers are ≥60 bp long, and consist of ≥20 bp with homology to the insert required for PCR amplification of the insert (grey line), followed by ≥40 bp with homology to pRS314 added as the primer tails (black line). The box contains the 40 bp tails from primers 5227 (top) and 5228 (bottom) that should be added to the primers to clone inserts into pDUP3-5/pDIS3-5 (Table 2). (f) Representation of the in vivo recombination between a SmaI-linearized pDIS vector and a PCR amplified insert containing 40 bp of homology to pDIS.

Fig. 3.

Inserts from pDUP/pDIS vectors are efficiently integrated and expressed from NEUT5L. (a) PCR from genomic DNA of NAT⁺ transformants showing proper integration into NEUT5L of the RBP1–NAT1 cassette from pDUP3/pDIS3-RBP1 and pDIS3-RBP1^G62D. (i) Primer set 3118+4969 was used to PCR amplify the genomic DNA of strain YJB12563 [rbp1Δ/Δ NEUT5L-RBP1 (pDUP)], while (ii) primer set 3118+4926 was used to PCR amplify the genomic DNA of strains YJB12565 [rbp1Δ/Δ NEUT5L-RBP1(pDIS)] and YJB12566 [rbp1Δ/Δ NEUT5L-RBP1^G62D (pDIS)]. (b) Phenotypic test showing that the RBP1 and RBP1^G62D constructs integrated via duplication (pDUP vector, column 3) or disruption (pDIS vectors, columns 4 and 5) of the NEUT5L region are expressed. Strains SC5314 (RBP1/RBP1), YJB12543 (rbp1Δ/Δ), and YJB12563-6 were plated onto YPAD medium and grown in the presence of discs embedded with 5 % DMSO (upper panels) or with 1 µg rapamycin ml⁻¹ in 5 % DMSO (bottom panels). Pictures showing the zone of growth inhibition were taken after 24 h of incubation at 30 °C. (c) and (d) Quantitative RT-PCR showing that RBP1 and RBP1^G62D (c) and URA3 (d) are expressed to native-locus levels when integrated at the NEUT5L region. RBP1 expression levels at its native locus in the wild-type SC5314 (RBP1/RBP1), the RBP1/rbp1Δ (YJB12982), and rbp1Δ/Δ (YJB12543) strains were compared with RBP1 expression at NEUT5L from strains YJB12563 [rbp1Δ/Δ NEUT5L-RBP1(pDUP)], YJB12565 [rbp1Δ/Δ NEUT5L-RBP1(pDIS)], and YJB12566 [rbp1Δ/Δ NEUT5L-RBP1^G62D (pDIS)]. URA3 expression levels at its native locus in the wild-type (CAF2-1) (URA3/URA3), the URA3/uraΔ (CAI4), and ura3Δ/Δ SC5314 strains were compared with URA3 expression at NEUT5L from strains YJB12984 [ura3Δ/Δ NEUT5L-URA3 (pDUP)] and YJB12985 [ura3Δ/Δ NEUT5L-URA3 (pDIS)]. qRT-PCR was performed on RNA samples obtained from the different strains grown to exponential phase in YPD at 30 °C. Shown is the mean±sd of the target gene (RBP1 or URA3)/reference gene (TEF1) ratio for each strain normalized to RBP1 or URA3 levels of expression of the wild-type strain of at least two independent experiments.