Shahi Ifrah, Dongas Sophia A, Ilmain Juliana K, Torres Victor J, Ratner Adam J
New York University Grossman School of Medicine, Department of Pediatrics, New York, NY.
New York University Grossman School of Medicine, Department of Microbiology, New York, NY.
bioRxiv. 2023 Jul 25:2023.06.21.545930. doi: 10.1101/2023.06.21.545930.
Cholesterol dependent cytolysins (CDCs) are a large family of pore forming toxins, produced by numerous gram-positive pathogens. CDCs depend on host membrane cholesterol for pore formation; some CDCs also require surface associated human CD59 (hCD59) for binding, conferring specificity for human cells. We purified a recombinant version of a putative CDC encoded in the genome of , tigurilysin (TGY), and used CRISPR/Cas9 to construct hCD59 knockout (KO) HeLa and JEG-3 cell lines. Cell viability assays with TGY on WT and hCD59 KO cells showed that TGY is a hCD59-dependent CDC. Two variants of TGY exist among genomes, only one of which is functional. We discovered that a single amino acid change between these two TGY variants determines its activity. Flow cytometry and oligomerization western blots revealed that the single amino acid difference between the two TGY isoforms disrupts host cell binding and oligomerization. Furthermore, experiments with hCD59 KO cells and cholesterol depleted cells demonstrated that TGY is fully dependent on both hCD59 and cholesterol for activity, unlike other known hCD59-dependent CDCs. Using full-length CDCs and toxin constructs differing only in the binding domain, we determined that having hCD59-dependence leads to increased lysis efficiency, conferring a potential advantage to organisms producing hCD59-dependent CDCs.
Cholesterol dependent cytolysins (CDCs) are produced by a variety of disease-causing bacteria, and may play a significant role in pathogenesis. Understanding CDC mechanisms of action provides useful information for developing anti-virulence strategies against bacteria that utilize CDCs and other pore-forming toxins in pathogenesis. This study describes for the first time a novel human-specific CDC with an atypical pore forming mechanism compared to known CDCs. In addition, this study demonstrates that human-specificity potentially confers increased lytic efficiency to CDCs. These data provide a possible explanation for the selective advantage of developing hCD59-dependency in CDCs and the consequent host restriction.
胆固醇依赖性细胞溶素（CDCs）是一大类孔形成毒素，由众多革兰氏阳性病原体产生。CDCs的孔形成依赖于宿主细胞膜胆固醇；一些CDCs还需要表面相关的人CD59（hCD59）进行结合，从而赋予对人细胞的特异性。我们纯化了在 基因组中编码的一种假定的CDC的重组形式，即tigurilysin（TGY），并使用CRISPR/Cas9构建了hCD59基因敲除（KO）的HeLa和JEG-3细胞系。用TGY对野生型和hCD59 KO细胞进行细胞活力测定表明，TGY是一种依赖hCD59的CDC。在 基因组中存在两种TGY变体，其中只有一种具有功能。我们发现这两种TGY变体之间的单个氨基酸变化决定了其活性。流式细胞术和寡聚化免疫印迹显示，两种TGY异构体之间的单个氨基酸差异破坏了宿主细胞结合和寡聚化。此外，用hCD59 KO细胞和胆固醇耗尽细胞进行的实验表明，与其他已知的依赖hCD59的CDCs不同，TGY的活性完全依赖于hCD59和胆固醇。使用仅在结合域不同的全长CDCs和毒素构建体，我们确定依赖hCD59会导致裂解效率提高，这赋予了产生依赖hCD59的CDCs的生物体潜在优势。
胆固醇依赖性细胞溶素（CDCs）由多种致病细菌产生，可能在发病机制中起重要作用。了解CDC的作用机制为开发针对在发病机制中利用CDCs和其他孔形成毒素的细菌的抗毒力策略提供了有用信息。本研究首次描述了一种与已知CDCs相比具有非典型孔形成机制的新型人类特异性CDC。此外，本研究表明人类特异性可能赋予CDCs更高的裂解效率。这些数据为在CDCs中发展对hCD59的依赖性及其随之而来的宿主限制的选择性优势提供了一种可能的解释。
