The hemagglutinating action of Vibrio vulnificus metalloprotease

Vibrio vulnificus protease (VVP), a 45-kDa zinc metalloprotease, consists of two functional domains: an N-terminal 35-kDa polypeptide having endoproteinase activity, and a C-terminal 10-kDa polypeptide that mediates the binding of VVP to the erythrocyte membrane. Therefore, VVP, but not its N-terminal endoproteinase domain alone, has agglutinating activity to rabbit erythrocytes. When a single zinc atom in the catalytic center was substituted by treatment with CuCl2 or NiCl2, proteolytic and hemagglutinating activities were reduced by Ni substitution but not by Cu substitution. Cu-treated 35-kDa polypeptide showed sufficient affinity of the catalytic center and weak binding ability to the erythrocyte membrane, but the Ni-treated polypeptide did not. These results suggest that the binding of endoproteinase domain to membrane is also necessary for hemagglutination.     

Actions of Vibrio vulnificus Metalloprotease on Human Plasma Proteinase-Proteinase Inhibitor Systems: A Comparative Study of Native Protease with Its Derivative Modified by Polyethylene Glycol

Vibrio vulnificus, an opportunistic human pathogen causing wound infection and septicemia, produces a metalloprotease (VVP) which is suspected to be a virulent determinant. The interactions of VVP, as well as its derivative (PEG₁-VVP) modified with polyethylene glycol, with a variety of human plasma proteins were investigated. We found that native VVP and its derivative were able to act directly on many biologically important human plasma proteins even in the presence of α-macroglobulin, the sole plasma inhibitor of native VVP. The activities of both classical and alternative pathways of the complement cascade system were drastically abolished by incubation with either VVP. Furthermore, these proteases rapidly digested the Aα-chain of human fibrinogen into fragment(s) with no clotting ability. Therefore both VVPs are thought to function as a fibrinogenolytic enzyme, causing delay of the coagulation reaction. VVP and PEG₁-VVP were also shown to destroy plasma proteinase inhibitors including α₁-proteinase inhibitor, a major inhibitor in human plasma. Because endogenous proteolytic enzymes and their inhibitors are indispensable in maintaining physiological homeostasis, these findings suggest that VVP (and PEG₁-VVP) may cause an imbalance of human plasma proteinase-proteinase inhibitor systems, thus eliciting an immunocompromised state in the host and facilitating the development of a systemic V. vulnificus infection such as septicemia.     

Siderophore-Mediated Utilization of Iron Bound to Transferrin by Vibrio parahaemolyticus

Vibrio parahaemolyticus produces a structurally novel type of siderophore, termed vibrioferrin, in response to iron-limitation. This study was performed to examine whether vibrioferrin can assimilate iron from human iron-binding proteins for growth. Comparison of the growth rates between V. parahaemolyticus AQ 3354 and its spontaneously arising, vibrioferrin-deficient mutant revealed that vibrioferrin was able to sequester iron from 30% iron-saturated human transferrin for growth, but not from human lactoferrin even if fully saturated with iron. In both strains, iron limitation induced two high-molecular-weight outer membrane proteins with apparent molecular masses of approximately 78 and 83 kDa. Since only the outer membrane fraction including these proteins showed a binding capacity to ferric vibrioferrin complex, either of them may function as its cell surface receptor. These results suggested that the organism might utilize such a source of host iron through the action of vibrioferrin during in vivo survival and proliferation, although its importance in pathogenesis is unknown.     

Simple Purification Method for a Vibrio vulnificus Hemolysin by a Hydrophobic Column Chromatography in the Presence of a Detergent

A Vibrio vulnificus hemolysin (VVH) was purified by two steps of hydrophobic column chromatography on Phenyl-Sepharose HP. The first chromatography was carried out at pH 6.0. In this pH condition, VVH efficiently bound to the column, but the hemolysin fraction eluted was accompanied with colored substance(s). To eliminate this colored substance, the second chromatography was carried out at pH 9.8 in the presence of 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent. Homogeneity of the hemolysin thus obtained was shown by polyacrylamide gel electrophoresis. The specific activity increased 33, 600 times and the yield was 35%. The method is simple and useful to supply enough VVH for study of the role of the hemolysin in the infection by V. vulnificus or on the mechanism of action of the hemolysin.     

Regulation systems of protease and hemolysin production in Vibrio vulnificus

Vibrio vulnificus, a gram‐negative halophilic estuarine bacterium, is an opportunistic human pathogen that causes rapidly progressive fatal septicemia and necrotizing wound infection. This species also causes hemorrhagic septicemia called vibriosis in cultured eels. It has been proposed that a range of virulence factors play roles in pathogenesis during human and/or eel infection. Among these factors, a metalloprotease (V. vulnificus protease [VVP]) and a cytolytic toxin (V. vulnificus hemolysin [VVH]) are of significant importance. VVP elicits the characteristic edematous and hemorrhagic skin damage, whereas VVH exhibits powerful hemolytic and cytolytic activities and contributes to bacterial invasion from the intestine to the blood stream. In addition, a few V. vulnificus strains isolated from diseased eels have recently been found to produce a serine protease designated as V. vulnificus serine protease (VvsA) instead of VVP. Similarly to VVP, VvsA may possess various toxic activities such as collagenolytic, cytotoxic and edema‐forming activity. In this review, regulation of V. vulnificus VVP, VVH and VvsA is clarified in terms of expression at the mRNA and protein levels. The explanation is given on the basis of the quorum sensing system, which is dependent on bacterial cell density. In addition, the roles of environmental factors and global regulators, such as histone‐like nucleoid structuring protein, cyclic adeno monophosphate receptor protein, RpoS, HlyU, Fur, ToxRS, AphB and LeuO, in this regulation are outlined. The cumulative impact of these regulatory systems on the pathogenicity of V. vulnificus is here delineated.     

Cytotoxicity of Vibrio vulnificus cytolysin on rat peritoneal mast cells

Histamine has been thought to be a permeability enhancing factor in Vibrio vulnificus infection. The injection of living bacteria or purified V. vulnificus cytolysin (VVC) can cause lethality in mice by inducing hemoconcentration and increased vascular permeability. In the present study, we tried to identify whether histamine release causes the increased vascular permeability that is responsible for the lethal effect of VVC. Treatment of rat peritoneal mast cells with high concentrations of VVC caused the release of whole cellular histamine and lactate dehydrogenase (LDH). At concentrations less than 10 HU/ml, histamine and LDH were not released whereas preloaded 2-deoxy-D-glucose was rapidly effluxed with the concomitant decrease in cellular ATP. VVC-treated mast cells were refractory to the stimulation of histamine secretion by Compound 48/80 but remained fully responsive to Ca2+ plus GTP-gamma-S. These results indicate that histamine can be released from mast cells only when the concentration of VVC is high enough to cause the lysis of cells. At low concentrations, VVC does not induce the release of stored histamine from damaged cells. The intravenous injection of 80 HU purified VVC to rats, which can produce the calculated blood concentration of about 3 HU/ml, caused a marked increase in pulmonary vascular permeability, hemoconcentration and death. However, no increase in blood histamine level was detected. This level of VVC in rat blood was enough to cause severe hemoconcentration and lethality but might not be enough to cause cytolysis of the mast cells and resulting histamine release.     

Production of Antigenically Related Exocellular Elastolytic Proteases Mediating Hemagglutination by Vibrios

Exocellular proteases produced by Vibrio fluvialis, V. furnissii, V. metschnikovii and V. campbellii were characterized and compared to those of V. mimicus protease (VMP) and V. vulnificus protease (VVP). These proteases possessed both elastolytic and hemagglutinating abilities and were identified, except that of V. metschnikovii, as metalloprotease. Conversely, V. metschnikovii protease failed to exhibit some of the salient features for metalloproteases suggesting the existence of protease(s) other than metalloprotease. However, antibodies against VVP cross-reacted to these proteases and to VMP indicating antigenic relatedness amongst vibrio proteases. This study, thus, demonstrated the prevalent distributions of antigenically related proteases both in pathogenic and non-pathogenic vibrios, bringing their status as a virulence determinant into question.     

Utilization of Iron Sources and Its Possible Roles in the Pathogenesis of Vibrio parahaemolyticus

Vibrio parahaemolyticus is an important enteropathogen in Japan, Taiwan and other coastal regions. The influence of the regulation of iron on the pathogenesis of this pathogen has not been well characterized. The growth of pathogenic and non-pathogenic strains of V. parahaemolyticus on iron-limited agar plates was stimulated by ferritin, lactoferrin and transferrin at 30 μM , and also by hemin, hemoglobin and ferric ammonium citrate at 100 μM . Spontaneous iron-utilizing mutant strains (mutants) were derived from a clinical strain, ST550. Compared with the parent strain, lowered virulence was demonstrated for these mutants, as assayed by adult mouse and suckling mouse models. The in vivo growth and enterotoxigenicity of these mutants were also lower in the suckling mice. Adherence of the mutants to excised mouse intestine was lower as demonstrated by scanning electron microscopy. The iron-regulated outer membrane protein profile also changed in selected mutants. These results indicate that iron-regulated outer membrane proteins and other unknown factors associated with iron utilization may have profound influences, besides iron acquisition, on the pathogenesis of V. parahaemolyticus.     

Mechanism of high susceptibility of iron-overloaded mouse to Vibrio vulnificus infection

Vibrio vulnificus produces fulminant septicemia in humans with underlying conditions, particularly those with diseases that elevate the iron level. The effect of a high iron level on the virulence of V. vulnificus was therefore investigated in mice treated with iron dextran. The mice loaded with iron became highly susceptible to V. vulnificus infection, the LD50 (50% lethal dose) decreased five logs when infected per peritoneum. However, when infected via the oral route, the LD50 was affected little unless the mouse was treated with an additional drug such as cyclophosphamide or D-galactosamine. Mice with or without iron-overloading died when the bacterial concentration in the blood reached 10(5) cfu/ml or above. Iron increased the growth rate of the bacteria, both inside and outside of the animal, quickly reaching a lethal concentration in the iron-overloaded mouse. V. vulnificus, grown with or without the addition of iron, showed strong cytotoxicity on the isolated cells or within the animal at high bacterial concentration. Iron overload stimulated the production of tumor necrosis factor alpha (TNF-alpha), a major factor of septic shock, in mice upon infection with the bacteria, probably caused by the endotoxin; however, the neutrophils, whose migration is effected by TNF-alpha, appeared to be less active. Taken together, the major virulence factor of V. vulnificus appeared to be the accelerated growth of bacteria to quickly reach the lethal level and the lower activity of immune cells including neutrophil as a result of iron-overloading. These two effects manifest other virulence factors, the host's as well as bacterial. Such factors, other than TNF-alpha stimulated by the endotoxin, enhanced cytotoxicity, which kills the host cells including the host's immune cells.     

Specificity of a heme-assimilating system of Vibrio vulnificus to synthetic heme compounds

Vibrio vulnificus strain L-180, a clinical isolate, can obtain iron from a synthetic heme, iron-tetra(4-sulfonatophenyl)porphyrin (Fe-TPPS), as well as from a natural heme, protoheme. This assimilation of iron bound to TPPS was demonstrated to be a common property of V. vulnificus by testing a total of 27 strains isolated from both clinical and environmental sources. Strain L-180 could also utilize Fe-TCPP, but not Fe-TMPyP, as a sole iron source. TPPS or its complex with a metal ion reduced bacterial multiplication in the broth containing a minimum dose of Fe-TPPS. When inoculated into human serum supplemented with Fe-TCPP, L-180 could grow only in the presence of a protease from the same bacterium. In both TPPS and TCPP, each side chain of a porphyrin ring has a negative charge. Therefore, this negative charge may be important for interaction with an outer membrane receptor involving in a heme-assimilating system of V. vulnificus.     

The cytotoxin-hemolysin genes of human and eel pathogenic Vibrio vulnificus strains: comparison of nucleotide sequences and application to the genetic grouping

Vibrio vulnificus can be divided into two groups on the basis of pathogenesis. Group 1 is pathogenic only to humans, whereas group 2 is pathogenic to eels and occasionally to humans. Although both groups produce a 50-kDa cytotoxin-hemolysin (V. vulnificus hemolysin; VVH), the toxins are different. In the present study, the nucleotide sequence of the toxin gene (vvhA ) of strain CDC B3547 (a group 2 strain) was determined, and the deduced amino acid sequence was compared to that of strain L-180 (a group 1 strain). The nucleotide sequence of vvhA of strain CDC B3547 was about 96% identical with that of strain L-180, which results in a difference of 3 amino acid residues in the C-terminal lectin domain of VVH. Nevertheless, two primer sets for polymerase chain reaction could be designed to differentiate the toxin gene of each strain. When 27 V. vulnificus clinical isolates were tested, group 1 strains (9 strains) were shown to react only to the primers designed for vvhA of strain L-180; whereas, the gene of group 2 strains (18 strains) could be amplified with the primers for vvhA of strain CDC B3547. These findings may lead to development of a novel genetic grouping system related to the virulence potential or to the host range.     

Existence of a Novel Hemagglutinin Having No Protease Activity in Vibrio mimicus

The protease elaborated by Vibrio mimicus is known to possess hemagglutinating ability to chicken erythrocytes, the well-known HA/protease. A non-protease hemagglutinin (HA) with strong agglutinating ability towards rabbit erythrocytes was obtained from 32 hr culture supernatant of a pathogenic environmental strain of V. mimicus. This HA (V. mimicus HA: VMHA) appeared stable at relatively higher temperature and agglutinated the erythrocytes from rabbit, guinea pig and mouse but not the erythrocytes from chicken, bovine, horse and sheep. Simple sugars, metal ions and chelating agents failed to inhibit the activity of VMHA. The activity of VMHA was found to be sensitive to digestion by proteolytic enzymes including HA/protease. These results provide evidence for the existence of novel HA other than HA/protease in V. mimicus.     

Protective effect of C-reactive protein against the lethality induced by Vibrio vulnificus lipopolysaccharide

Vibrio vulnificus infection has attracted special interest because of its high mortality. A strong clinical association exists between hepatic dysfunction and increased morbidity and mortality from V. vulnificus infection. In this study, the effect of C-reactive protein (CRP), a typical hepatogenic acute phase protein, on the lethality induced by V. vulnificus lipopolysaccharide (LPS) was investigated in galactosamine-sensitized mice. The pretreatment of CRP, in a dose of at least 2 mg/kg, 2 hr before the challenge of LPS completely protected mice against the lethality by V. vulnificus LPS. The elevation of serum tumor necrosis factor-alpha (TNF-alpha) induced by LPS administration was not affected by CRP pretreatment. However, the LPS- or TNF-alpha-induced hepatotoxicity was completely prevented by CRP. These results indicate that CRP does not prevent the synthesis, but prevents the hepatotoxic action of TNF-alpha. The possibility that impaired production of acute phase proteins in patients with pre-existing hepatic dysfunction may predispose the higher risk of V. vulnificus infection needs to be evaluated further.     

变性高效液相色谱技术对创伤弧菌检测的研究

应用PCR结合变性高效液相色谱技术对创伤弧菌进行检测,建立创伤弧菌快速准确的检测新方法。经过DHPLC分析条件优化,在DHPLC非变性温度下分析创伤弧菌特异性PCR扩增产物。同时进行方法特异性、灵敏度、重复性实验。实验结果表明所建立的创伤弧菌PCR-DHPLC检测方法特异性强、灵敏度高、重现性好、结果稳定可靠、检测时间短,检测低限可达到124 CFU/mL,是创伤弧菌快速检测的新技术。     

Isolation and characterization of hemolysin mutants of Vibrio vulnificus

Abstract The importance of the cytolysin/hemolysin in the virulence of Vibrio vulnificus was investigated using both the naturally occuring virulent and avirulent colony variants and ethylmethane-sulfonate generated mutants. Both virulent and avirulent isogenic morphotypes produced similar amounts of hemolysin. Two mutants deficient in the production of hemolysin and negative for CHO cell activity were characterized and their virulence for mice was examined. Non-hemolytic mutants were found to be as virulent as their parent strain. It is concluded that the hemolysin produced by V. vulnificus is not required for the full virulence of this pathogen.     

Non-thiol-activated property of a cholesterol-binding hemolysin produced by Vibrio vulnificus

Abstract Vibrio vulnificus hemolysin (VVH) and streptolysin O (SLO) are both cholesterol-binding hemolysins. Both hemolysins were inactivated with H₂O₂, but the lost activity of SLO was restored by addition of thiol compounds, whereas that of VVH was not. Moreover, the activity of VVH was lowered by thiol compounds but not by thiolblocking agents, whereas the latter produced a decrease in SLO activity. These results suggest that VVH is not a thiol-activated hemolysin, in spite of its cholesterol-binding property.     

Structure of vulnibactin,a new polyamine-containing siderophore from Vibrio vulnificus

A new siderophore named vulnibactin has been isolated from low iron cultures of Vibrio vulnificus, a human pathogen. The structure was established as N-[3-(2,3-dihydroxybenzamido)propyl]-1,3-bis[2-(2-hydroxyphenyl)-trans-5-methyl-2-oxazoline-4-carboxamido]propane by a combination of acid hydrolysis, nuclear magnetic resonance spectroscopy and positive fast atom bombardment mass spectrometry. Vulnibactin is characterized as containing one residue of 2,3-dihydroxybenzoic acid as well as two residues of salicylic acid, both of which are involved in the formation of oxazoline rings with l-threonine bound to a norspermidine backbone. In addition, two other compounds with siderophore activity were purified and their structures were also determined. These two compounds provided further support for the structure of vulnibactin.     

Ferric-reductase activities in Vibrio vulnificus biotypes 1 and 2

In this paper, the ferric-reductase activities of Vibrio vulnificus were investigated. This species comprises two biotypes pathogenic for humans and eels that are able to express different mechanisms for iron acquisition. All strains of both biotypes used in this study were able to reduce ferric citrate, irrespective of the iron levels in the growth medium. Some variation in the degree of reduction was observed among the strains, with the highest values corresponding to one acapsulated environmental strain of biotype 1. When cell fractions were tested, only those from periplasm and cytoplasm showed reductase activity whereas no activity was detected in membranes. Low temperatures inhibited these activities in both whole cells and cell fractions. At least six bands with ferric-reductase activity were identified in all strains using native polyacrylamide gels. These data demonstrate that the two biotypes of V. vulnificus produce similar ferric-reductases mainly located in the periplasm and cytoplasm and these could be involved in iron acquisition.     

水产品中创伤弧菌和副溶血弧菌双重PCR检测方法的建立

目的 建立一种同步检测创伤弧菌和副溶血弧菌的双重PCR方法。方法 选择副溶血弧菌tlh基因和创伤弧菌vvhA基因作为靶序列各设计一对引物。用合成的引物对副溶血弧菌和创伤弧菌进行双重PCR扩增,确定特异性和最低检出限。然后用此方法对53株副溶血弧菌和7株创伤弧菌进行检测。结果 确定了双重PCR检测创伤弧菌和副溶血弧菌的最优反应条件,其中退火温度为60 ℃,方法具有较好的特异性。对副溶血弧菌的最低限为1.0×102 CFU/mL,创伤弧菌最低限为4.2×104 CFU/mL。双重PCR对分离株检测符合率达100%。结论 建立的双重PCR方法简便、快速、特异性好,可同时检测副溶血弧菌和创伤弧菌,为水产品中病原菌的基层检测提供解决方案。