Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, MA 02115.
Cambridge Institute for Medical Research, Cambridge, CB2 OXY, United Kingdom.
Proc Natl Acad Sci U S A. 2017 Oct 31;114(44):E9356-E9365. doi: 10.1073/pnas.1711310114. Epub 2017 Oct 19.
During malaria blood-stage infections, parasites interact with the RBC surface to enable invasion followed by intracellular proliferation. Critical factors involved in invasion have been identified using biochemical and genetic approaches including specific knockdowns of genes of interest from primary CD34 hematopoietic stem cells (cRBCs). Here we report the development of a robust in vitro culture system to produce RBCs that allow the generation of gene knockouts via CRISPR/Cas9 using the immortal JK-1 erythroleukemia line. JK-1 cells spontaneously differentiate, generating cells at different stages of erythropoiesis, including terminally differentiated nucleated RBCs that we term "jkRBCs." A screen of small-molecule epigenetic regulators identified several bromodomain-specific inhibitors that promote differentiation and enable production of synchronous populations of jkRBCs. Global surface proteomic profiling revealed that jkRBCs express all known host receptors in a similar fashion to cRBCs and that multiple strains invade jkRBCs at comparable levels to cRBCs and RBCs. Using CRISPR/Cas9, we deleted two host factors, basigin (BSG) and CD44, for which no natural nulls exist. BSG interacts with the parasite ligand Rh5, a prominent vaccine candidate. A BSG knockout was completely refractory to parasite invasion in a strain-transcendent manner, confirming the essential role for BSG during invasion. CD44 was recently identified in an RNAi screen of blood group genes as a host factor for invasion, and we show that knockout results in strain-transcendent reduction in invasion. Furthermore, we demonstrate a functional interaction between these two determinants in mediating erythrocyte invasion.
在疟疾的血期感染中，寄生虫与 RBC 表面相互作用，以实现入侵和随后的细胞内增殖。已经通过生化和遗传方法确定了参与入侵的关键因素，包括从原代 CD34 造血干细胞（cRBC）中特异性敲低感兴趣基因。在这里，我们报告了一种强大的体外培养系统的开发，该系统可产生 RBC，允许通过 CRISPR/Cas9 在永生化 JK-1 红细胞白血病系中产生基因敲除。JK-1 细胞自发分化，产生处于不同红细胞生成阶段的细胞，包括终末分化的有核 RBC，我们将其称为“jkRBC”。小分子表观遗传调节剂的筛选鉴定出几种溴结构域特异性抑制剂，可促进分化并实现 jkRBC 的同步群体产生。全局表面蛋白质组学分析显示，jkRBC 以类似于 cRBC 的方式表达所有已知的宿主受体，并且多种寄生虫以类似于 cRBC 和 RBC 的水平入侵 jkRBC。使用 CRISPR/Cas9，我们删除了两个宿主因子，basigin（BSG）和 CD44，它们都没有天然的缺失。BSG 与寄生虫配体 Rh5 相互作用，Rh5 是一种重要的疫苗候选物。BSG 敲除完全抵抗寄生虫入侵，具有跨株的抗性，这证实了 BSG 在入侵过程中的重要作用。CD44 最近在 RNAi 筛选血型基因中被鉴定为入侵的宿主因子，我们表明，敲除导致入侵的跨株减少。此外，我们证明了这两个决定因素在介导红细胞入侵中的功能相互作用。
