Identification of a Vibrio furnissii oligopeptide permease and characterization of its in vitro hemolytic activity.
We describe purification and characterization of an oligopeptide permease protein (Hly-OppA) from Vibrio furnissii that has multifaceted functions in solute binding, in in vitro hemolysis, in antibiotic resistance, and as a virulence factor in bacterial pathogenesis. The solute-binding function was revealed by N-terminal and internal peptide sequences of the purified protein and was confirmed by discernible effects on oligopeptide binding, by accumulation of fluorescent substrates, and by fluorescent substrate-antibiotic competition assay experiments. The purified protein exhibited host-specific in vitro hemolytic activity against various mammalian erythrocytes and apparent cytotoxicity in CHO-K1 cells. Recombinant Hly-OppA protein and an anti-Hly-OppA monoclonal antibody exhibited and neutralized the in vitro hemolytic activity, respectively, which further confirmed the hemolytic activity of the gene product. In addition, a V. furnissii hly-oppA knockout mutant caused less mortality than the wild-type strain when it was inoculated into BALB/c mice, indicating the virulence function of this protein. Finally, the in vitro hemolytic activity was also confirmed with homologous ATP-binding cassette-type transporter proteins from other Vibrio species.
Wu
Tung-Kung
TK
Department of Biological Science and Technology, National Chiao Tung University, Hsin-Chu, Taiwan, 300, Republic of China. tkwmll@mail.nctu.edu.tw
Wang
Yu-Kuo
YK
Chen
Yi-Chin
YC
Feng
Jen-Min
JM
Liu
Yen-Hsi
YH
Wang
Ting-Yi
TY
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018441:Biofilms; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D006461:Hemolysis; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D014733:Vibrio / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D014735:Vibrio Infections / Q000382:microbiology; D014774:Virulence
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; C037230:oligopeptide permease, Bacteria