# Stepwise assembly of α-hemolysin from intermediates to the mature pore in native erythrocytes

## TL;DR

This study reveals how the α-hemolysin toxin from Staphylococcus aureus forms pores in red blood cells, using detailed structural snapshots and lipid interaction analysis.

## Contribution

The study provides high-resolution structures of α-HL in native erythrocytes and identifies lipid interactions crucial for pore formation.

## Key findings

- Structures of α-HL intermediates and mature pores were resolved at 3.1–3.8 Å resolution in native erythrocytes.
- Shorter or unsaturated lipid chains and phosphatidylcholine modulate α-HL pore formation.
- Mass spectrometry identified key erythrocyte lipid components interacting with α-HL.

## Abstract

Chatterjee et al. reveal that structural snapshots of different conformations of α-HL toxin from Staphylococcus aureus, such as arc-like intermediates, heptameric prepores, and pore, and octameric species, identified from the prehemolytic and posthemolytic stages, offer step-by-step oligomerization of α-HL during erythrocyte membrane lysis.

Alpha-hemolysin (α-HL) is a small pore-forming toxin secreted by pathogenic Staphylococcus aureus, inducing cell death process by forming pores in membrane. So far, detergents or artificial lipid environments have been utilized to characterize the toxin structure. The toxin-induced changes in the membrane, membrane remodeling after toxin treatment, and the role of the toxin during pore formation process remain ambiguous. Thus, understanding pore formation in the cellular environment, including the roles of the plasma membrane and lipid composition, is crucial for drug development. In this study, we captured step-by-step oligomerization of α-HL and membrane rupture of erythrocyte cells using confocal microscopy, cryo-EM imaging, and single-particle analysis. We resolved 3.1–3.8 Å resolution structures of pore, prepore, and immature pore conformations in cellular environment. Furthermore, mass spectrometry analysis demonstrated key erythrocyte lipid components interacting with α-HL. Our findings indicate that shorter or unsaturated lipid chains facilitate pore formation and the role of phosphatidylcholine with varying physical properties in modulating the toxin’s activity.