Purification and Characterization of a Hemolysin Produced by Vibrio parahaemolyticus

Zen-Yoji, Hiroshi; Hitokoto, Hiroshi; Morozumi, Satoshi; Clair, R. A. Le

doi:10.1093/infdis/123.6.665

Cited by 54 publications

(39 citation statements)

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“…In similar experiments using human blood, pinpoint zones of hemolysis were detected ( Table 1). The different hemolytic intensities among RBC species may explain the previously reported negative results since RBCs from different animal species vary in sensitivity to bacterial hemolysins, including Leptospira interrogans (25), Staphylococcus aureus (4), certain Vibrio species (14,18,35,36), and Borrelia burgdorferi (34). Concerning M. penetrans hemolytic activity, the molecular basis of the difference in RBC sensitivity is unknown.…”

Section: Resultsmentioning

confidence: 99%

Hemolytic and Hemoxidative Activities inMycoplasma penetrans

Kannan

Baseman

2000

Infect Immun

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Mycoplasma penetrans is a newly isolated Mollicute from the urine of patients infected with human immunodeficiency virus that demonstrates the capacity to adhere to and invade human cells. A previous report, based on assays with mouse red blood cells (RBCs), indicated that M. penetrans lacked hemolytic activity. In our studies, we incubated different isolates of M. penetrans with various RBC species and observed hemolytic zones surrounding individual mycoplasma colonies. All M. penetrans strains displayed hemolysis after 2 to 3 days of incubation. Hemolytic activity diffused from single colonies, eventually causing complete lysis. Hemolysis was most pronounced with sheep RBCs, followed by horse, chicken, and human cells. Furthermore, hemolytic activity was demonstrable in both intact mycoplasma cell preparations and spent culture supernatant. However, unlike intact mycoplasmas, the hemolytic activity in the supernatant was dependent on the reducing agent, cysteine. In addition to hemolysis, a brown precipitate was closely associated with mycoplasma colonies, suggesting oxidation of hemoglobin. Absorption spectra indicated that hemoglobin was oxidized to methemoglobin, and the addition of catalase demonstrated H 2 O 2 -mediated hemoxidation. Other experiments suggested that hemoxidation enhanced total hemolysis, providing the first evidence of both hemolytic and hemoxidative activities in M. penetrans.Mycoplasmas, members of the cell wall-less Mollicutes and considered among the smallest self-replicating cells, have been detected in a wide range of hosts, including humans, other vertebrates, invertebrates, and plants (2,19,20,27). In previous studies of pathogenic mycoplasmas, hemadsorption and cytadherence activities were closely associated with virulence potential (2, 30). For example, hydrogen peroxide-mediated and membrane-associated hemolytic activities have been found in numerous Mycoplasma species (15,24,30), and analysis of mycoplasma genomic sequences revealed the presence of a hemolysin-like gene (VXpSPT7_orf424) in Mycoplasma pneumoniae (13) and a homologous gene (MG146) in Mycoplasma genitalium (10). It is well known that bacterial hemolysins lyse erythrocytes (RBCs) and a variety of other cell types, including mast cells, neutrophils, and polymorphonuclear cells, which enables hemolytic microorganisms to directly damage host tissues as well as induce inflammatory responses (5). However, a new species, Mycoplasma penetrans, which was isolated from the urine of patients infected with human immunodeficiency virus (22), was shown to actively invade mammalian cells in culture (21) and cause cytopathology in experimentally infected chicken embryos (12) but lack hemolytic activity (24). In this report we describe hemolytic activity in all isolates of M. penetrans and show that H 2 O 2 -mediated hemoxidative activity contributes to total mycoplasma-mediated hemolysis. MATERIALS AND METHODSBacterial strains and medium. M. penetrans cells (GTU-54 and isolates from France and Texas) (11) were obtained from J. T...

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Section: Resultsmentioning

confidence: 99%

Hemolytic and Hemoxidative Activities inMycoplasma penetrans

Kannan

Baseman

2000

Infect Immun

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“…This phenomenon is known as the Kanagawa phenomenon (KP) and is considered a good marker to distinguish between pathogenic and nonpathogenic V. parahaemolyticus strains (25,37). Thermostable direct hemolysin (TDH), which is responsible for the KP (28,43), has multiple biological activities, including hemolysis, enterotoxicity, cytotoxicity, and cardiotoxicity (11-13, 26, 29, 32, 34, 38). For this reason, TDH has been considered a major virulence factor of V. parahaemolyticus.…”

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confidence: 99%

Contribution of Vibrio parahaemolyticus Virulence Factors to Cytotoxicity, Enterotoxicity, and Lethality in Mice

Hiyoshi¹,

Kodama

Iida³

et al. 2010

Infect Immun

199

226

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Vibrio parahaemolyticus, one of the human-pathogenic vibrios, causes three major types of clinical illness: gastroenteritis, wound infections, and septicemia. Thermostable direct hemolysin (TDH) secreted by this bacterium has been considered a major virulence factor of gastroenteritis because it has biological activities, including cytotoxic and enterotoxic activities. Previous reports revealed that V. parahaemolyticus strain RIMD2210633, which contains tdh, has two sets of type III secretion system (T3SS) genes on chromosomes 1 and 2 (T3SS1 and T3SS2, respectively) and that T3SS1 is responsible for cytotoxicity and T3SS2 is involved in enterotoxicity, as well as in cytotoxic activity. However, the relative importance and contributions of TDH and the two T3SSs to V. parahaemolyticus pathogenicity are not well understood. In this study, we constructed mutant strains with nonfunctional T3SSs from the V. parahaemolyticus strain containing tdh, and then the pathogenicities of the wild-type and mutant strains were evaluated by assessing their cytotoxic activities against HeLa, Caco-2, and RAW 264 cells, their enterotoxic activities in rabbit ileal loops, and their lethality in a murine infection model. We demonstrated that T3SS1 was involved in cytotoxic activities against all cell lines used in this study, while T3SS2 and TDH had cytotoxic effects on a limited number of cell lines. T3SS2 was the major contributor to V. parahaemolyticus-induced enterotoxicity. Interestingly, we found that both T3SS1 and TDH played a significant role in lethal activity in a murine infection model. Our findings provide new indications that these virulence factors contribute to and orchestrate each distinct aspect of the pathogenicity of V. parahaemolyticus.Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that inhabits estuarine and coastal waters and can be isolated from seafood (6,7,9). It causes acute gastroenteritis in humans after they consume contaminated raw or undercooked seafood. Although this microorganism is better known for causing gastroenteritis, it also can cause wound infections and septicemia (5,23,36).Most clinical isolates of V. parahaemolyticus from patients with diarrhea show ␤-hemolysis on Wagatsuma agar (37). This phenomenon is known as the Kanagawa phenomenon (KP) and is considered a good marker to distinguish between pathogenic and nonpathogenic V. parahaemolyticus strains (25, 37). Thermostable direct hemolysin (TDH), which is responsible for the KP (28, 43), has multiple biological activities, including hemolysis, enterotoxicity, cytotoxicity, and cardiotoxicity (11-13, 26, 29, 32, 34, 38). For this reason, TDH has been considered a major virulence factor of V. parahaemolyticus.Whole-genome sequencing of a KP-positive V. parahaemolyticus strain revealed that this strain contains two sets of gene clusters for the type III secretion system (T3SS), T3SS1 and T3SS2, one on each of its two chromosomes (21). T3SS gene clusters have been detected in numerous Gram-negative animal and plant pathogens, wh...

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“…The hemolytic character appears to be associated consistently with strains isolated from patients. Such hemolysins have not, however, satisfactorily demonstrated the capacity to elicit pathologic changes in laboratory animals which could be correlated with human disease (17,22,25,26,(42)(43)(44). It is believed by some investigators that if enterotoxins were produced by V. parahaemolyticus, such toxins would be quite dif-ferent from those of V. cholerae (18).…”

Section: Discussionmentioning

confidence: 99%

“…Because Kanagawa-phenomenon positive strains are isolated predominantly from patients, hemolytic strains are generally considered to be pathogenic, and the non-hemolytic, Kanagawa-phenomenon negative strains non-pathogenic (13,23,32). To date, studies of V. parahaemolyticus toxins have been restricted to isolation of hemolysins from hemolytic Kanagawa-phenomenon positive strains (16,17,20,22,25,26,(42)(43)(44). Attempts have not been made to isolate toxin(s) from Kanagawa-phenomenon negative strains, despite the fact that such strains have been isolated from gastroenteritis victims (3).…”

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Toxin Isolation from a Kanagawa‐Phenomenon Negative Strain of Vibrio parahaemolyticus

Sochard

Colwell

1977

Microbiology and Immunology

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Production of a toxin by Vibrio parahaemolyticus Kanagawa-phenomenon negative strains was examined. Ammonium sulfate fractions of broth culture filtrates were dialyzed, concentrated by lyophilization, and tested for toxic effects by mouse intraperitoneal injection. One fraction, which we think is a toxin, was isolated from a broth culture filtrate of V. parahaemolyticus FC 1011 (a Kanagawaphenomenon negative strain) and consistently produced lethal effects in mice at high concentrations and diarrhea in lower concentrations. The toxin was assayed for mouse LD50 and ability to produce diarrhea via forced feeding in mice. V. parahaemolyticus FC 1011 toxin was found to be protein, to be inactivated by heat or trypsin hydrolysis, and to produce positive skin permeability reactions in rabbits. However, it failed to induce fluid accumulation in ligated ileal loops in rabbits.Prominent among symptoms recorded for victims of Vibrio parahaemolyticusassociated food poisoning are diarrhae, abdominal pain and vomiting, with chills, fever, headache and bloody stools less frequently reported (2,3,7,8,10,19,33,(36)(37)(38). V. parahaemolyticus illnesses have frequently been diagnosed as dysentery and non-typhoidal salmonellosis (1-3). Bacterial-induced diarrhea is currently thought to be mediated by at least three mechanisms : (i) cell-free protein enterotoxins, liberated by nonpenetrating organisms and initiating fluid accumulation in the gut, viz., Vibrio cholerae (12), Escherichia coli (29) and Clostridium perfringens (10) , (ii) invasion by organisms, such as Shigella (15) and E. coli (11), manifesting cytotoxic properties which interfere with host cell functions and cause diarrhea; and (iii) a combination of these. Because of the variety of symptoms reported by V. parahaemolyticus gastroenteritis victims and the conflicting pathology data, it is not clear whether the disease is a manifestation of effects of enterotoxin on the host, invasion of the causative agent via the gut, or to toxic components produced only by certain serotypes of V. parahaemolyticus. Another member of the genus Vibrio, V. cholerae, is known to produce an exo-enterotoxin, associated with symptoms elicited by the organism (12). Because of the strong similarity of V. parahaemolyticus-associated disease with cholera, there is reason to suspect that V. parahaemolyticus may produce an enterotoxin (6, 40).

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Purification and Characterization of a Hemolysin Produced by Vibrio parahaemolyticus

Cited by 54 publications

References 5 publications

Hemolytic and Hemoxidative Activities inMycoplasma penetrans

Hemolytic and Hemoxidative Activities inMycoplasma penetrans

Contribution of Vibrio parahaemolyticus Virulence Factors to Cytotoxicity, Enterotoxicity, and Lethality in Mice

Toxin Isolation from a Kanagawa‐Phenomenon Negative Strain of Vibrio parahaemolyticus

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