Data_Sheet_1_The F1Fo-ATP Synthase β Subunit Is Required for Candida albicans Pathogenicity Due to Its Role in Carbon Flexibility.doc

Previous work has explored link between mitochondrial biology and fungal pathogenicity in F1Fo-ATP synthase in Candida albicans. In this work we have detailed the more specific roles of the F1Fo-ATP synthase β subunit, a key protein subunit of F1Fo-ATP synthase. The ability to assimilate alternative carbons in glucose-limited host niches is known to be a critical factor for infection caused by opportunistic pathogens including C. albicans. The function of the F1Fo-ATP synthase β subunit was characterized through the construction of an ATP2 gene null mutant (atp2Δ/Δ) and the gene-reconstituted strain (atp2Δ/ATP2) in order to understand the link between carbon metabolism and C. albicans pathogenesis. Cell growth, viability, cellular ATP content, mitochondrial membrane potential (ΔΨm), and intracellular ROS were compared between null mutant and control strain. Results showed that growth of the atp2Δ/Δ mutant in synthetic medium was slower than in complex medium. However, the synthetic medium delayed the onset of reduced cell viability and kept cellular ATP content from becoming fully depleted. Consistent with these observations, we identified transcriptional changes in metabolic response that activated other ATP-generating pathways, thereby improving cell viability during the initial phase. Unlike glucose effects, the atp2Δ/Δ mutant exhibited an immediate and sharp reduction in cell viability on non-fermentable carbon sources, consistent with an immediate depletion of cellular ATP content. Along with a reduced viability in non-fermentable carbon sources, the atp2Δ/Δ mutant displayed avirulence in a murine model of disseminated candidiasis as well as lower fungal loads in mouse organs. Regardless of the medium, however, a decrease in mitochondrial membrane potential (ΔΨm) was found in the atp2Δ/Δ mutant but ROS levels remained in the normal range. These results suggest that the F1Fo-ATP synthase β subunit is required for C. albicans pathogenicity and operates by affecting metabolic flexibility in carbon consumption.

先前的研究已经探讨了线粒体生物学与白色念珠菌中F1Fo-ATP合酶真菌致病性之间的联系。在本研究中，我们详细阐述了F1Fo-ATP合酶β亚单位在F1Fo-ATP合酶这一关键蛋白亚单位中更为特定的作用。在葡萄糖受限的宿主生境中，同化替代碳源的能力被认为是包括白色念珠菌在内的机会性病原体引起感染的关键因素。通过构建ATP2基因敲除突变体（atp2Δ/Δ）及其基因重组成株（atp2Δ/ATP2），我们表征了F1Fo-ATP合酶β亚单位的职能，旨在揭示碳代谢与白色念珠菌致病性之间的关联。我们比较了突变体与对照株在细胞生长、存活率、细胞内ATP含量、线粒体膜电位（ΔΨm）以及细胞内活性氧（ROS）水平之间的差异。结果显示，在合成培养基中，atp2Δ/Δ突变体的生长速度慢于在复杂培养基中。然而，合成培养基延缓了细胞存活率降低的开始，并防止了细胞内ATP含量的完全耗竭。与这些观察结果一致，我们发现了代谢响应中的转录变化，这些变化激活了其他ATP生成途径，从而在初始阶段提高了细胞存活率。与葡萄糖效应不同，atp2Δ/Δ突变体在非发酵性碳源上表现出细胞存活率的立即且急剧下降，这与细胞内ATP含量的立即耗竭一致。与在非发酵性碳源中的降低存活率相伴，atp2Δ/Δ突变体在播散性念珠菌病小鼠模型中表现出非致病性，以及小鼠器官中的真菌负荷减少。然而，无论在何种培养基中，atp2Δ/Δ突变体的线粒体膜电位（ΔΨm）均有所下降，但ROS水平仍处于正常范围内。这些结果表明，F1Fo-ATP合酶β亚单位对于白色念珠菌的致病性至关重要，并通过影响碳消耗的代谢灵活性来实现其作用。