In a recent issue of Biochemical Journal, Kathuria et al. [Biochem. J. (2018) 475, 3039–3055] report that membrane binding of the pore-forming toxin Vibrio cholerae cytolysin (VCC) is facilitated by the presence of cholesterol, and the presence of this sterol within the lipid bilayer is key for the formation of a functional pore. Yet, in the presence of accessory non-lipid components, VCC retains its membrane-binding capability likely through membrane lipid raft structures. In light of their results, the authors provide new insights into the roles of cholesterol and of membrane microstructures in the binding, the oligomeric assembly and the cytolytic pore formation of VCC which all take place following infection by V. cholerae.

Vibrio cholerae is the etiologic agent of the disease cholera, a condition in humans characterized by voluminous aqueous diarrhea and rapid dehydration occurring shortly following infection, resulting in hypotensive shock and death if left untreated [1]. It affects 3–5 million people each year, killing over 120 000 [2]. Aside from the major virulence factor Cholera toxin, the pathogenicity of the Gram-negative bacteria includes the production and secretion of other accessory toxins that target cell membranes. Among them, the pore-forming toxin (PFT) Vibrio cholerae cytolysin (VCC) is a potent cell-damaging toxin that is capable of permeabilizing intestinal and immune cells to facilitate colonization of the human host.

Based on their structural features, PFTs are classified into two classes: (i) α-PFT that adopts α-helical secondary structures to span the targeted membrane and (ii) β-PFT that assembles into β-barrel structures to form the cytolytic pore (reviewed by Iacovache et al. [3]). VCC belongs to the latter class by organizing into a heptameric β-barrel [4,5] on the targeted cell membrane upon proteolytic activation of its monomeric soluble form [6]. The sequence of the assembly events includes (i) the binding of the monomeric water-soluble form of VCC to membranes, (ii) the oligomerization of VCC into a heptameric prepore intermediate and (iii) the insertion of the β-barrel superstructure into the cellular membrane [7,8].

It is now well established that both the recognition of glycans and the association with lipids and cholesterol are key features required for the membrane-binding and pore formation activities of VCC [913]. However, the exact role of the individual biochemical components is less clear. In a recent paper published in the Biochemical Journal, Kathuria et al. [14] attempt to address this issue with respect to cholesterol. They first show, using liposomes, that the presence of cholesterol was sufficient for the proper targeting of VCC to lipid bilayers and for pore-forming activity in a dose-dependent manner. This strengthens the hypothesis that non-lipid components are not required for efficient binding of VCC to cholesterol-containing liposomes and for pore formation. Then, by chemically depleting cholesterol from human erythrocytes, the authors demonstrated that despite the efficient binding of VCC to these membranes, no pore-forming activity was measured. Such abortive oligomeric form of VCC was then able to recover its activity when cholesterol was artificially reintroduced in the culture med.

It was previously considered by De and Olson [5], based on biochemical data from a homologous system [15] and on the crystal structure of VCC in its heptameric form [5], that the longer stem structure, compared with the length of the stem from the staphylococcal α-hemolysin [16], could favor a stronger affinity for thicker membrane microdomains such as in the case of cholesterol-rich lipid rafts [1721]. In one of the key points of their study, Kathuria et al. [14] confirmed this hypothesis by letting recombinant VCC protein interact with human erythrocytes. Then, they performed a subcellular fractionation and using raft-specific markers, the authors were able to recover VCC in the cholesterol and lipid raft marker-rich fractions. Perturbation of the cholesterol-rich membrane microdomains by chemically depleting the cells of cholesterol abrogated the specific interaction of VCC with these lipid rafts. Finally, the authors show that while impairing the cholesterol-binding capability of VCC via a single-point mutation [22], the toxin was still able to associate with cholesterol-rich lipid raft, indicating that cholesterol does not appear to be responsible for the specific targeting of VCC on the host cell membranes.

This last result raises the question whether the presence of cholesterol is the sole target of VCC for proper anchoring and pore formation activity or whether the structural integrity of the cholesterol-rich lipid raft microdomains dictates the efficiency with which VCC binds and penetrates the cell membrane. Kathuria et al. [14] hypothesize that cholesterol is the key molecule enabling the pore-forming cytolytic activity of VCC, as observed by the initial liposome experiments. However, in a biological context, VCC will face a complex membrane environment and it is likely that the presence of cholesterol will modulate the fluidity of the membrane at specific regions also rich in other factors having a certain affinity for the bacterial toxin and thus enabling VCC to insert in the membrane bilayer. An appropriate structural context in the membrane bilayer, which would include the presence of cholesterol, is likely to be the key for an optimal cytolytic activity of VCC.

Further structural studies on both the membrane lipid structure and the abortive form of VCC present in the absence of cholesterol and identified here will enable a better understanding of the role of each component in mediating the cytotoxic role of VCC on the human host cells and possibly accelerate the design of therapies capable of mitigating the detrimental effect of the V. cholerae cytolysin.

Abbreviations

     
  • PFT

    pore-forming toxin

  •  
  • VCC

    Vibrio cholerae cytolysin

Competing Interests

The Author declares that there are no competing interests associated with this manuscript.

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