Identification of a Vibrio furnissii Oligopeptide Permease and Characterization of Its In Vitro Hemolytic Activity

Abstract

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 substrateantibiotic 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.

The Vibrio cholerae Pst2 Phosphate Transport System Is Upregulated in Biofilms and Contributes to Biofilm-Induced Hyperinfectivity

“…OppA has been associated with biofilm production in other Vibrio species (24,47), and therefore we investigated an oppA deletion mutant. We determined that oppA is required for the growth of V. cholerae on peptides as the sole carbon source and that it contributes slightly to biofilm production, but the ⌬oppA mutant displayed no colonization phenotype (data not shown).…”

An extracellular oligopeptide permease may be a potential virulence factor of Vibrio harveyi

“…A clear zone formed around the protein was considered as positive results. All the samples were dealt with filtration sterilization before the assay (Wu et al, 2007).…”

“…Some Gram-negative bacteria such as E. coli have three peptide uptake systems with varying substrate specificities, allowing them to utilize a broader range of peptides for nutrition purposes: dipeptide permease (Dpp), tripeptide permease (Tpp) and oligopeptide permease (Opp). Opp handles peptides of two to five residues (Wu et al, 2007;Yvonne et al, 2007). Opp is an ATP-binding cassette transporter in bacteria, and composed of five subunits: two transmembrane proteins (oppB and oppC), two ATP binding proteins (oppD and oppE) and an oligopeptide binding protein (OppA) (Tame et al, 1995;Claverys et al, 2000).…”

“…(Lee et al, 2004), and Mycoplasma homini (Hopfe et al, 2008). Direct evidence was obtained by Wu et al (2007), who identified an SBP (designated as hly-OppA) from the extracellular media of V. furnissii, which showed both solute-binding and hemolytic activity function in vitro. The hly-OppA knockout mutant caused less mortality than the wild-type strain when it was inoculated into BALB/c mice (Wu et al, 2007).…”

“…Direct evidence was obtained by Wu et al (2007), who identified an SBP (designated as hly-OppA) from the extracellular media of V. furnissii, which showed both solute-binding and hemolytic activity function in vitro. The hly-OppA knockout mutant caused less mortality than the wild-type strain when it was inoculated into BALB/c mice (Wu et al, 2007). Some ABC type transporter proteins of bacteria have been reported to provide protection against experimental challenge, including Yersinia pestis (Tanabe et al, 2006), Haemophilus parasuis (Hong et al, 2011), Moraxella catarrhalis (Yang et al, 2011) and Chlamydia pneumoniae (Li et al, 2010).…”