Bipartite pore-forming toxin YaxAB: understanding pore formation using live-cell imaging

Abstract

Bacteria employ a variety of strategies to invade and colonise the host tissue, including the secretion of toxins to overcome the cellular barrier. Bacterial pore-forming toxins (PFTs) disrupt the lipid bilayer by forming nano-sized pores that permeabilise the host cell membrane to ions and small molecules. In response to PFTs, the host cell engages in membrane repair mechanisms to overcome the damage inflicted and to maintain cellular homeostasis. Although the lytic action of single-component toxins has been widely investigated, the mechanism of bi-partite toxin activity, which involves the binding and oligomerisation of two protein components, is not well understood. This work aims to elucidate the pore-forming mechanism of bipartite PFT YaxAB produced from Yersinia enterocolitica, a human pathogen causing fever, diarrhoea, and occasionally sepsis.

YaxA and YaxB subunits were purified and shown to act sequentially, with YaxA binding to the membrane and YaxB associating thereafter to effect cell lysis. Mutants of YaxA and YaxB were generated that possessed a single surface-exposed cysteine residue to fluorescently tag the monomers using cysteine-specific maleimide dyes. These fluorescently tagged toxins were used to study self-assembly and pore-formation on HeLa cells using live-cell HILO (highly inclined and laminated optical sheet) microscopy.

A previous study suggested that the YaxA subunit binds to the lipid bilayer and remains monomeric until it binds to its lytic partner YaxB, thus forming a dimer and driving further oligomerisation, leading to a full pore complex. However, our results showed that YaxA alone could form pre-pore complexes on the membrane of HeLa cells. Further incubation with YaxB resulted in the formation of a full pore complex bearing both subunits on the cell surface. Upon tracking these YaxA pre-pore complexes and YaxAB full pore complexes on the cell membrane, we observed that alongside Brownian motion, they also displayed anomalous diffusive behaviour, which could be the consequence of membrane heterogeneity.

Most previous studies on PFTs have been conducted using artificial membrane systems, such as liposomes and supported bilayers. This is the first study investigating the pore-forming mechanism of a bipartite PFT on living cells, providing a novel insight into the role of YaxA in pre-pore complex formation. The functional heterogeneity of YaxA and YaxB, where YaxA binds to the membrane and forms pre-pore complexes, while YaxB subsequently associates to induce cell lysis, could be leveraged for the development of cell-specific therapeutic intervention.