摘要
恶性疟原虫（Plasmodium falciparum）和弓形虫（Toxoplasma gondii）是顶复门原虫的两个代表虫种，是专性细胞内寄生虫，可以感染真核生物的多种宿主细胞。疟原虫裂殖子在血细胞期主要侵入宿主的红细胞，并在红细胞内发育繁殖。感染恶性疟原虫的红细胞可以与宿主毛细血管的内皮细胞粘附，进而逃避脾脏的清除。而弓形虫可以侵入任何有核细胞，但两种虫体入侵宿主细胞的分子机制尚不完全清楚。研究发现，顶复门寄生虫侵入宿主细胞是一个连续的过程，侵入的必要条件是虫体的多种分子（配体）与宿主细胞表面受体粘附。肝素类糖胺聚糖分子是虫体侵入细胞的主要受体，肝素类分子能够抑制虫体对细胞的侵入功能，但是与该类受体相互作用的关键分子及其作用机制目前还不清楚。而对这些分子的阐明不但可以解释疟原虫、弓形虫的分子致病机理，还有利于寻找阻断虫体在宿主体内繁殖的药物靶标和疫苗候选抗原。
迄今为止，在疟原虫和弓形虫中确定的与肝素结合的蛋白质非常有限。早期研究结果表明，肝素能够抑制恶性疟原虫侵入宿主红细胞，并认为肝素通过与裂殖子表面蛋白质MSP1相互作用，进而阻断了裂殖子与红细胞表面的粘附。而目前发现的与肝素结合的刚地弓形虫蛋白质有5个，他们分别是棒状体蛋白质ROP2和ROP4，致密颗粒蛋白GRA2，表面蛋白质SAG1和SAG3。但这些结果并不能完全解释肝素抑制虫体入侵的全部机理。
在本研究中，我们提出了一种新的研究方法，即基于受体的亲和纯化，再结合高通量蛋白质质谱分析的方法，首次对恶性疟原虫和刚地弓形虫宿主细胞侵入相关的蛋白质组进行了全面和深入的研究，在鉴定了两种虫体与肝素结合的蛋白质组的基础上，比较两种虫体与肝素结合的蛋白质的功能特征。最终用大量的实验数据揭示了肝素抑制恶性疟原虫和刚地弓形虫侵入宿主细胞的作用机理。我们认为，肝素主要是通过与疟原虫、弓形虫表面及顶复合体中的棒状体分子结合，阻断这些细胞黏附分子与宿主细胞表面受体的作用，进而抑制虫体对宿主细胞的侵入。
1.肝素类分子抑制虫体侵入的分析
我们通过体外抑虫实验，将不同浓度的肝素类似物（包括肝素、硫化肝素和硫酸葡聚糖）加入虫体培养基中，比较这些分子对虫体侵入细胞的抑制能力。在我们的实验中，所有的样品都进行了三次重复实验，在每次重复实验中设有三个平行实验。结果显示：随着肝素、硫化肝素、硫酸葡聚糖浓度的增加，裂殖子的相对侵入率下降，抑制作用逐渐增强，而硫酸软骨素没有抑制作用。硫化肝素的抑制作用较弱，只有当其浓度达到1000μg/mL时，才有抑制作用，而低浓度时没有抑制作用。结果进一步证实了肝素类分子能够抑制虫体侵入。
2.恶性疟原虫、弓形虫肝素结合蛋白质组的鉴定及比较分析
本研究首先用人的红细胞培养恶性疟原虫3D7株，用昆明鼠培养弓形虫RH株速殖子，然后利用Percoll梯度离心的方法纯化疟原虫裂殖体和弓形虫速殖子。将纯化后的虫体悬浮于含有蛋白酶抑制剂的磷酸盐缓冲液中，反复冻融几次后高速离心。将含有虫体可溶性蛋白质的上清液与Heparin-sepharose混合，然后洗脱掉未结合的蛋白质，用SDS-PAGE和Western-Blot方法鉴定与肝素结合的蛋白质。结果显示：无论是疟原虫还是弓形虫，均有多种蛋白质与Heparin-sepharose结合。进一步实验结果表明，高浓度的肝素可以竞争性抑制虫体蛋白与Heparin-sepharose结合，而硫酸软骨素A完全没有抑制作用，且未偶联肝素的Sepharose与虫体蛋白不结合，由此说明这些蛋白质与肝素的结合具有特异性。
为了鉴定与肝素结合的恶性疟原虫、弓形虫的蛋白质组，我们采用亲和层析的方法，首先纯化与Heparin-sepharose特异性结合的蛋白质，然后将蛋白质酶解成肽段，进行二维液相色谱与质谱串联分析。我们根据胰蛋白酶的酶切规律，对混合数据库中的参考序列进行模拟酶切，与生成的质谱峰文件进行比对，鉴定出匹配的肽段。结果显示：我们总共鉴定到了6062个独有肽段，通过与疟原虫数据库比对，共鉴定出811个恶性疟原虫蛋白质。其中99%的多肽来源于恶性疟原虫3D7，仅有87个多肽与人和牛的蛋白质匹配。用同样的方法，我们从弓形虫的肝素结合蛋白质中鉴定到了6252个独有肽段，通过与NCBI数据库比对，共鉴定出956个弓形虫蛋白质和188个小鼠蛋白质。研究进一步说明，肝素可以与恶性疟原虫裂殖子和刚地弓形虫速殖子的多种蛋白质相互作用。
我们将鉴定到的恶性疟原虫和弓形虫的蛋白质组按照分布部位和生物学功能进行分类。结果显示：蛋白质的亚细胞定位分布相当广泛，包括细胞质、细胞核、线粒体，以及棒状体、微线体等16类。通过GO分析和相关文献查询，可以进一步将这些肝素结合蛋白质分为14个生物学功能亚类，且恶性疟原虫和弓形虫的蛋白质组在相同的亚细胞定位和生物学功能分类中，数量相差不大，由此说明顶复门寄生虫可能具有相似的宿主细胞侵入机制。此外，还发现了很多功能未知的蛋白质，且弓形虫组显著高于恶性疟原虫组，这说明了多数弓形虫蛋白质的功能尚不清楚。在这些功能相关的蛋白质中与红细胞侵入相关的恶性疟原虫蛋白质有47个，与有核细胞侵入相关的弓形虫蛋白质有33个，其中多数为棒状体相关蛋白质。棒状体是顶复门寄生虫独有的细胞器，该细胞器分泌的一些蛋白质参与了虫体与宿主细胞间移动接头的形成。在裂殖子侵入宿主细胞的过程中，肝素通过与棒状体蛋白质相互作用，可能抑制了移动接头的组装，或者阻断了他们与宿主细胞膜受体的结合。更为重要的是，在这些肝素结合蛋白质中有170个蛋白质在疟原虫和弓形虫中呈现相同的亚细胞定位和生物学功能。由此说明两种虫体具有相似的细胞侵入机理。
3.恶性疟原虫重组蛋白质的肝素抑制实验及体外抑虫实验
我们将与肝素结合的16个蛋白质分别克隆到pGEX-4T-1或pET32a载体上，用重组蛋白质进一步验证肝素结合的特异性。结果显示：16个分子中有14个蛋白质片段成功地获得了可溶性表达，可以进行后续的肝素结合实验。其中，有9个蛋白质片段与Heparin-sepharose结合，5个蛋白质片段不与Heparin-sepharose结合，这说明了蛋白质与肝素的结合是特异的。
最后，为了进一步验证这些蛋白质的功能，我们通过抑虫实验检测了恶性疟原虫四个重组蛋白质抑制虫体侵入的能力。结果显示：棒状体蛋白3-48，丝氨酸重复抗原5-50和裂殖子表面蛋白1-42均能抑制虫体侵入红细胞，而裂殖子表面蛋白1-33和谷胱甘肽-S-转移酶没有抑制作用。因此说明与肝素结合的裂殖子相关蛋白质在虫体侵入细胞过程中发挥了重要作用。
综上所述，本研究通过受体亲和层析结合高通量蛋白质组学分析方法及一系列验证实验，发现了恶性疟原虫和刚地弓形虫与肝素相互作用的多种蛋白质分子，其中与侵入相关的分子主要分布在棒状体细胞器中。该研究结果不但确定了肝素类分子是疟原虫和弓形虫侵入宿主细胞的重要受体，还揭示了肝素抑制虫体侵入宿主细胞的作用机理，也进一步证实了肝素衍生物作为抗虫药物的可行性。
Plasmodium falciparum and Toxoplasma gondii are the representative strains ofapicomplexan parasites capable of infecting a broad range of eukaryotic host cells asobligate intracellular pathogens. P. falciparum merozoites invade and multiply withinhost erythrocytes during the blood-stage of their life cycle and the infected red bloodcells adhere to host endothelial line to avoid spleen-mediated clearance. T. gondii isunique as it can invade virtually any nucleated cells, although the mechanisms havenot been completely understood. The prerequisite for host invasion of allapicomplaxan parasites is the attachment of the parasite ligands to the host cellreceptors. Heparin-like molecules, such as heparan sulfate, are widely distributed oncell surface, which have been regarded as important receptors for invasion. Heparinand its derivatives can block the invasion of the P. falciparm merozoites and T. gondiitachyzoites into host cells, albeit the parasite ligands in the host-parasite interplay aswell as the molecular mechanisms remain poorly understood. Identification of theparasite ligands can not only explain the molecular pathogenesis of P. falciparm and T.gondii, but also can falicitate the discovry of novel drugs that can block parasitepropagation in host cell.
To date, only a limited number of heparin-binding proteins derived from both P.falciparum and T. gondii has been revealed. Previous studies suggested that theinhibitory effect of heparin on RBC invasion by P. falciparum was due to itsinteraction with merozoite surface protein1. Heparin was found to be able to blockthe attachment of P. falciparm merozoite to RBC surface. Further，only5T.gondii-derived proteins were found to bind heparin, such as rhoptry protein2, rhoptryprotein4, dense granule protein2, the surface protein1and surface protein3. Theresults indicated that heparin can interact with multiple parasite proteins.
In this study, we presented a novel approach by combination of receptor-basedpurification with high-through-put proteomic identification for a systematic andcomprehensive research of both P. falciparum merozoite-and T. gondii tachyzoite-derived heparin-binding proteome. The data to a large extent explains themechanism of host cell invasion inhibition by heparin.
1. Analysis of invasion inhibition with heparin-like molecules
To confirm the inhibitory effect on RBC invasion of P. falciparum, we addeddifferent concentrations of heparin, heparan sulfate, chondroitin sulfate A and dextransulfate in the culture of P. falciparum to compare inhibition activity according to thereports earlier. All samples were tested in triplicate for three times. We found thatheparin, heparan sulfate, and dextran sulfate inhibited merozoite invasion in aconcentration dependent manner, while chondroitin sulfate A showed no effect oninvasion. Heparan sulfate had a weaker effect, showing invasion inhibitory effect onlyat the concentration of1000μg/mL, with no effect at lower concentrations. The datafurther confirmed that heparin-like molecular can specifically inhibit parasiteinvasion.
2. Identified and comparative analysis of heparin-binding proteome betweenPlasmodium falciparum and Toxoplasma gondii
The highly synchronous P. falciparum (3D7line) parasites were cultured inhuman erythrocytes according to standard methods. Tachyzoites of T. gondii RH strainwere harvested from the abdominal cavities of Kunming strain mice under anesthesia2or3days post-inoculation. The schizonts and tachyzoites were further purified bygradient Percoll centrifugation. The purified parasite suspended in phosphate bufferedsaline containing a complete cocktail of protease inhibitors were freeze-thawedseveral times and centrifuged at a high speed for15min. The supernatant of parasitelysate was mixed with Heparin-sepharose. The bound proteins were analyzed bySDS-PAGE and Western-blot. Multiple polypeptides from both P. falciparum and T.gondii were detected to bind to Heparin-sepharose. Further, the binding of the proteinsto Heparin-sepharose could only be competed out by soluble heparin at relatively highconcentrations, chondroitin sulfate A did not show any inhibitory effect on the bindingof the proteins to Heparin-sepharose and no binding was seen to un-conjugatedSepharose suggesting the binding of a large number of the polypeptides to be specificfor heparin.
To identify the heparin-binding proteome of P.falciparum and T.gondii, the elutedproteins from the Heparin-sepharose were further analyzed by multi-dimensionalliquid chromatography tandem mass spectrometry after trypsinization. The spectra were searched against a combined database of all possible predicted tryptic peptides.A total of6,062unique peptides mapped to811P. falciparum3D7proteins wereidentified by searching the P. falciparum proteins database. More than99%peptideswere of P. falciparum origin with only87peptides matched to human-or bovine-proteins. A total of6252unique peptides mapped to956T. gondii proteins wereidentified by searching the NCBI Toxoplasma gondii database. The analysis alsoresulted in identification of more than188mouse proteins. The data further suggestedthat the binding to heparin was specific and heparin can interact with multiple P.falciparum and T. gondii-derived ligands.
The proteins identified were categorized according to their subcellularlocalisation and biological function. First, the proteins were found to originated froma wide range of subcellular localisation including cytoplasma, nuclear, mitochondrion,rhoptry, microneme and so on. Secondly, they could be mainly categorized intofunctional14groups according to GO annotation and previous studies. However,proteins of unknown function constitute the largest category in that identified from T.gondii, a reflection of the incomplete understanding of the roles fulfilled byToxoplasma products. Strikingly, in the functional categorization of theheparin-binding proteome,47P. falciparum-derived proteins and33T. gondii-derivedproteins associated with the cell invasion. Most proteins located in rhoptry organelle,which is a unique structure contains several critical cell adhesins and functionalized informing the junction between the invading parasite and host cell. The consequence ofsuch interaction between heparin and rhoptry proteins can either be an inhibition ofjunction assembly or blockage of their binding to receptors on host cell membrane inthe sequential process of parasite invasion. More importantly,170heparin-bindingproteins of P. falciparum and T. gondii were found in the same subcellular localisationand biological function, suggesting invasion mechanism of P. falciparum and T.gondii is similar.
3. Confirmation of heparin-binding and invasion inhibition with recombinant proteins
Genes encoding the16proteins of P. falciparum with prominent heparin-bindingproperties were cloned into pGEX-4T-1or pET32a expression vectors and therecombinant proteins were tested to obtain more evidence for heparin-binding. In total14protein fractions from16molecules were successfully expressed. Of these proteinfraction tested,9showed specific binding to Heparin-sepharose，but5did not, indicating the binding was specific.
Lastly, four recombinant merozoite-associated proteins were further tested fortheir invasion blocking/inhibitory effects in vitro. Rhoptry neck proteins3-48, serinerepeat antigen5-50and merozoite surface protein1-42did show invasion inhibitoryeffect in a concentration dependent way, while merozoite surface protein1-33andglutathione S-transferase did not. The data collectively suggested that themerozoite-associated heparin-binding proteins are functionally important inerythrocyte invasion.
In summary, both P. falciparum merozoite-and T. gondii tachyzoite-derivedheparin-binding proteome were identified by combination of receptor-basedpurification with high-through-put proteomic identification. The proteins associatedwith the cell invasion were found to be predominantly located in the rhoptry organelle.The data not only revealed the mechanism of heparin inhibition on P. falciparum andT. gondii invasion, but also further supports the exploration of heparin derivatives asanti-parasite drugs.
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