Glycogen synthase activity in Candida albicans is partly controlled by the functional ortholog of Saccharomyces cerevisiae Gac1p.

IF 3.7 2区 生物学 Q2 MICROBIOLOGY
mSphere Pub Date : 2024-10-29 Epub Date: 2024-09-24 DOI:10.1128/msphere.00575-24
Jian Miao, David L Williams, Michael D Kruppa, Brian M Peters
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引用次数: 0

Abstract

To adapt to various host microenvironments, the human fungal pathogen Candida albicans possesses the capacity to accumulate and store glycogen as an internal carbohydrate source. In the model yeast Saccharomyces cerevisiae, ScGlc7p and ScGac1p 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 C. albicans, 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 C1_01140C, and not the currently annotated C. albicans 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 C. albicans. Fluorescent protein tagging and microscopy indicated that C1_01140Cp-GFPy colocalized with CaGlc7p-tdTomato and CaGsy1p-tdTomato accordingly. Co-immunoprecipitation assays further confirmed that C1_01140Cp associates with CaGlc7p and CaGsy1p during glycogen synthesis. Lastly, c1_01140cΔ/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 ScGac1p in C. albicans.IMPORTANCEThe capacity to synthesize glycogen offers microbes metabolic flexibility, including the fungal pathogen Candida albicans. In Saccharomyces cerevisiae, dephosphorylation of glycogen synthase by the ScGlc7p-containing phosphatase is a critical rate-limiting step in glycogen synthesis. Subunits, including ScGac1p, target ScGlc7p to α-1,4-glucosyl primers for efficient ScGsy2p synthase activation. However, this process in C. albicans had not been delineated. Here, we show that the C. albicans genome encodes for two homologous phosphatase-binding subunits, annotated CaGac1p and uncharacterized C1_01140Cp, both containing a GVNK motif required for polysaccharide affinity. Surprisingly, loss of CaGac1p only moderately reduced glycogen accumulation, whereas loss of C1_01140Cp ablated it. Fluorescence microscopy and co-immunoprecipitation approaches revealed that C1_01140Cp associates with CaGlc7p and CaGsy1p 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 C. albicans and C1_01140Cp is the functional ortholog of ScGac1p.

白色念珠菌中糖原合成酶的活性部分受控于酿酒酵母 Gac1p 的功能直向同源物。
为了适应各种宿主微环境,人类真菌病原体白色念珠菌具有积累和储存糖原作为内部碳水化合物来源的能力。在模式酵母酿酒酵母中,ScGlc7p 和 ScGac1p 是丝氨酸/苏氨酸 1 型蛋白磷酸酶催化和调节亚基,它们通过改变糖原合成酶 Gsy2p 的磷酸化状态来控制糖原合成。尽管最近对白僵菌糖原合成途径进行了描述,但驱动合成酶活化的分子事件目前仍未确定。在这项研究中,我们结合使用了微生物学和遗传学技术,确定了未定性基因 C1_01140C 所编码的蛋白质,而不是目前已注释的白僵菌 Gac1p,是参与糖原合成的主要调控亚基。C1_01140Cp 包含一个在不同物种的多个淀粉/糖原结合蛋白中观察到的保守 GVNK 基序,该基序中每个残基的丙氨酸替换都会显著影响白僵菌的糖原累积。荧光蛋白标记和显微镜检查表明,C1_01140Cp-GFPy 与 CaGlc7p-tdTomato 和 CaGsy1p-tdTomato 有相应的共定位。共免疫沉淀试验进一步证实,C1_01140Cp 在糖原合成过程中与 CaGlc7p 和 CaGsy1p 结合。最后,c1_01140cΔ/Δ 在小鼠外阴阴道念珠菌病模型中表现出定植缺陷。总之,我们的数据表明,未表征的 C1_01140Cp 是白念珠菌中 PPP1R 亚基 ScGac1p 的功能直向同源物。重要意义合成糖原的能力为微生物提供了新陈代谢的灵活性,包括真菌病原体白念珠菌。在酿酒酵母中,糖原合成酶被含有 ScGlc7p 的磷酸酶去磷酸化是糖原合成的关键限速步骤。包括 ScGac1p 在内的亚基将 ScGlc7p 与 α-1,4-葡萄糖基引物连接,以有效激活 ScGsy2p 合成酶。然而,白僵菌中的这一过程尚未被明确。在这里,我们发现白僵菌基因组编码两个同源的磷酸酶结合亚基,即已注释的 CaGac1p 和未表征的 C1_01140Cp,两者都含有多糖亲和性所需的 GVNK 基序。令人惊讶的是,CaGac1p 的缺失只会适度减少糖原累积,而 C1_01140Cp 的缺失则会消除糖原累积。荧光显微镜和共免疫沉淀方法显示,C1_01140Cp 在糖原合成过程中与 CaGlc7p 和 CaGsy1p 结合。此外,在小鼠阴道炎期间,C1_01140Cp 对阴道粘膜的真菌适应性也有贡献。因此,这项工作证明糖原合成酶的调控在白僵菌中是保守的,而 C1_01140Cp 是 ScGac1p 的功能直向同源物。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
mSphere
mSphere Immunology and Microbiology-Microbiology
CiteScore
8.50
自引率
2.10%
发文量
192
审稿时长
11 weeks
期刊介绍: mSphere™ is a multi-disciplinary open-access journal that will focus on rapid publication of fundamental contributions to our understanding of microbiology. Its scope will reflect the immense range of fields within the microbial sciences, creating new opportunities for researchers to share findings that are transforming our understanding of human health and disease, ecosystems, neuroscience, agriculture, energy production, climate change, evolution, biogeochemical cycling, and food and drug production. Submissions will be encouraged of all high-quality work that makes fundamental contributions to our understanding of microbiology. mSphere™ will provide streamlined decisions, while carrying on ASM''s tradition for rigorous peer review.
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