Phenotypic Analysis and Virulence of Candida albicans LIG4 Mutants

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.

既往研究中，我们曾报道白色念珠菌（Candida albicans）编码DNA连接酶的基因的分离与初步表征。该基因（LIG4）是酵母与人类连接酶IV（ligase IV）的结构与功能同源物，参与DNA双链断裂（DNA double-strand breaks）的非同源末端连接（nonhomologous end joining, NHEJ）修复过程。本研究证实，白色念珠菌中不存在其他LIG4同源基因。为探究LIG4在形态发生与毒力中的功能，我们构建了基因敲除突变体。杂合子菌株的LIG4转录水平显著降低，而纯合敲除株中则完全无法检测到该转录本。与此相应，杂合子菌株的菌丝形成（myceliation）能力存在明显缺陷，纯合敲除株的缺陷程度更为显著。该表型在多种固体与液体培养基中均得到验证，包括Spider培养基、199培养基以及2%葡萄糖-1%酵母提取物-2% Bacto Peptone培养基，且在不同pH条件下均成立。将野生型等位基因（wild-type allele）回补至纯合敲除突变体后，细胞形成菌丝的能力得到部分恢复。与LIG4在形态发生中的正向调控作用一致，我们在形态转变过程中检测到LIG4的mRNA水平显著上调。LIG4并非DNA复制以及电离辐射（ionizing radiation）、紫外线（UV light）诱导的DNA损伤修复所必需，这提示此类DNA损伤主要通过同源重组（homologous recombination）通路完成修复。然而，我们的研究数据表明，白色念珠菌的非同源末端连接复合物（NHEJ apparatus）可能在该二倍体生物中调控形态发生过程。此外，敲除单拷贝或双拷贝LIG4基因均会导致念珠菌病小鼠模型（murine model of candidiasis）中的毒力衰减。