Phenotypic analysis and virulence of Candida albicans LIG4 mutants

Abstract

In previous studies, we reported the isolation and preliminary characterization of a DNA ligase-encoding gene of Candida albicans. This gene (LIG4) is the structural and functional homologue of both yeast and human ligase IV, which is involved in nonhomologous end joining (NHEJ) of DNA double-strand breaks. In the present study, we have shown that there are no other LIG4 homologues in C. albicans. In order to study the function of LIG4 in morphogenesis and virulence, we constructed gene deletions. LIG4 transcript levels were reduced in the heterozygote and were completely absent in null strains. Concomitantly, the heterozygote showed a pronounced defect in myceliation, which was slightly greater in the null strain. This was true with several solid and liquid media, such as Spider medium, medium 199, and 2% glucose-1% yeast extract-2% Bacto Peptone, at several pHs. Reintroduction of the wild-type allele into the null mutant partially restored the ability of cells to form hyphae. In agreement with the positive role of LIG4 in morphogenesis, we detected a significant rise in mRNA levels during the morphological transition. LIG4 is not essential for DNA replication or for the repair of DNA damage induced by ionizing radiation or UV light, indicating that these lesions are repaired primarily by homologous recombination. However, our data show that the NHEJ apparatus of C. albicans may control morphogenesis in this diploid organism. In addition, deletion of one or both copies of LIG4 resulted in attenuation of virulence in a murine model of candidiasis.

FIG. 1

Determination of LIG4 homologues in C. albicans. Genomic DNAs from strains 1001 (lanes 1), 3153A (lanes 2), and 4918 (lanes 3) were cut with the indicated restriction enzymes, electrophoresed, and subjected to Southern hybridization using the XhoI/XhoI internal fragment of C. albicans LIG4 (see Fig. 2). Abbreviations: B, BamHI; E, EcoRI; H, HindIII; X, XhoI. Size markers are indicated.

FIG. 2

Restriction map of C. albicans (Ca) LIG4 and scheme showing the replacement of wild-type LIG4 (top [above arrow]) as well as the construction of the revertant allele (bottom [below arrow]). An internal PstI/SacI fragment from LIG4 was replaced with the hisG-URA3-hisG cassette. Following selection on 5′-FOA plates, URA3 and one copy of hisG were deleted in the LIG4/lig4 heterozygote before replacement of the second wild-type allele. Wild-type LIG4 was reintroduced into the null mutant lig4/lig4 (Ura3⁻) to construct the revertant, as indicated in the scheme (bottom). Abbreviations: B, BamHI; E, EcoRI; H, HindIII; K, KpnI; N, NsiI; P, PstI; Sc, SacI; Sl, SalI; X, XhoI; Xb, XbaI; Se, SceI.

FIG. 3

Analysis of LIG4 mutants. (A and B) Southern blots of LIG4 deletion mutants with the whole deletion cassette as a probe (Fig. 2, top). (A) Candida genomic DNA digested with EcoRI. Lane 1, parental strain CAI4 yields fragments of 1.4 and 3.7 kb. Lane 2, heterozygous LIG4/lig4::URA3 (single) disruptant (CEA1); note that, in addition to the bands from the wild-type allele, two new bands of 3.5 and 5.5 kb are present. Lane 3, heterozygous LIG4/lig4 strain after 5′-FOA selection (CEA1.5); the 3.5- and 5.5-kb bands are resolved into a new, 5.4-kb band. Lane 4, lig4::URA3/lig4 double disruptant (CEA2); note the 3.5- and 5.5-kb bands from the second disruption with hisG-URA3-hisG and the 5.4-kb fragment from the first allele disruption. (B) Like panel A, but digested with XhoI. Lane 1, parental strain CAI4 yields a 1.3-kb fragment and two large fragments. Lane 2, heterozygous LIG4/lig4::URA3 (CEA1); note that in addition to the bands arising from the wild-type allele, a new, 4.5-kb band indicates the insertion of the cassette in the other allele. Lane 3, heterozygous LIG4/lig4 strain after 5′-FOA selection (CEA1.5); The 4.5-kb band is reduced to a 1.6-kb fragment because of the loss of the URA3-hisG part of the cassette. Lane 4, lig4::URA3/lig4 double disruptant (CEA2). (C) Verification of LIG4 mutants and the revertant by PCR analysis. Shown in an agarose gel are PCR products obtained by amplification of genomic DNA from CAI4 (lane 1), the heterozygote (LIG4/lig4) (lane 2), the null mutant (lig4/lig4) (lane 4), and the revertant (lane 3) with the oligonucleotides indicated in Materials and Methods. (D) Top Northern analysis of LIG4 expression in wild-type CAI4 (lane 1), the heterozygote (LIG4/lig4) (CEA1) (lane 2), and the null strain (CEA2) (lane 3) with the XhoI/XhoI fragment as a probe. All the strains are Ura3⁺. (Bottom) rRNAs from the same samples stained with methylene blue. (E) Electrophoretic karyotypes of strains CAI4 (lane 1), CEA1 (LIG4/lig4::URA3) before (lane 2) and after (lane 3) treatment with SceI, and CEA2 (lig4::URA3/lig4) after treatment with SceI (lane 4), as shown by ethidium bromide staining (a) and Southern blot hybridization with the whole disruption cassette as a probe (b).

FIG. 4

Effect of LIG4 mutations on hyphal development. The indicated strains were grown on or in each medium for 5 days (solid media, panel A) or 36 h (liquid media, panel B). (A) Growth of strains in Spider medium at pH 6.8 (row 1) or pH 7.3 (row 2). The edges of colonies from each strain in Spider medium at pH 7.3 were photographed (magnification, ∼×15) (row 3). The growth of each strain on M-199 agar is also shown (row 4). (B) Microscopic growth of parental strain (CAF2) and CEA2 (null mutant) in liquid Spider medium and M-199 at 30°C. Pictures were taken with a Normarski objective (magnification, ∼×27).

FIG. 5

Northern analysis of the LIG4 transcript during the induction of germination. Cells from C. albicans 3153A grown in YPD medium for 2 days at 28°C were transferred to prewarmed YPD medium and incubated at 37°C. Samples were taken at the indicated times (minutes). (A) Schematic representation of the germination process. (B) Northern analysis of the samples shown in panel A with the XhoI/XhoI fragment as a probe (Fig. 2). (C) 23S rRNA from the same samples stained with methylene blue for quantification purposes.

FIG. 6

Sensitivities of C. albicans lig4 and S. cerevisiae rad52/rad52 mutants to 0.005% MMS (for details, see the text). Left to right, growth at fivefold dilutions.

FIG. 7

Survival of mice following infection with C. albicans Δlig4 mutants (CEA1 [▴], CEA2 [■], and CEA3 [×]) and parental strain CAF2 (⧫). Moribund animals were monitored daily for 21 days.

FIG. 8

Histological presentation of kidneys obtained from mice 24 h after infection with C. albicans CAF2 (A), CEA1 (B), CEA2 (C), and CEA3 (D). Original magnification, ×150. Sections were stained with Gomori's methenamine-silver nitrate.