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Functional Analysis of Rhomboid Proteases during Toxoplasma Invasion

机译：弓形虫侵袭期间菱形蛋白酶的功能分析

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Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T.?gondii tachyzoites. Analysis of these mutants demonstrated that ROM4 is the primary protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible roles in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the existence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not absolutely essential, it is important for efficient host cell invasion by T.?gondii. >IMPORTANCE Apicomplexan parasites such as Toxoplasma gondii express surface proteins that bind host cell receptors to aid invasion. Many of these adhesins are subject to cleavage by rhomboid proteases (ROMs) within their transmembrane segments during invasion. Previous studies have demonstrated the importance of adhesin cleavage for parasite invasion and proposed that the ROMs responsible for processing would be essential for parasite survival. In T.?gondii, ROM5 was thought to be the critical ROM for adhesin shedding due to its robust protease activity in vitro and posterior localization on the parasite surface. Here, we knocked out all three ROMs in T.?gondii tachyzoites and found that ROM4, but not ROM5, was key for adhesin cleavage. However, none of the ROMs individually or in combination was essential for cell entry, further emphasizing that essential pathways such as invasion typically rely on redundant pathways to ensure survival.
机译：弓形虫和其他apicomplexan寄生虫入侵宿主细胞需要跨膜粘附素，该粘附素介导与底物和宿主细胞上的受体结合，以促进运动和侵袭。菱形蛋白酶（ROM）被认为可以在其跨膜片段内切割粘附素，从而使寄生虫与受体脱离并完全进入宿主细胞。为了检查单个ROM在入侵过程中的特定作用，我们针对在 T.?gondii 速殖子中表达的三个ROM生成了单，双和三敲除。对这些突变体的分析表明，ROM4是参与粘附素加工和宿主细胞入侵的主要蛋白酶，而ROM1或ROM5在这些过程中的作用微不足道。 ROM4的缺失阻止了黏附素如MIC2（微nemene蛋白2）的脱落，导致它们积聚在细胞外寄生虫的表面。表面粘附素的增加导致非生产性附着，滑行运动性改变，活动结形成受损以及入侵效率降低。尽管ROM4对于有效入侵很重要，但缺少所有三个ROM的突变体仍然可行，并且MIC2仍可从入侵的突变体寄生虫表面有效去除，这意味着存在入侵过程中不依赖ROM的粘附素去除机制。总的来说，这些结果表明，尽管粘附素的ROM处理不是绝对必要的，但对于 T.?gondii 的有效宿主细胞入侵而言，它却很重要。 > IMPORTANCE （Apocomplexan）寄生虫，例如 Toxoplasma gondii ，表达结合宿主细胞受体的表面蛋白以帮助入侵。这些黏附素中的许多黏附素在侵袭过程中会受到菱形蛋白酶（ROM）跨膜区段内的裂解作用。先前的研究表明，粘附素裂解对于寄生虫入侵的重要性，并提出负责处理的ROM对于寄生虫生存至关重要。在 T.gondii 中，ROM5被认为是粘附素脱落的关键ROM，这是由于其强大的体外蛋白酶活性和寄生虫表面的后部定位。在这里，我们剔除了 T.gondii 速殖子中的所有三个ROM，发现ROM4（而不是ROM5）是粘附素裂解的关键。但是，ROM的单独或组合都不是细胞进入必不可少的，进一步强调了诸如入侵之类的重要途径通常依赖于冗余途径来确保存活。

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