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Background <p>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 <i>Toxoplasma gondii</i> 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.</p> Methods <p>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 <i>T. gondii</i> tachyzoites.</p> Results <p>AlphaFold2 structural predictions revealed that TgSABP1 and its homologues contain a conserved globular domain (pLDDT &gt; 90) with significant structural homology (with root-mean-square deviation [RMSD] &lt; 4&#xa0;Å) to a <i>Plasmodium falciparum</i> 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 (<i>K</i><sub>D</sub> = 0.02 ± 0.01&#xa0;M) compared to SA (<i>K</i><sub>D</sub> = 2.07 ± 0.45&#xa0;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<sub>bind</sub> = −8.3&#xa0;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 <i>T. gondii</i> tachyzoites. </p> Conclusions <p>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 <i>T. gondii</i> 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. </p> Graphical abstract <p></p>

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A sialic acid-binding protein in Toxoplasma gondii contains a conserved globular domain in apicomplexan parasites

  • 期刊论文

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  • 发表日期：

    2025年7月1日

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  • Xiaoyu Sang,
  • Yize Liu,
  • Yiwei Zhang,
  • Ran Chen,
  • Ying Feng,
  • Ning Jiang,
  • Qijun Chen

摘要

Background

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.

Methods

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.

Results

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 (KD = 0.02 ± 0.01 M) compared to SA (KD = 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 (ΔGbind = −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.

Conclusions

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.

Graphical abstract