Structure and Functional Characterization of Vibrio parahaemolyticus Thermostable Direct Hemolysin

Abstract

Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic foodborne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.5 Å resolution. The TDH tetramerformsacentralporewithdimensionsof23Å indiameterand ϳ50 Å in depth. -Cation interactions between protomers comprising the tetramer were indispensable for hemolytic activity of TDH. The N-terminal region was intrinsically disordered outside of the pore. Molecular dynamic simulations suggested that water molecules permeate freely through the central and side channel pores. Electron micrographs showed that tetrameric TDH attached to liposomes, and some of the tetramer associated with liposome via one protomer. These findings imply a novel membrane attachment mechanism by a soluble tetrameric pore-forming toxin.Vibrio parahaemolyticus is a Gram-negative marine bacterium known to be one of the major causes of pandemic seafoodborne gastroenteritis. V. parahaemolyticus possesses two circular replicons of 3.2 and 1.9 megabase pairs, which might confer an advantage by enabling DNA replication in seawater of lower temperature and/or low nutritional value (1, 2). Such an advantage would potentially increase risks of food intoxication by allowing explosive expansion of the population of the microorganism. Hemolysis on Wagatsuma agar (a blood agar), known as the Kanagawa phenomenon, is associated with human pathogenic strains of V. parahaemolyticus. A major virulence factor of this pathogen is the thermostable direct hemolysin (TDH) 7 (3-5), which has a variety of biological activities including hemolytic activity, cardiotoxicity, and enterotoxicity. There are two copies of the tdh gene (or its homologue trh) in pathogenic V. parahaemolyticus, indicating the importance of this exotoxin for survival of the organism (2). The mature form of TDH consists of 165 amino acids, including a single intramolecular disulfide bond, but no close homologue of TDH has been found in other organisms. The significance of Arg 46 , Gly 62 , Trp 65 , and Gly 90 residues on hemolysis was determined by site-directed mutagenesis (3, 6).The common features of the bacterial pore-forming toxin are as follows. 1) It is released as a soluble monomer into the extra-bacterial space. 2) It oligomerizes to form a pore at the host cell membrane (7,8). An earlier study reported that TDH acted as a pore-forming toxin, creating a functional pore ϳ20 Å in diameter (reviewed in Ref. 3). We previously constructed a low resolution C 4 symmetric model of tetrameric TDH in solution based on small angle x-ray scattering (SAXS), transmission electron microscopy (TEM), and analytical ultracentrifugation (9). However, the precise structure and the mechanism for its pore-forming toxicity are still unknown. Several bacterial toxins, including TDH, show paradoxical responses to heat treatment, known as the Arrhenius effect (10, * This study was s...

High Frequency of Virulence Factor Genes tdh , trh , and tlh in Vibrio parahaemolyticus Strains Isolated from a Pristine Estuary

“…This necessitated band purification and reamplification before sequence data could be obtained to confirm the identities of these weak amplicons. All 23 tdh sequences recovered using primers tdh86F and tdh331R, when translated, produced an 80-amino-acid segment of the TDH protein that contained the conserved residues Arg 46 , Gly 62 , and Trp 65 (7). Sequences recovered using tdh86F/331R had on average 95% identity to the reference tdh sequence of V. parahaemolyticus ATCC 33846.…”

“…TDH and TRH embed in and disrupt host cell membranes, acting as porins (2,6,7), with channels of approximately 23-Å diameter (7). Insertion of these toxin proteins into the membranes causes a nonspecific efflux of divalent cations and influx of water molecules (7,8).…”

Characterization of a new phage, termed ϕA318, which is specific for Vibrio alginolyticus

“…The complex was uniform, with four dense domains holding together tightly, and these were tetramers of thermostable direct hemolysin, formed by four subunits of a 21.6-kDa protein of 189 amino acids. In whole genome analysis, it has been predicted that V. alginolyticus ATCC 17749 and some strains of V. parahaemolyticus can produce it [23]. Using bioinformatics to find sequences with homology to thermostable direct hemolysin (TDH) sequences in V. alginolyticus (GenBank accession no.…”

Genes Similar to the Vibrio parahaemolyticus Virulence-Related Genes tdh , tlh , and vscC2 Occur in Other Vibrionaceae Species Isolated from a Pristine Estuary

“…Genes encoding the thermostable direct hemolysin (TDH) and the homologous thermostable direct hemolysin-related hemolysin (TRH), tdh and trh, respectively, have been implicated in V. parahaemolyticus virulence (19,20,21). TDH and TRH are tetrameric proteins that act as porins and facilitate efflux of divalent cations and other solutes from and influx of water molecules into intestinal cells (11,22,23). Occurrence of tdh is correlated with the Kanagawa phenomenon (KP), a beta-hemolytic reaction on saline blood agar (Wagatsuma agar) (19).…”