α-hemolysin (HlyA) is a major exotoxin secreted by uropathogenic Escherichia coli (UPEC), known for its ability to lyse red blood cells (RBCs). Although its lytic effects are well characterized, the nonlytic alterations on RBCs, such as increased permeability to Ca2+, osmotic imbalance, and morphological alterations, remain less understood and may be critical in UPEC pathogenesis. This study investigates the impact of these nonlytic alterations on the rheology and mechanics of RBCs using two biomimetic microfluidic devices that model key aspects of RBCs’ circulation. In the first device, which mimics the mechanical deformation of RBCs in narrow capillaries, HlyA sublytic concentrations were found to significantly impair RBC deformability. These changes were accompanied by an increase in cytosolic Ca2+ and volume expansion. In contrast, the nonacylated protoxin ProHlyA neither impaired the deformability of RBCs nor triggered changes in cytosolic Ca2+ or cell volume. The second device, which simulates the RBCs’ filtration by the spleen’s red pulp, revealed that HlyA, but not ProHlyA, increased RBCs’ retention in small gaps resembling splenic fenestrations. The extent of RBCs’ retention was partially mitigated by blocking purinergic signaling, indicating a contribution of the HlyA-induced volume increase in this process. Our results suggest that the increase in cytosolic Ca2+ elicited by HlyA impacts RBCs’ circulation by decreasing RBCs’ deformability and increasing spleen retention. However, this impairment of RBCs’ performance can function as a defense mechanism to aid in the retention of HlyA-bound RBCs, removing them from circulation, and potentially preventing vascular hemolysis.

中文翻译：

---

大肠埃希菌 α-溶血素在微流控脾样装置中诱导红细胞潴留


α-溶血素 （HlyA） 是由尿路致病性大肠杆菌 （UPEC） 分泌的一种主要外毒素，以其裂解红细胞 （RBC） 的能力而闻名。尽管其裂解作用已得到充分表征，但红细胞的非溶解性改变，例如对 Ca2+ 的通透性增加、渗透失衡和形态学改变，仍然知之甚少，并且在 UPEC 发病机制中可能至关重要。本研究使用两种模拟红细胞循环关键方面的仿生微流控装置来调查这些非溶解性改变对红细胞流变学和力学的影响。在第一个模拟 RBC 在狭窄毛细血管中机械变形的装置中，发现 HlyA 亚溶浓度会显着损害 RBC 的变形能力。这些变化伴随着胞质 Ca2+ 的增加和体积扩大。相比之下，非酰化原毒素 ProHlyA 既不损害红细胞的变形性，也不触发胞质 Ca2+ 或细胞体积的变化。第二个装置模拟红细胞被脾脏红髓过滤，揭示了 HlyA（而不是 ProHlyA）增加了红细胞在类似于脾开窗的小间隙中的保留。通过阻断嘌呤能信号传导，部分减轻了 RBC 的保留程度，表明 HlyA 诱导的体积增加在此过程中做出了贡献。我们的结果表明，HlyA 引发的胞质 Ca2+ 的增加通过降低红细胞的变形性和增加脾脏保留来影响红细胞的循环。然而，红细胞性能的这种损害可以作为一种防御机制，帮助保留 HlyA 结合的红细胞，将它们从循环中清除，并可能防止血管溶血。