Aerolysin

Aerolysin is a toxin made by the bacterium Aeromonas hydrophila, a species associated with diarrheal disease and deep wound infections.^[1] It is secreted as a soluble protein. On a target cell, seven copies join into a narrow channel through the membrane, and the cell loses its contents. In the laboratory the same channel, set in a lipid bilayer, is used as a biological nanopore: it resolves oligonucleotides that differ by a single base and peptides that differ by a single amino acid.

The name comes from a paper of 1974 on a hemolytic substance in the culture fluid of A. hydrophila. The authors purified it by salt fractionation and gel filtration, estimated its molecular weight at 50,000 and wrote that it was "here termed 'aerolysin'".^[2]

A gene named aerA codes for a chain of 493 residues. A signal peptide of 23 residues is removed on export, and what remains is proaerolysin, a protein of about 52,000 daltons that occurs as a dimer.^[3] Proaerolysin binds proteins that hang on the outer face of a cell membrane by a glycosylphosphatidylinositol anchor.^[4] Then a protease of the host such as furin cuts a loop near the C terminus after an arginine, and only the cut protein can form oligomers and channels.^[5] A ring of seven cut molecules is the prepore. It inserts into the membrane as a β-barrel, which leaves a channel through the wall of the cell.

The first structure, of the proaerolysin precursor, came from X-ray crystallography at 2.8 Å resolution in 1994. The fold had not been seen before. Electron micrographs of the oligomer gave the authors enough to build a model of the membrane channel.^[1] In 2016 cryo-electron microscopy gave structures of three intermediates and of the finished pore. The pore consists of two concentric β-barrels held together by hydrophobic contacts, a fold the authors link to the "prion-like ultrastability" of the channel. Aerolysin is the founding member of a family of pore-forming toxins found in all kingdoms of life.^[6]

Aerolysin differs from the other pores in use mainly by its length. A 2019 study put it at about 10 nm, against about 8.5 nm for alpha-hemolysin and about 8.0 nm for MspA, and noted that the greater length together with a narrower bore slows a molecule down without help from a motor or an adapter. By mutating residues at the sensing region, near positions 220 and 238, the authors varied the sensing diameter from 5 Å to 15 Å.^[7] Alpha-hemolysin is 1.4 nm wide at its narrowest point.

Alpha-hemolysin gave the first single strands of DNA in 1996, and aerolysin received much less attention as a sensor. In 2016 a group showed that a wild-type aerolysin pore, with no motor enzyme and no engineering, could separate oligonucleotides of 2 to 10 bases from one another, tell them apart in a mixture and follow exonuclease I as it cut a strand step by step.^[8]

Peptides followed. The wild-type pore separated short homopeptides, mixed in solution, that differed in length by one amino acid, and it found a few dozen impurities in a commercial peptide sample of high purity.^[9] In 2020, with a short polycationic carrier attached, all twenty proteinogenic amino acids gave a signal, and 13 of them could be identified by their current blockades, a result the authors suggest may lead toward nanopore protein sequencing. In simulations, a carrier-bound amino acid stayed inside the sensing region of the pore for long enough to be measured.^[10]

JeoPOREdy!, the company's quiz game, has a category called Aerolysin Alley. It contains one clue, and the answer is "What is aerolysin?" The clue says that two research groups turned the Aeromonas hydrophila pore into "a champion sensor, even though in nature it punches holes in your gut cells".^[11]

• Alpha-hemolysin
• Biological nanopore
• Nanopore protein sequencing
• JeoPOREdy!