DataSheet_1_Mid-Liver Stage Arrest of Plasmodium falciparum Schizonts in Primary Porcine Hepatocytes.docx

During co-evolution Plasmodium parasites and vertebrates went through a process of selection resulting in defined and preferred parasite-host combinations. As such, Plasmodium falciparum (Pf) sporozoites can infect human hepatocytes while seemingly incompatible with host cellular machinery of other species. The compatibility between parasite invasion ligands and their respective human hepatocyte receptors plays a key role in Pf host selectivity. However, it is unclear whether the ability of Pf sporozoites to mature in cross-species infection also plays a role in host tropism. Here we used fresh hepatocytes isolated from porcine livers to study permissiveness to Pf sporozoite invasion and development. We monitored intra-hepatic development via immunofluorescence using anti-HSP70, MSP1, EXP1, and EXP2 antibodies. Our data shows that Pf sporozoites can invade non-human hepatocytes and undergo partial maturation with a significant decrease in schizont numbers between day three and day five. A possible explanation is that Pf sporozoites fail to form a parasitophorous vacuolar membrane (PVM) during invasion. Indeed, the observed aberrant EXP1 and EXP2 staining supports the presence of an atypical PVM. Functions of the PVM include the transport of nutrients, export of waste, and offering a protective barrier against intracellular host effectors. Therefore, an atypical PVM likely results in deficiencies that may detrimentally impact parasite development at multiple levels. In summary, despite successful invasion of porcine hepatocytes, Pf development arrests at mid-stage, possibly due to an inability to mobilize critical nutrients across the PVM. These findings underscore the potential of a porcine liver model for understanding the importance of host factors required for Pf mid-liver stage development.

在协同进化过程中，疟原虫与脊椎动物经历了选择过程，形成了明确且具有偏好性的寄生虫-宿主组合。据此，恶性疟原虫（Plasmodium falciparum，下文简称Pf）子孢子可感染人类肝细胞，却似乎无法适配其他物种的宿主细胞机制。寄生虫入侵配体与其对应的人类肝细胞受体之间的相容性，是Pf宿主选择性的关键驱动因素。然而，目前尚不明确Pf子孢子在跨物种感染中的成熟能力，是否也在宿主嗜性的形成中发挥作用。本研究利用从猪肝中分离的新鲜肝细胞，探究其对Pf子孢子入侵与发育的容许性。我们借助靶向HSP70、MSP1、EXP1及EXP2的抗体，通过免疫荧光技术监测肝内发育进程。实验数据显示，Pf子孢子可入侵非人肝细胞并完成部分成熟过程，但在感染后第3天至第5天期间，裂殖体数量显著减少。一种可能的解释是，Pf子孢子在入侵过程中无法形成寄生泡膜（parasitophorous vacuolar membrane，下文简称PVM）。实际观测到的EXP1与EXP2异常染色，也证实了非典型PVM的存在。寄生泡膜的功能包括营养转运、废物排出，以及为抵御细胞内宿主效应因子提供保护性屏障。因此，非典型PVM很可能会造成功能缺陷，进而在多个层面对寄生虫发育产生不利影响。综上，尽管Pf子孢子可成功入侵猪肝肝细胞，但其发育会停滞在肝内中期阶段，这可能是由于无法通过PVM转运关键营养物质。本研究结果凸显了猪肝模型在解析Pf肝内中期发育所需宿主因子重要性方面的应用潜力。