Pang Li Mei, Zeng Guisheng, Chow Eve Wai Ling, Xu Xiaoli, Li Ning, Kok Yee Jiun, Chong Shu Chen, Bi Xuezhi, Gao Jiaxin, Seneviratne Chaminda Jayampath, Wang Yue
A*STAR Infectious Diseases Labs (A*STAR ID Labs), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
Singapore Oral Microbiomics Initiative, National Dental Research Institute Singapore, National Dental Center Singapore, Singapore, Singapore.
mBio. 2025 Feb 5;16(2):e0328324. doi: 10.1128/mbio.03283-24. Epub 2024 Dec 17.
, the most frequently isolated fungal pathogen in humans, forms biofilms that enhance resistance to antifungal drugs and host immunity, leading to frequent treatment failure. Understanding the molecular mechanisms governing biofilm formation is crucial for developing anti-biofilm therapies. In this study, we conducted a genetic screen to identify novel genes that regulate biofilm formation in . One identified gene is , a homolog of the gene. The ∆/∆ mutant exhibited severe defects in biofilm formation and significantly reduced chitin content in the cell wall. Overexpression of the constitutively active version of the Rho1 GTPase Rho1, an upstream activator of the protein kinase C (PKC)-mitogen-activated protein kinase (MAPK) cell-wall integrity pathway, rescued these defects. Affinity purification, mass spectrometry, and co-immunoprecipitation revealed Sdd3's physical interaction with Bem2, the GTPase-activating protein of Rho1. Deletion of significantly reduced the amount of the active GTP-bound form of Rho1, thereby diminishing PKC-MAPK signaling and downregulating chitin synthase genes and . Taken together, our studies identify a new biofilm regulator, Sdd3, in that modulates Rho1 activity through its inhibitory interaction with Bem2, thereby regulating the PKC-MAPK pathway to control chitin biosynthesis, which is critical for biofilm formation. As an upstream component of the pathway and lacking a homolog in mammals, Sdd3 has the potential to serve as an antifungal target for biofilm infections.IMPORTANCEThe human fungal pathogen is categorized as a critical priority pathogen on the World Health Organization's Fungal Priority Pathogens List. A key virulence attribute of this pathogen is its ability to form biofilms on the surfaces of indwelling medical devices. Fungal cells in biofilms are highly resistant to antifungal drugs and host immunity, leading to treatment failure. This study conducted a genetic screen to discover novel genes that regulate biofilm formation. We found that deletion of the gene caused severe biofilm defects. Sdd3 negatively regulates the Rho1 GTPase, an upstream activator of the protein kinase C-mitogen-activated protein kinase pathway, through direct interaction with Bem2, the GTPase-activating protein of Rho1, resulting in a significant decrease in chitin content in the fungal cell wall. This chitin synthesis defect leads to biofilm formation failure. Given its essential role in biofilm formation, Sdd3 could serve as an antifungal target for biofilm infections.
白色念珠菌是人类最常分离出的真菌病原体，它会形成生物膜，增强对抗真菌药物和宿主免疫的抵抗力，导致治疗频繁失败。了解生物膜形成的分子机制对于开发抗生物膜疗法至关重要。在本研究中，我们进行了基因筛选，以鉴定调控白色念珠菌生物膜形成的新基因。一个鉴定出的基因是Sdd3，它是RHO1基因的同源物。∆sdd3/∆sdd3突变体在生物膜形成方面表现出严重缺陷，且细胞壁中几丁质含量显著降低。组成型激活的Rho1 GTP酶Rho1是蛋白激酶C（PKC）-丝裂原活化蛋白激酶（MAPK）细胞壁完整性途径的上游激活剂，其过表达挽救了这些缺陷。亲和纯化、质谱分析和免疫共沉淀揭示了Sdd3与Bem2（Rho1的GTP酶激活蛋白）的物理相互作用。删除SDD3显著降低了活性GTP结合形式的Rho1的量，从而减少了PKC-MAPK信号传导并下调了几丁质合酶基因CHS2和CHS3。综上所述，我们的研究在白色念珠菌中鉴定出一种新的生物膜调节因子Sdd3，它通过与Bem2的抑制性相互作用调节Rho1活性，从而调节PKC-MAPK途径以控制几丁质生物合成，这对生物膜形成至关重要。作为该途径的上游成分且在哺乳动物中缺乏同源物，Sdd3有潜力成为生物膜感染的抗真菌靶点。重要性人类真菌病原体白色念珠菌被列入世界卫生组织真菌重点病原体清单中的关键优先病原体。该病原体的一个关键毒力属性是其在植入式医疗设备表面形成生物膜的能力。生物膜中的真菌细胞对抗真菌药物和宿主免疫具有高度抗性，导致治疗失败。本研究进行了基因筛选以发现调控生物膜形成的新基因。我们发现删除SDD3基因会导致严重的生物膜缺陷。Sdd3通过与Rho1的GTP酶激活蛋白Bem2直接相互作用，负向调节蛋白激酶C-丝裂原活化蛋白激酶途径的上游激活剂Rho1 GTP酶，导致真菌细胞壁中几丁质含量显著降低。这种几丁质合成缺陷导致生物膜形成失败。鉴于其在生物膜形成中的关键作用，Sdd3可作为生物膜感染的抗真菌靶点。
