Pneumolysin-damaged cells benefit from non-homogeneous toxin binding to cholesterol-rich membrane domains

Highlights

• Pneumolysin binds to distinct plasmalemmal domains of nucleated cells.

• Pneumolysin targets cholesterol-rich domains on phase-separated vesicles.

• Plasmalemmal repair benefits from the inhomogeneous distribution of pneumolysin.

Abstract

Nucleated cells eliminate lesions induced by bacterial pore-forming toxins, such as pneumolysin via shedding patches of damaged plasmalemma into the extracellular milieu. Recently, we have shown that the majority of shed pneumolysin is present in the form of inactive pre-pores. This finding is surprising considering that shedding is triggered by Ca²⁺-influx following membrane perforation and therefore is expected to positively discriminate for active pores versus inactive pre-pores.

Here we provide evidence for the existence of plasmalemmal domains that are able to attract pneumolysin at high local concentrations. Within such a domain an immediate plasmalemmal perforation induced by a small number of pneumolysin pores would be capable of triggering the elimination of a large number of not yet active pre-pores/monomers and thus pre-empt more frequent and perilous perforation events. Our findings provide further insights into the functioning of the cellular repair machinery which benefits from an inhomogeneous plasmalemmal distribution of pneumolysin.

Introduction

Streptococcus pneumoniae is a potent human pathogen [1,2]. Its cholesterol-dependent cytolysin (CDC), pneumolysin (PLY) is instrumental for breaching the host's epithelial barrier and for the incapacitation of the immune system [[3], [4], [5]]. CDCs are secreted as soluble monomers and bind to cholesterol within eukaryotic plasmalemma [6,7]. After oligomerization, they initially form inactive pre-pores that eventually undergo a transition to active membrane-perforating pores [6,8].

The plasmalemmal lipid bilayer is believed to display an inhomogeneous distribution of different lipid species [9,10]. Aided by protein-lipid and lipid-lipid interactions, cholesterol-rich microdomains embedded in a saturated lipid environment (lipid rafts) alternate with regions of low cholesterol surrounded by unsaturated phospholipids [[11], [12], [13], [14]]. Remaining beyond the resolution limit of conventional light microscopy [9], short-living and highly dynamic, lipid rafts could be stabilized by protein-ligands, which, in turn, could trigger raft coalescence [15].

While playing an important role in cellular homeostasis [4], membrane rafts are also hijacked by pathogens to harm a host cell: plasmalemmal binding of CDCs and their oligomerization is potentiated within membrane lipid rafts [2,[15], [16], [17], [18], [19]].

Nucleated cells are capable of eliminating PLY-induced lesions by shedding damaged, PLY- containing plasmalemmal patches into the extracellular milieu in form of microvesicles [1,20,21]. Recently, we have shown that the vast majority of shed, microvesicle-associated PLY is present in the form of inactive pre-pores; whereas active PLY-pores featuring a perforated membrane account for only 10% of the total PLY [22]. This finding is surprising considering that the process of shedding is triggered by Ca²⁺-entry from the extracellular milieu following plasmalemmal perforation and is therefore expected to positively discriminate for active pores versus inactive pre-pores [21,22].

Here, using live-cell imaging in combination with model membrane experiments under microfluidic control we have analyzed potential mechanisms that might be responsible for the preferential shedding of inactive PLY-pre-pores in the process of plasmalemmal repair. We provide evidence for the existence of distinct plasmalemmal domains that are capable to attract PLY at a high local concentration. Furthermore, we show that in artificial membranes PLY preferentially targets cholesterol-rich domains and their boundaries. It is feasible that such domains might be either permanently present within the plasmalemma of nucleated cells as a result of protein-driven lipid segregation, or are a result of PLY-induced self-association of nanoscale, lipid-driven lipid inhomogeneities such as membrane rafts. Our findings provide further insights into the functioning of the cellular repair machinery that benefits from an inhomogeneous distribution of PLY on the plasmalemma and triggers an effective antibacterial defense [1,20].