Pharmaceutical Sciences Program, College of Graduate Health Sciences, University of Tennessee Health Science Center, Memphis, Tennessee, USA.
Department of Surgery, Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee, USA.
mSphere. 2024 Oct 29;9(10):e0057524. doi: 10.1128/msphere.00575-24. Epub 2024 Sep 24.
To adapt to various host microenvironments, the human fungal pathogen possesses the capacity to accumulate and store glycogen as an internal carbohydrate source. In the model yeast , Glc7p and Gac1p are the serine/threonine type 1 protein phosphatase catalytic and regulatory subunits that control glycogen synthesis by altering the phosphorylation state of the glycogen synthase Gsy2p. Despite recent delineation of the glycogen synthesis pathway in , the molecular events driving synthase activation are currently undefined. In this study, using a combination of microbiologic and genetic techniques, we determined that the protein encoded by uncharacterized gene , and not the currently annotated Gac1p, is the major regulatory subunit involved in glycogen synthesis. C1_01140Cp contains a conserved GVNK motif observed across multiple starch/glycogen-binding proteins in various species, and alanine substitution of each residue in this motif significantly impaired glycogen accumulation in . Fluorescent protein tagging and microscopy indicated that C1_01140Cp-GFPy colocalized with Glc7p-tdTomato and Gsy1p-tdTomato accordingly. Co-immunoprecipitation assays further confirmed that C1_01140Cp associates with Glc7p and Gsy1p during glycogen synthesis. Lastly, Δ/Δ exhibited colonization defects in a murine model of vulvovaginal candidiasis. Collectively, our data indicate that uncharacterized C1_01140Cp is the functional ortholog of the PPP1R subunit Gac1p in .IMPORTANCEThe capacity to synthesize glycogen offers microbes metabolic flexibility, including the fungal pathogen . In , dephosphorylation of glycogen synthase by the Glc7p-containing phosphatase is a critical rate-limiting step in glycogen synthesis. Subunits, including Gac1p, target Glc7p to α-1,4-glucosyl primers for efficient Gsy2p synthase activation. However, this process in had not been delineated. Here, we show that the genome encodes for two homologous phosphatase-binding subunits, annotated Gac1p and uncharacterized C1_01140Cp, both containing a GVNK motif required for polysaccharide affinity. Surprisingly, loss of Gac1p only moderately reduced glycogen accumulation, whereas loss of C1_01140Cp ablated it. Fluorescence microscopy and co-immunoprecipitation approaches revealed that C1_01140Cp associates with Glc7p and Gsy1p during glycogen synthesis. Moreover, C1_01140Cp contributed to fungal fitness at the vaginal mucosa during murine vaginitis. Therefore, this work demonstrates that glycogen synthase regulation is conserved in and C1_01140Cp is the functional ortholog of Gac1p.
为了适应各种宿主微环境，人类真菌病原体具有积累和储存糖原作为内部碳水化合物来源的能力。在模式酵母中，Glc7p 和 Gac1p 是丝氨酸/苏氨酸 1 型蛋白磷酸酶的催化亚基和调节亚基，通过改变糖原合酶 Gsy2p 的磷酸化状态来控制糖原合成。尽管最近已经确定了 在中的糖原合成途径，但目前尚不清楚驱动合酶激活的分子事件。在这项研究中，我们使用微生物学和遗传学技术的组合，确定了未被描述的基因编码的蛋白，而不是当前注释的 Gac1p，是参与糖原合成的主要调节亚基。C1_01140Cp 包含一个保守的 GVNK 基序，在各种物种的多个淀粉/糖原结合蛋白中都有观察到，并且该基序中每个残基的丙氨酸取代显著削弱了 在中的糖原积累。荧光蛋白标记和显微镜观察表明，C1_01140Cp-GFPy 与 Glc7p-tdTomato 和 Gsy1p-tdTomato 相应地共定位。免疫共沉淀实验进一步证实，C1_01140Cp 在糖原合成过程中与 Glc7p 和 Gsy1p 结合。最后，Δ/Δ 在阴道念珠菌病的小鼠模型中表现出定植缺陷。总的来说，我们的数据表明，未被描述的 C1_01140Cp 是 在中的 PPP1R 亚基 Gac1p 的功能同源物。
合成糖原的能力为微生物提供了代谢灵活性，包括真菌病原体 。在 中，Glc7p 包含的磷酸酶对糖原合酶的去磷酸化是糖原合成的关键限速步骤。包括 Gac1p 在内的亚基将 Glc7p 靶向到α-1,4-葡萄糖基引物，以有效激活 Gsy2p 合酶。然而，这一过程在 中尚未被描绘。在这里，我们表明 基因组编码两个同源的磷酸酶结合亚基，注释的 Gac1p 和未被描述的 C1_01140Cp，都包含一个 GVNK 基序，该基序是多糖亲和力所必需的。令人惊讶的是，Gac1p 的缺失仅适度降低了糖原的积累，而 C1_01140Cp 的缺失则使其完全消除。荧光显微镜和免疫共沉淀方法表明，C1_01140Cp 在糖原合成过程中与 Glc7p 和 Gsy1p 结合。此外，C1_01140Cp 在小鼠阴道炎期间有助于阴道黏膜上的真菌适应性。因此，这项工作表明糖原合酶的调节在 中是保守的，C1_01140Cp 是 Gac1p 的功能同源物。
