<b><i>Escherichia coli α-hemolysin induces red blood cell retention in a microfluidic spleen-like device</i></b>

α-hemolysin (HlyA) is a major exotoxin secreted by uropathogenic <i>Escherichia coli</i> (UPEC), known for its ability to lyse red blood cells (RBCs). While its lytic effects are well characterized, the non-lytic 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 non-lytic 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 non-acylated 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.