A Cellular Metalloproteinase Activates Vibrio cholerae Pro-cytolysin

Abstract

Many strains of Vibrio cholerae produce a cytolysin (VCC) that forms oligomeric transmembrane pores in animal cells. The molecule is secreted as a procytolysin (pro-VCC) of 79 kDa that must be cleaved at the N terminus to generate the active 65-kDa toxin. Processing can occur in solution, and previous studies have described the action of mature VCC thus generated. However, little is known about the properties of pro-VCC itself. In this study, it is shown that pro-VCC exist as a monomer in solution and binds as a monomer to eukaryotic cells. Bound pro-VCC can then be activated either by exogenous, extracellular, or by endogenous, cellbound proteases. In both cases, cleavage generates the 65-kDa VCC that oligomerizes to form transmembrane pores. A wide variety of exogenous proteinases can mediate activation. In contrast, the activating cellular protease is selectively inhibited by the hydroxamate inhibitor TAPI, and thus probable candidates are members of the ADAM-metalloproteinase family. Furin, MMP-2, MMP-9, and serine proteinases were excluded. Cells over-expressing ADAM-17, also known as tumor necrosis factor ␣ converting enzyme, displayed increased activation of VCC, and knockout cells lacking ADAM-17 had a markedly decreased capacity to cleave the protoxin. The possibility is raised that pro-VCC is targeted to membrane sites that selectively contain or are accessible to cellular ADAM-metalloproteinases. Although many microbial toxins are activated by furin, this is the first evidence for processing by a cellular metalloproteinase. We identified ADAM-17 as a potent activator of pro-VCC.

Photobacterium damselae subsp. damselae Major Virulence Factors Dly, Plasmid-Encoded HlyA, and Chromosome-Encoded HlyA Are Secreted via the Type II Secretion System

“…More pertaining to our study, V. cholerae VCC is secreted as an inactive protoxin that is matured after protease cleavage in the extracellular milieu (36,37). It thus might be argued that the observed decrease in the hemolytic activity in epsL mutants could be due to a defect of epsL mutants to secrete a factor responsible for maturating the hemolysins, rather than to a defect in secreting the hemolysins themselves.…”

Unsaturated Fatty Acids Drive Disintegrin and Metalloproteinase (ADAM)-dependent Cell Adhesion, Proliferation, and Migration by Modulating Membrane Fluidity

“…Reagents and Antibodies-VCC and polyclonal rabbit antisera against VCC were produced as described (15). Monoclonal antibodies against the cytoplasmic domain of E-cadherin were purchased from BD Bioscience.…”

“…For these analyses, a novel erythrocyte-based model was first introduced based on the unique property of the bacterial exotoxin Vibrio cholerae cytolysin (VCC) to be cleaved by ADAM10 that is present on rabbit erythrocytes (14,15). Generation of mature VCC resulted in hemolysis, which thus provided a simple and rapid readout.…”

Binding of Escherichia coli Hemolysin and Activation of the Target Cells Is Not Receptor-dependent

“…Toxins used for comparison of cytolytic activity were streptolysin O (20), Staphylococcus aureus ␣-toxin (21), and Vibrio cholerae cytolysin (22).…”