Prieto Daniel, Román Elvira, Correia Inês, Pla Jesus
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, Madrid, Spain.
PLoS One. 2014 Jan 27;9(1):e87128. doi: 10.1371/journal.pone.0087128. eCollection 2014.
The opportunistic pathogen Candida albicans is a frequent inhabitant of the human gastrointestinal tract where it usually behaves as a harmless commensal. In this particular niche, it needs to adapt to the different micro environments that challenge its survival within the host. In order to determine those factors involved in gut adaptation, we have used a gastrointestinal model of colonization in mouse to trace the behaviour of fungal cells. We have developed a genetic labelling system based on the complementary spectral properties of the fluorescent proteins GFP and a new C. albicans codon-adapted RFP (dTOM2) that allow a precise quantification of the fungal population in the gut via standard in vitro cultures or flow cytometry. This methodology has allowed us to determine the role of the three MAP kinase pathways of C. albicans (mediated by the MAPK Mkc1, Cek1 or Hog1) in mouse gut colonization via competitive assays with MAPK pathway mutants and their isogenic wild type strain. This approach reveals the signalling through HOG pathway as a critical factor influencing the establishment of C. albicans in the mouse gut. Less pronounced effects for mkc1 or cek1 mutants were found, only evident after 2-3 weeks of colonization. We have also seen that hog1 mutants is defective in adhesion to the gut mucosa and sensitive to bile salts. Finally, we have developed a genetic strategy for the in vivo excision (tetracycline-dependent) of any specific gene during the course of colonization in this particular niche, allowing the analysis of its role during gut colonization.
机会性致病原白色念珠菌是人类胃肠道的常见寄居菌，在胃肠道中它通常作为无害的共生菌存在。在这个特定的生态位中，它需要适应不同的微环境，这些微环境对其在宿主体内的生存构成挑战。为了确定参与肠道适应的因素，我们利用小鼠胃肠道定植模型来追踪真菌细胞的行为。我们基于荧光蛋白GFP和一种新的白色念珠菌密码子优化的RFP（dTOM2）的互补光谱特性，开发了一种基因标记系统，该系统可通过标准体外培养或流式细胞术对肠道中的真菌群体进行精确量化。这种方法使我们能够通过与丝裂原活化蛋白激酶（MAPK）途径突变体及其同基因野生型菌株的竞争试验，确定白色念珠菌的三条MAPK途径（由MAPK Mkc1、Cek1或Hog1介导）在小鼠肠道定植中的作用。该方法揭示了通过高渗甘油（HOG）途径的信号传导是影响白色念珠菌在小鼠肠道中定植的关键因素。对于mkc1或cek1突变体，发现其影响不太明显，仅在定植2至3周后才明显。我们还发现hog1突变体在黏附于肠道黏膜方面存在缺陷，并且对胆盐敏感。最后，我们开发了一种基因策略，用于在这个特定生态位的定植过程中对任何特定基因进行体内切除（四环素依赖性），从而能够分析其在肠道定植过程中的作用。
