Cytotoxicity and Enterotoxicity of the Thermostable Direct Hemolysin‐Deletion Mutants of Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus, one of the human-pathogenic vibrios, is a Gram-negative halophilic bacterium that naturally inhabits marine and estuarine environments. The bacterium causes food-borne gastroenteritis, most frequently associated with the consumption of raw or undercooked seafood (1). Consumption of sufficiently high numbers of the organism can cause watery diarrhea, septicemia and even death (7,10). Almost all the clinical isolates of V. parahaemolyticus show β-hemolysis on Wagatsuma agar, a special blood agar medium (24). The hemolysis has been called the Kanagawa-phenomenon (KP), and is considered a good marker to differentiate human pathogenic V. parahaemolyticus from the non-pathogenic strains. To date, the thermostable direct hemolysin (TDH), has been shown to have a role in KP (6,27). In 1988, Honda et al. (9) discovered a new hemolysin in KP-negative clinical V. parahaemolyticus isolates. This hemolysin was referred to as TDH-related hemolysin (TRH). It is immunologically similar to TDH, and shares approximately 67% identity with TDH at the amino acid level (9). TDH and TRH show several common biological properties, such as hemolytic activity, enterotoxicity and cytotoxicity (5,6,8,10,16,17,23,25,29,31). V. parahaemolyticus strains isolated from patients with diarrhea produce TDH or TRH or both, whereas environmental isolates rarely produce these proteins (6, 27). Thus, TDH and TRH have been considered to be involved in the gastrointestinal disorders caused by V. parahaemolyticus (6).Although live V. parahaemolyticus shows cytotoxicity and enterotoxicity (2, 15, 25), the virulence factors responsible for these activities have not been fully elucidated. Recently, we completed the genome sequencing of a KP-positive (TDH-producing) V. parahaemolyticus RIMD2210633 strain (13). In this study, we constructed tdh-deletion mutants from the sequenced RIMD2210633 strain, and examined the hemolytic activity, cytotoxicity and enterotoxicity of the mutant strains Abstract: The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of Vibrio parahaemolyticus. We have recently completed the genome sequence of a TDH-producing V. parahaemolyticus strain, RIMD2210633. In this study, we constructed tdh-deletion mutants from the sequenced strain by homologous recombination and analyzed their phenotypes. Although the deletion of both copies of tdh completely abolished the hemolytic activity of the wild-type strain, the deletion did not affect the cytotoxicity to HeLa cells. Enterotoxicity, assayed by the rabbit ileal loop test, was lowered by tdh deletion, but the mutant still showed partial fluid accumulation in rabbit intestine. These results indicate that the cytotoxicity and enterotoxicity of TDH-producing V. parahaemolyticus are not explained by TDH alone, and suggest that an unknown virulence factor(s) could be involved in these pathogenic activities.

Arp2/3-independent assembly of actin by Vibrio type III effector VopL

“…pCMV5-C3 was a kind gift from P. C. Sternweis (University of Texas Southwestern Medical Center). V. para strains POR-1, POR-2, and POR-3 were obtained from T. Iida and T. Honda (Osaka University, Osaka, Japan) (3,22). Expression plasmids containing dominant negative [pcDNA3-(HA) 3 Pyrene Actin Assembly Assays.…”

“…V. para strains POR-1, POR-2, and POR-3 were obtained from T. Iida and T. Honda (Osaka University, Osaka, Japan) (3,22). Expression plasmids containing dominant negative [pcDNA3-(HA) 3 Pyrene Actin Assembly Assays. Actin polymerization assays were performed as described previously (36).…”

“…Sequencing of the V. para genome identified two pathogenicity islands encoding type III secretion systems, TTSS1 and TTSS2, the latter associated with virulent, clinical isolates of V. para (2). Iida and colleagues (3,4) have demonstrated that the TTSS on chromosome one (TTSS1) is linked to cytotoxicity in infected tissue culture cells, whereas the TTSS on chromosome two (TTSS2) is associated with enterotoxicity in the rabbit ileal loop model.…”

Vibrio parahaemolyticus orchestrates a multifaceted host cell infection by induction of autophagy, cell rounding, and then cell lysis

“…TDH and TRH are reversible amyloid toxins that cause ␤-hemolysis on Wagatsuma agar, known as the ''Kanagawa phenomenon.'' However, infection with ⌬tdh and ⌬trh strains of V. parahaemolyticus results in rapid and acute cell death in a tissue culture model (3). This cell death is associated with the presence of a type III secretion system (T3SS) (3).…”

“…However, infection with ⌬tdh and ⌬trh strains of V. parahaemolyticus results in rapid and acute cell death in a tissue culture model (3). This cell death is associated with the presence of a type III secretion system (T3SS) (3). Bacterial T3SSs deliver proteins, called ''effectors,'' into the cytosol of host cells during infection (4).…”