Virulence and karyotype analyses of rad52 mutants of Candida albicans: regeneration of a truncated chromosome of a reintegrant strain (rad52/RAD52) in the host

Abstract

The virulence of Candida albicans mutants lacking one or both copies of RAD52, a gene involved in homologous recombination (HR), was evaluated in a murine model of hematogenously disseminated candidiasis. In this study, the virulence of the rad52Delta mutant was dependent upon the inoculum concentration. Mice survived at a cell inoculum of 1 x 10(6), but there was a decrease in survival time at dosages of 1.5 x 10(6) and especially at 3 x 10(6) cells per animal. The heterozygote RAD52/rad52 behaved like wild type, whereas a reintegrant strain was intermediate in its ability to cause death compared to these strains and to the avirulent rad52/rad52 null at inocula of 1 x 10(6) and 1.5 x 10(6) cells. A double mutant, lig4/lig4/rad52/rad52, was avirulent at all inocula used. PCR analysis of the RAD52 and/or LIG4 loci showed that all strains recovered from animals matched the genotype of the inoculated strains. Analysis of the electrophoretical karyotypes indicated that the inoculated, reintegrant strain carried a large deletion in one copy of chromosome 6 (the shortest homologue, or Chr6b). Interestingly, truncated Chr6b was regenerated in all the strains recovered from moribund animals using the homologue as a template. Further, regeneration of Chr6b was paralleled by an increase in virulence that was still lower than that of wild type, likely because of the persistent loss of heterozygosity in the regenerated region. Overall, our results indicate that systemic candidiasis can develop in the absence of HR, but simultaneous elimination of both recombination pathways, HR and nonhomologous end-joining, suppresses virulence even at very high inocula.

FIG. 1.

Growth rate of wild-type and rad52 mutant strains. Heterozygote TCR1 (▾), null TCR2.2 (•), revertant TCR3.2.1 (▿), and the double mutant EAT2 (○) were incubated in YPD at 30°C, and CFU were determined as indicated in Materials and Methods. Results are the means of two parallel cultures. For each strain, regression lines are shown.

FIG. 2.

A. Virulence of the C. albicans rad52 and lig4 mutants compared to CAF2-1 (wild-type) cells. All strains, CAF2-1, TCR1 (RAD52/rad52), TCR2.2 (rad52/rad52), TCR 3.2.1 (rad52/RAD52), CEA2 (lig4/lig4), and EAT2 (lig4lig4/rad52rad52), were used to infect mice at an inoculum of 1 × 10⁶ cells per animal. For strain TCR2.2, another group of animals was infected with 1.5 × 10⁶ yeast cells per animal. The percent survival of mice infected with each strain was determined 28 days postinfection. B. Virulence of C. albicans strains CAF2-1, TCR2.2 (rad52/rad52), CEA2 (lig4/lig4), and EAT2 (lig4/rad52) was evaluated in mice infected with 3 × 10⁶ CFU per mouse.

FIG. 3.

Log₁₀ CFU/g of tissue in mice infected with all strains. At 24, 48, and 72 h postinfection, moribund mice were sacrificed and the CFU of each strain were determined by plating homogenates of kidneys on YPD agar. Cultures were incubated at 30°C, and colonies were counted after a 48-h incubation. Strain TCR3.2.1 is the original reintegrant strain, and TCR3.2.1r1 is the strain recovered from infected animals. Inocula are indicated for all strains except CAF2-1, TCR1, and TCR3.2.1 strains, which were used at 1 × 10⁶ cells per mouse.

FIG. 4.

Periodic acid-Schiff-stained kidney sections from mice infected with 3 × 10⁶ CFU for 48 h with CAF2-1, TCR2.2, EAT2, and CEA2. Arrows indicate the location of hyphae.

FIG. 5.

PCR genotyping of inoculated (i) and recovered (r) mutant strains. A. Verification of the presence of the hisG-URA3-hisG-disrupted RAD52 allele in the inoculated strain (TCR1i; lanes 1and 6) and one recovered strain (TCR1r1; lanes 2 and 7) of TCR1 as well as in the inoculated (TCR2.2i; lanes 3 and 8) and two recovered (TCR2.2r1 and TCR2.2r2; lanes 4 to 5 and 9 to 10) strains of TCR2.2 (rad52Δ/rad52Δ Ura⁺). PCR analysis used oligonucleotides RV2-URA3.1 (lanes 1 to 5) and RV1-URA3.2 (lanes 6 to 10), which amplify fragments of 1.8 and 1.3 kb, respectively. B. Verification of both wild-type and hisG-disrupted RAD52 alleles in parental CAI4 (RAD52; lane 1), TCR2.2.1 (rad52::hisG/rad52::hisG; lane2), TCR2.2 inoculated (TCR2.2i; rad52::hisG/rad52::hisG-URA3-hisG; lane 3), two TCR2.2 strains recovered from animals (TCR2.2r1 and TCR2r2; lanes 4 and 5), TCR1.1 (RAD52/rad52::hisG; lane 6), revertant TCR3.2.1 inoculated (TCR3.2.1i; lane 7), and two TCR3.2.1 strains recovered from the animals (TCR3.2.1r1 and TCR3.2.1r2; lanes 8 and 9). PCR analysis used oligonucleotides RV1 and RV2, which flank the disrupted region of the RAD52 ORF and amplify fragments of 1.5 and 1.4 kb for wild-type and hisG-disrupted alleles, respectively (8). Note that these oligonucleotides do not amplify the hisG-URA3-hisG-disrupted RAD52 due to the large size of the fragment. C. Verification of the presence of the hisG-URA3-hisG- and hisG-disrupted LIG4 alleles in the inoculated (CEA2i) and two recovered (CEA2r1 and CEA2r2) strains of CEA2. PCR analysis using the oligonucleotides LIG4.3-URA3.1 (lanes 1 to 3) and LIG4.4-URA3.2 (lanes 4 to 6), which are supposed to amplify fragments of 1.8 and 1.3 kb, respectively, at the hisG-URA3-hisG locus, and oligonucleotides LIG4.3 and LIG4.4, which amplify fragments of 1.4 kb and 1.2 kb for the LIG4::hisG (lanes 8 to 10) and LIG4 (CAI control; lane 7) loci, respectively. D. (Upper panel) Verification of the presence of the hisG-URA3-hisG-disrupted RAD52 allele in the inoculated (EAT2i; lanes 1 and 5) and three recovered (EAT2r1, EAT2r2, and EAT2r3; lanes 2 to 4 and 6 to 8) strains of the double mutant EAT2. PCR analysis using the oligonucleotides RV2-URA3.1 (lanes 1 to 4) and RV1-URA3.2 (lanes 5 to 8), which amplify fragments of 1.8 and 1.3 kb, respectively. (Lower panel) Verification of the presence of both the rad52::hisG allele using the oligonucleotides RV1-RV2 (lanes 1 to 4; a 1.4-kb band) and the lig4::hisG allele using oligonucleotides LIG4.3 and LIG4.4 in the same strains (lanes 5 to 8; a 1.4-kb band). Strain designations are as follows: CAF2-1, wild type; TCR1, heterozygote RAD52/rad52::hisG-URA3-hisG; TCR2.2, homozygous null rad52::hisG/rad52hisG-URA3; TCR3.2.1, reintegrant rad52::hisG/rad52::(RAD52)_n-hisG-URA3; CEA2, homozygous null lig4::hisG/lig4::hisG-URA3-hisG; EAT2, double mutant lig4::hisG/lig4::hisG rad52::hisG/rad52::hisG-URA3-hisG.

FIG. 6.

Karyotypes of the inoculated and recovered strains. A. Pulsed-field gel electrophoresis (PFGE) under standard conditions that separate the eight chromosomes of strains inoculated and recovered from infected mice. Lanes 1 (CAF2-1i), 3 (TCR1i), 7 (TCR3.2.1i), 12 (TCR2.2i), and 16 (EAT2i) represent the karyotypes of inoculated strains. Lanes 2 (CAF2-1r1), 4 to 6 (TCR1r1, TCR1r2, and TCR1r3), 8 to 11 (TCR3.2.1r1, TCR3.2.1r2, TCR3.2.1r3, and TCR3.2.1r4), 13 to 15 (TCR2.2r1, TCR2.2r2, and TCR2.2r3), and 17 to 19 (EAT2r1, EAT2r2, and EAT2r3) are karyotypes of the matched set of strains recovered from animals at 48 h postinfection. B. PFGE under conditions that separate both homologues of the smaller chromosomes. Lanes are as in panel A, but the inoculated rad52 strain (lane 12) was duplicated to flank the recovered isogenic strains. SN1 and SN2 stand for supernumerary chromosomes 1 and 2, respectively. C. Karyotype and Southern blot using the COX12 probe of CAF2 (lane 1), inoculated revertant (lane 2), and one recovered revertant (lane 3).

FIG. 7.

Survival of mice infected with 1 × 10⁶ cells per mouse of strains CAF2-1, TCR1.1, TCR3.2.1 (original), and TCR3.2.1r1 (recovered) strains. See the legend for Fig. 2A for the origin of the original and recovered strains.