Study of the hemolysin mechanism by functionalized nanopatterned surfaces.

SAFFIOTI NICOLÁS ANDRÉS1, LEAL DENIS MARÍA FLORENCIA2, GUGLIELMOTTI VICTORIA1, ROSS BERALDI ALEJANDRA2, HERLAX VANESA V. 3, PALLAROLA DIEGO1, SCHWARZBAUM P J

Congreso; Reunion anual SAB 2019; 2019

The Uropathogenic Escherichia coli (UPEC) is the main etiological factor of urinaryinfections1. UPEC produces the α-hemolysin (HlyA) toxin which plays an important roleduring the infection process2. HlyA exerts sublytic signaling on red blood cells (RBC)which involves Ca2+ entrance and may ultimately lead to hemolysis. Although HlyA wasshown to form oligomers in the cell membrane, the role of oligomerization in toxinactivity remains controversial. To address this question, we are exposing human RBC tocustom-built nanopatterned surfaces where HlyA can be attached at precise locationsand densities. As a first approach, we tested the best conditions for toxin attachmentusing linkers and continuous gold surfaces.First, the gold surface was modified with cystamine. Then, it reacted with a polyethyleneglycol polymer linker that has an N-hydroxysuccinimide ester on one end. The linker hasa maleimide group on the other end which reacts with HlyA in a third step. The proteinattachment was followed using a quartz microbalance. When HlyA was added to thesurface we detected a significant change in resonance frequency if the linker previouslyreacted with the gold in a non-aqueous medium. This change does not revert withwashing indicating covalent binding of the protein.To measure the HlyA activity, RBC were loaded with Fluo 4 and incubated on surfacesfunctionalized with HlyA or Pro-HlyA (a precursor of HlyA without hemolytic activity). Thesurfaces with HlyA elicited Ca2+ entrance in 3,6 ± 0.24 % of total cells compared with0.62 ± 0.1 % in the Pro-HlyA bound surfaces. This would indicate that the protein boundto the surface keeps its activity. The low number of affected RBC is related to low proteinstability and loss activity during the binding process.Future experiments will be directed towards increasing protein stability in the bindingprocess and employment of non-continuous surfaces to modify toxin density, controllingthe extent of oligomerization, and its effect on HlyA activity.