Intracellular pathogens like Toxoplasma gondii often target proteins and pathways critical for host cell survival and stress response. Molecular chaperones encoded by the evolutionary conserved Heat shock proteins (Hsps) maintain proteostasis and are vital to cell survival following exposure to any form of stress. A key protein of this family is Hsp70, an ATP-driven molecular chaperone, which is stress inducible and often indiscernible in normal cells. Role of this protein with respect to intracellular survival and multiplication of protozoan parasite like T. gondii remains to be examined. We find that T. gondii infection upregulates expression of host Hsp70. Hsp70 selective inhibitor 2-phenylethynesulfonamide (PES) attenuates intracellular T. gondii multiplication. Biotinylated PES confirms selective interaction of this small molecule inhibitor with Hsp70. We show that PES acts by disrupting Hsp70 chaperone function which leads to dysregulation of host autophagy. Silencing of host Hsp70 underscores its importance for intracellular multiplication of T. gondii, however, attenuation achieved using PES is not completely attributable to host Hsp70 indicating the presence of other intracellular targets of PES in infected host cells. We find that PES is also able to target T. gondii Hsp70 homologue which was shown using PES binding assay. Detailed molecular docking analysis substantiates PES targeting of TgHsp70 in addition to host Hsp70. While establishing the importance of protein quality control in infection, this study brings to the fore a unique opportunity of dual targeting of host and parasite Hsp70 demonstrating how structural conservation of these proteins may be exploited for therapeutic design.

像弓形虫一样的细胞内病原体通常靶向宿主细胞存活和应激反应的关键蛋白和途径。由进化保守的热休克蛋白（Hsps）编码的分子伴侣可以维持蛋白稳态，并且在暴露于任何形式的压力后对于细胞存活至关重要。该家族的关键蛋白是Hsp70，一种由ATP驱动的分子伴侣，它在正常细胞中是应激诱导的，通常是不可识别的。该蛋白相对于刚体弓形虫的原生动物寄生虫的细胞内存活和繁殖的作用尚待检验。我们发现刚地弓形虫感染上调了宿主Hsp70的表达。Hsp70选择性抑制剂2-苯基乙炔磺酰胺（PES）减弱细胞内T. gondii乘法。生物素化的PES证实了这种小分子抑制剂与Hsp70的选择性相互作用。我们表明，PES通过破坏Hsp70伴侣功能导致宿主自噬失调而起作用。宿主Hsp70的沉默强调了其对弓形虫细胞内增殖的重要性，但是，使用PES获得的减毒并不完全归因于宿主Hsp70，表明感染的宿主细胞中存在PES的其他胞内靶标。我们发现PES也能够针对弓形虫使用PES结合测定法显示的Hsp70同源物。除了宿主Hsp70外，详细的分子对接分析还证实了TgHsp70对PES的靶向作用。在确立蛋白质质量控​​制在感染中的重要性的同时，这项研究为宿主和寄生虫Hsp70的双重靶向提供了独特的机会，证明了如何将这些蛋白质的结构保守性用于治疗设计。