The role of microsporidian polar tube protein 4 (PTP4) in host cell infection

Microsporidia have been identified as pathogens that have important effects on our health, food security and economy. A key to the success of these obligate intracellular pathogens is their unique invasion organelle, the polar tube, which delivers the nucleus containing sporoplasm into host cells during invasion. Due to the size of the polar tube, the rapidity of polar tube discharge and sporoplasm passage, and the absence of genetic techniques for the manipulation of microsporidia, study of this organelle has been difficult and there is relatively little known regarding polar tube formation and the function of the proteins making up this structure. Herein, we have characterized polar tube protein 4 (PTP4) from the microsporidium Encephalitozoon hellem and found that a monoclonal antibody to PTP4 labels the tip of the polar tube suggesting that PTP4 might be involved in a direct interaction with host cell proteins during invasion. Further analyses employing indirect immunofluorescence (IFA), enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays confirmed that PTP4 binds to mammalian cells. The addition of either recombinant PTP4 protein or anti-PTP4 antibody reduced microsporidian infection of its host cells in vitro. Proteomic analysis of PTP4 bound to host cell membranes purified by immunoprecipitation identified transferrin receptor 1 (TfR1) as a potential host cell interacting partner for PTP4. Additional experiments revealed that knocking out TfR1, adding TfR1 recombinant protein into cell culture, or adding anti-TfR1 antibody into cell culture significantly reduced microsporidian infection rates. These results indicate that PTP4 is an important protein competent of the polar tube involved in the mechanism of host cell infection utilized by these pathogens.

微孢子虫（Microsporidia）已被证实为一类对人类健康、粮食安全及经济发展具有重要影响的病原体。这类专性胞内病原体（obligate intracellular pathogens）得以成功侵染宿主的关键在于其独特的侵染细胞器——极丝（polar tube）：在侵染过程中，极丝可将携带孢原质（sporoplasm）的细胞核注入宿主细胞内。由于极丝体积微小、弹射与孢原质转运过程极为迅速，且目前尚无成熟的微孢子虫遗传操作技术，对该细胞器的研究始终存在诸多困难，学界对极丝的形成机制及其结构蛋白的功能认知仍相对匮乏。本研究对脑炎微孢子虫（Encephalitozoon hellem）的极丝蛋白4（polar tube protein 4, PTP4）进行了功能鉴定，发现靶向PTP4的单克隆抗体（monoclonal antibody）可标记极丝的尖端，提示PTP4可能在侵染过程中直接与宿主细胞蛋白发生相互作用。后续通过间接免疫荧光试验（indirect immunofluorescence, IFA）、酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）及荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）等方法开展的进一步分析证实，PTP4可结合哺乳动物细胞。在体外实验中，无论是添加重组PTP4蛋白还是抗PTP4抗体，均可降低微孢子虫对宿主细胞的侵染效率。通过免疫沉淀（immunoprecipitation）纯化结合于宿主细胞膜的PTP4并进行蛋白质组学分析，本研究鉴定出转铁蛋白受体1（transferrin receptor 1, TfR1）为PTP4的潜在宿主互作伴侣蛋白。额外实验结果显示，敲除TfR1基因、向细胞培养体系中添加重组TfR1蛋白或抗TfR1抗体，均可显著降低微孢子虫的侵染率。上述结果表明，PTP4作为极丝的关键组成蛋白，参与了这类病原体介导的宿主细胞侵染机制。