Identification of an iron-regulated hemin-binding outer membrane protein, HupO, in Vibrio fluvialis: effects on hemolytic activity and the oxidative stress response.
In pathogenic bacteria, iron acquisition is important for colonization and proliferation in the host under iron-limited conditions. The ability of Vibrio spp. to acquire iron is often critical to their virulence, causing gastroenteritis or excessive watery diarrhea in humans. In the study described here, we cloned the 2,100-bp heme utilization protein gene hupO in Vibrio fluvialis. HupO had high homology to iron-regulated outer membrane receptor proteins in Vibrio sp. and contained motifs that are common to bacterial heme receptors, including a consensus TonB box, a FRAP domain, and an NPNL domain. To characterize the hemin-binding activity of HupO, we purified the recombinant HupO protein (rHupO) from Escherichia coli by using an overexpression system. HupO was found to bind to hemin but not to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting demonstrated that the 77-kDa outer membrane protein HupO of V. fluvialis was induced under iron-restricted conditions. We constructed a hupO mutant, HP1, to investigate the biochemical function of HupO in V. fluvialis. The hemolytic activity of HP1 was reduced compared to that of wild-type cells and, when exposed to hydrogen peroxide, significantly lower numbers of HP1 survived than was the case in the wild type. These results suggest that HupO is associated with virulence expression in V. fluvialis through stimulation of hemolysin production and resistance to oxidative stress. In experimentally infected mice, the 50% lethal dose value of the wild-type was lower than that of the mutant, HP1.
Ahn
Sun-Hee
SH
Department of Biotechnology and Bioengineering, Pukyong National University, Busan 608-737, Republic of Korea.
Han
Jeong-Hyun
JH
Lee
Jong-Hee
JH
Park
Kee-Jai
KJ
Kong
In-Soo
IS
D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006427:Hemin / Q000378:metabolism*; D006461:Hemolysis / Q000502:physiology*; D007501:Iron / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D018384:Oxidative Stress / Q000502:physiology*; D016415:Sequence Alignment; D017385:Sequence Homology; D014733:Vibrio / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D014735:Vibrio Infections / Q000378:metabolism; D014774:Virulence / Q000235:genetics / Q000502:physiology
D001425:Bacterial Outer Membrane Proteins; C497367:HupO protein, Vibrio fluvialis; D006427:Hemin; D007501:Iron