Abstract
Cholesterol-dependent cytolysins (CDCs), of which intermedilysin (ILY) is an archetypal member, are a group of pore-forming toxins secreted by a large variety of pathogenic bacteria. These toxins, secreted as soluble monomers, oligomerize upon interaction with cholesterol in the target membrane and transect it as pores of diameters of up to 100 to 300 Å. These pores disrupt cell membranes and result in cell lysis. The immune receptor CD59 is a well-established cellular factor required for intermedilysin pore formation. In this study, we applied genome-wide CRISPR-Cas9 knock-out screening to reveal additional cellular co-factors essential for ILY-mediated cell lysis. We discovered a plethora of genes previously not associated with ILY, many of which are important for membrane constitution. We show that heparan sulfates facilitate ILY activity, which can be inhibited by heparin. Furthermore, we identified hits in both protein and lipid glycosylation pathways and show a role for glucosylceramide, demonstrating that membrane organization is important for ILY activity. We also cross-validated identified genes with vaginolysin and pneumolysin and found that pneumolysin's cytolytic activity strongly depends on the asymmetric distribution of membrane phospholipids. This study shows that membrane-targeting toxins combined with genetic screening can identify genes involved in biological membrane composition and metabolism.
Highlights
Bacteria use a variety of strategies to exploit hosts, and one of the prominent ways to achieve this is by toxins
The advent of genetic approaches such as genome-wide CRISPR knock-out screening has allowed researchers to unravel novel mechanisms in different fields of research. This has led to unprecedented growth in knowledge about a wide variety of biological phenomena
The findings described in this study are relevant to the advancement of our understanding of bacterial pore-forming toxins, in addition to revealing genetic details of membrane domain formation, which are targeted by toxins
Summary
Bacteria use a variety of strategies to exploit hosts, and one of the prominent ways to achieve this is by toxins. Unique among pore-forming toxins are cholesterol-dependent cytolysins (CDCs), which are the main virulence factors of a variety of pathogenic bacteria[4,5,6,7,8] that form pores (~100–300 Å diameter). Cells can repair membrane disruption caused by toxins through exocytosis or shedding of the damaged membranes[11], and some cells are more resistant to these toxins than others[12, 13]. Because of their inherent ability to bind to the membranes, non-toxic mutants of CDCs are used to study membrane domains[14,15,16]
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