The variant undecapeptide sequence of the Arcanobacterium pyogenes haemolysin, pyolysin, is required for full cytolytic activity

Abstract

The cholesterol-dependent cytolysins (CDCs) are characterized by an undecapeptide sequence (ECTGLAWEWWR) that is located near the C terminus and within domain 4 of these proteins. Pyolysin (PLO), the CDC of Arcanobacterium pyogenes, has a variant undecapeptide sequence (EATGLAWDPWW). Site-directed mutants were constructed in undecapeptide residues in a recombinant PLO molecule containing a hexahistidine tag (His-PLO). Mutations in each of the three undecapeptide tryptophan residues resulted in low haemolytic activity, confirming the importance of these residues in the protein. Deletion of a proline residue (P 499 ), inserted in PLO, or substitution of this residue with either phenylalanine or glycine resulted in mutant proteins with undetectable or low haemolytic activities, indicating that P 499 is essential for His-PLO haemolytic activity. Substitution of the PLO undecapeptide sequence with a consensus undecapeptide resulted in a His-PLO protein with only 01 % activity, confirming that the variant PLO undecapeptide is required for the full cytolytic activity of this toxin. The presence of the conserved undecapeptide cysteine residue either alone (His-PLO.C 492 ) or in a consensus sequence resulted in His-PLO molecules which were activated in the presence of reducing compounds, confirming the importance of this residue in the thiol-activated nature of many CDC toxins. The ability of His-PLO mutant proteins to bind cholesterol mimicked haemolytic activity, with the exception of His-PLO.C 492 , which, despite having reduced haemolytic activity, showed an increased ability to bind cholesterol compared to His-PLO. Despite reductions in haemolytic activity and cholesterol-binding, all mutant proteins were still able to bind to erythrocyte membranes, suggesting that other regions of PLO may recognize host-cell membranes, through receptors other than cholesterol.

The Human‐Specific Action of Intermedilysin, a Homolog of Streptolysin O, Is Dictated by Domain 4 of the Protein

“…It was also revealed that the C residue is important in the high affinity to cholesterol and the second W residue in the region also plays a key role in binding to cholesterol, especially to membrane-embedded cholesterol. Previously, however, the C residue of this region in more typical CDCs such as PLY and SLO has been shown to be non-essential for activity (25,30), but to increase binding affinity to cholesterol in the case of PLO (6). In addition mSLO(GAP) with the complete ILY undecapeptide sequence still exhibited cholesterol sensitivity whereas mILY(EDW), with the complete PLO undecapeptide sequence, did not.…”

Replacing the 238th aspartic acid with an arginine impaired the oligomerization activity and inflammation-inducing property of pyolysin

“…The resulting pET-30a (+)-plo P499F encoded rPLO P499F with proline (P) at position 499 of immature PLO replaced with phenylalanine (F). rPLO P499F (named His-PLO.F 499 in Billington [35]) reportedly lost its cell membrane binding capacity compared with rPLO and was used as a control in the current study. pET-30a (+)-plo D238R and pET-30a (+)-plo N376R encoded rPLO D238R and rPLO N376R, respectively.…”

The Aromatic Ring of Phenylalanine 334 Is Essential for Oligomerization ofVibrio vulnificusHemolysin

“…It is also well known that VVH binds to cholesterol in vitro (10,35), but the cholesterol binding site has not yet been defined. Interestingly, VVH does not have the tryptophan-rich motif that is highly conserved in gpCDCs and is involved in membrane recognition (3,9,27). Therefore, there is a great interest in understanding whether or not both cholesterol and carbohydrate are required for VVH to bind to susceptible cells.…”

“…For instance, LukF has a tyrosine residue at the bottom of the rim domain, and this tyrosine residue participates in the first attachment of LukF to the cellular membrane (34). It is also well known that the tryptophan-rich motif of gpCDCs, which is necessary to recognize and/or span the cellular membrane, exists at the bottom of the CDC protein (3,9,27). According to our model of VVH, some hydrophobic residues were present at the bottom of the rim domain (data not shown).…”

“…(i) There is no similarity in amino acid sequences. (ii) gpCDCs have a highly conserved tryptophan-rich motif, which is involved in membrane recognition (3,9,27), whereas this motif does not exist in Vibrionaceae hemolysins. (iii) gpCDCs, such as perfringolysin and intermedilicine, are composed of four domains, whereas Vibrionaceae hemolysins are composed of two or three domains (21,24,25).…”