Sang Xiaoyu, Liu Yize, Zhang Yiwei, Chen Ran, Feng Ying, Jiang Ning, Chen Qijun
Key Laboratory of Livestock Infectious Diseases, Ministry of Education, and Key Laboratory of Ruminant Infectious Disease Prevention and Control (East), Ministry of Agriculture and Rural Affairs, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, 120 Dongling Road, Shenyang, 110866, China.
Research Unit for Pathogenic Mechanisms of Zoonotic Parasites, Chinese Academy of Medical Sciences, 120 Dongling Road, Shenyang, 110866, China.
Parasit Vectors. 2025 Jul 1;18(1):239. doi: 10.1186/s13071-025-06845-5.
Apicomplexan protozoans employ an intricate invasion mechanism involving dynamic interactions with host cells, characterized by sequential secretion of adhesins and lectins. Our laboratory previously identified TgSABP1, a novel Toxoplasma gondii adhesin, demonstrating specific binding affinity for sialic acid (SA) receptors on host cell surfaces. However, the structural determinants governing SA recognition by this adhesin remain undefined.
Three-dimensional structural predictions of TgSABP1 and homologous proteins were generated using AlphaFold2. Bio-layer interferometry (BLI) quantified the binding affinities between the recombinant proteins and ligands. Competitive BLI assays evaluated small molecules that potentially inhibit the TgSABP1-sialyllactose interactions. Molecular docking simulations employing AutoDock Vina software elucidated ligand-binding site interactions. In vitro invasion inhibition assays were performed to assess the therapeutic potential of lead compounds targeting TgSABP1 against T. gondii tachyzoites.
AlphaFold2 structural predictions revealed that TgSABP1 and its homologues contain a conserved globular domain (pLDDT > 90) with significant structural homology (with root-mean-square deviation [RMSD] < 4 Å) to a Plasmodium falciparum invasion-related protein PfIMP2 (PDB: 5LG9). BLI quantification demonstrated the micromolar binding affinities of the recombinant proteins for 3'-sialyllactose-polyacrylamide (PAA) and 6'-sialyllactose (6'SL)-PAA. Intriguingly, although recombinant TgSABP1 showed stronger lactose binding (K = 0.02 ± 0.01 M) compared to SA (K = 2.07 ± 0.45 M), only the latter exhibited an inhibition on the TgSABP1-6'SL-PAA interaction. Virtual screening of Food and Drug Administration (FDA)-approved compounds identified eltrombopag as a high-affinity molecule (ΔG = -8.3 kcal/mol) targeting the SA-binding pocket in TgSABP1. Functional validation demonstrated that eltrombopag effectively blocked the TgSABP1/6'SL-PAA interaction and significantly decreased host cell invasion of T. gondii tachyzoites.
Our study reveals a conserved globular domain of apicomplexan parasites as a novel SA-binding domain. Structural and functional characterization demonstrates its critical role in mediating TgSABP1-host cell interactions. Targeting this SA-binding pocket with eltrombopag effectively decreased T. gondii tachyzoite invasion, suggesting its therapeutic potential as an anti-invasion target. These findings not only elucidate a conserved mechanism underlying host receptor recognition in apicomplexans, but also establish a structural framework for the rational design of broad-spectrum inhibitors targeting invasion-related lectin domains.
顶复门原生动物采用一种复杂的入侵机制，涉及与宿主细胞的动态相互作用，其特征是依次分泌粘附素和凝集素。我们实验室先前鉴定出TgSABP1，一种新型的刚地弓形虫粘附素，它对宿主细胞表面的唾液酸（SA）受体具有特异性结合亲和力。然而，这种粘附素识别SA的结构决定因素仍不明确。
使用AlphaFold2生成TgSABP1和同源蛋白的三维结构预测。生物层干涉术（BLI）定量重组蛋白与配体之间的结合亲和力。竞争性BLI分析评估可能抑制TgSABP1 - 唾液酸乳糖相互作用的小分子。使用AutoDock Vina软件进行分子对接模拟，以阐明配体结合位点相互作用。进行体外入侵抑制试验，以评估靶向TgSABP1的先导化合物对刚地弓形虫速殖子的治疗潜力。
AlphaFold2结构预测显示，TgSABP1及其同源物包含一个保守的球状结构域（pLDDT>90），与恶性疟原虫入侵相关蛋白PfIMP2（PDB：5LG9）具有显著的结构同源性（均方根偏差[RMSD]<4Å）。BLI定量表明重组蛋白对3'-唾液酸乳糖 - 聚丙烯酰胺（PAA）和6'-唾液酸乳糖（6'SL）-PAA具有微摩尔级的结合亲和力。有趣的是，尽管重组TgSABP1与SA（K = 2.07±0.45 M）相比显示出更强的乳糖结合（K = 0.02±0.01 M），但只有后者对TgSABP1 - 6'SL - PAA相互作用表现出抑制作用。对美国食品药品监督管理局（FDA）批准的化合物进行虚拟筛选，确定艾曲泊帕是一种靶向TgSABP1中SA结合口袋的高亲和力分子（ΔG = -8.3 kcal/mol）。功能验证表明，艾曲泊帕有效地阻断了TgSABP1/6'SL - PAA相互作用，并显著降低了刚地弓形虫速殖子对宿主细胞的入侵。
我们的研究揭示了顶复门寄生虫的一个保守球状结构域作为一种新型的SA结合结构域。结构和功能表征证明了其在介导TgSABP1 - 宿主细胞相互作用中的关键作用。用艾曲泊帕靶向这个SA结合口袋可有效降低刚地弓形虫速殖子的入侵，表明其作为抗入侵靶点的治疗潜力。这些发现不仅阐明了顶复门寄生虫宿主受体识别的保守机制，还为合理设计靶向入侵相关凝集素结构域的广谱抑制剂建立了结构框架。
