JB Accepts, published online ahead of print on 6 November 2009

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J. Bacteriol. doi:10.1128/JB.01049-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Aromatic ring of phenylalanine 334 is essential for oligomerization of Vibrio vulnificus hemolysin

Takashige Kashimoto^*, Shunji Ueno, Takeshi Koga, Shinji Fukudome, Hayato Ehara, Mayumi Komai, Hiroyuki Sugiyama, and Nobuyuki Susa

Laboratory of Veterinary Public Health, School of Veterinary Medicine, Kitasato University, Higashi 23-35-1, Towada Aomori 034-8628, Japan

^* To whom correspondence should be addressed. Email: kashimot{at}vmas.kitasato-u.ac.jp.

Vibrio vulnificus hemolysin (VVH) is thought to be a member of the cholesterol dependent cytolysin (CDC) family of pore-forming toxins. To date, structure function relationships of CDCs produced by Gram-negative bacteria remain largely unknown. We show here the aromatic ring of phenylalanine residue conserved in Vibrionaceae hemolysins is essential for oligomerization of Vibrio vulnificus hemolysin (VVH). We generated the VVH mutants substituted Phe 334 to Ile (F334I), Ala (F334A), Tyr (F334Y), or Trp (F334W) and tested their binding and oligomerizing activity on Chinese hamster ovary cells. Binding in all mutants fell by approximately 50% compared with the wild type. Oligomerizing activities were completely eliminated in F334I and F334A, whereas this ability was partially retained in F334Y and F334W. These findings indicated that both hydrophobicity and an aromatic ring residue at the 334th position was needed for full binding activity, and that the oligomerizing activity of this toxin was dependent on the existence of an aromatic ring residue at the 334th position. Our findings might help further understanding of the structure and function relationships in Vibrionancae hemolysins.