Use of a marker plasmid to examine differential rates of growth and death between clinical and environmental strains of Vibrio vulnificus in experimentally infected mice

Vibrio vulnificus is Gram-negative bacterium that contaminates oysters, causing highly lethal sepsis after consumption of raw oysters and wound infection. We previously described two sets of V. vulnificus strains with different levels of virulence in subcutaneously inoculated iron dextran-treated mice. Both virulent, clinical strains and attenuated, environmental strains could be recovered in high numbers from skin lesions and livers; however, the attenuated environmental strains required significantly higher numbers of colony-forming units (cfu) in the inoculum to produce lethal infection. Using some of these strains and an additional clinical strain, we presently asked if the different abilities to cause infection between the clinical and environmental strains were due to differences in rates of growth or death of the bacteria in the mouse host. We therefore constructed a marker plasmid, pGTR902, that functions as a replicon only in the presence of arabinose, which is not present in significant levels in animal tissues. V. vulnificus strains containing pGTR902 were inoculated into iron dextran-treated and untreated mice. Measuring the proportion of bacteria that had maintained the marker plasmid recovered from mice enabled us to monitor the number of in vivo divisions, hence growth rate; whereas measuring the number of marker plasmid-containing bacteria recovered enabled the measurement of death of the vibrios in the mice. The numbers of bacterial divisions in vivo for all of the strains over a 12-15 h infection period were not significantly different in iron dextran-treated mice; however, the rate of death of one environmental strain was significantly higher compared with the clinical strains. Infection of non-iron dextran-treated mice with clinical strains demonstrated that the greatest effect of iron dextran-treatment was increased growth rate, while one clinical strain also experienced increased death in untreated mice. V. vulnificus inoculated into iron dextran-treated mice replicated extremely rapidly over the first 4 h of infection with doubling times of approximately 15-28 min. In contrast, one of the environmental strains exhibited a reduced early growth rate. These results demonstrate that differences in virulence among naturally occurring V. vulnificus can be explained by diverse abilities to replicate rapidly in or resist defences of the host. The marker plasmid pGTR902 should be useful for examining virulence of bacteria in terms of differentiating growth verses death in animal hosts for most Gram-negative bacteria.     

Essential role of an adenylate cyclase in regulating Vibrio vulnificus virulence

Vibrio vulnificus, a halophilic estuarine bacterium, causes a fatal septicemia and necrotizing wound infection. To investigate the role of cAMP in V. vulnificus virulence regulation, an in-frame deletion mutant of the cya gene encoding adenylate cyclase was constructed. The cya null mutation resulted in a pleiotropic change of virulence phenotypes. The production of hemolysin and protease, the motility, and the cytotoxicity were decreased by the cya mutation. The defects in the cya mutant were functionally complemented in trans by a plasmid carrying the wild type cya allele. The V. vulnificus cya mutant exhibited a 100-fold increase in LD50 to mice. The result indicates that cAMP plays an essential role in the global regulation of V. vulnificus virulence.     

The ttpC gene is contained in two of three TonB systems in the human pathogen Vibrio vulnificus, but only one is active in iron transport and virulence

The TonB system of proteins is required for the energy-dependent active transport of iron-bound substrates across the outer membrane of gram-negative bacteria. We have identified three TonB systems within the human pathogen Vibrio vulnificus. The TonB1 system contains the TonB1, ExbD1, and ExbB1 proteins, whereas both the TtpC2-TonB2 and TtpC3-TonB3 systems contain an additional fourth protein, TtpC. Here we report that TtpC3, although highly related to TtpC2, is inactive in iron transport, whereas TtpC2 is essential for the function of the TtpC2-TonB2 system in V. vulnificus. This protein, together with TonB2, is absolutely required for both the uptake of endogenously produced iron-bound siderophores as well as siderophores produced from other organisms. Through complementation we show that V. vulnificus is capable of using different TtpC2 proteins from other Vibrio species to drive the uptake of multiple siderophores. We have also determined that aerobactin, a common bacterial siderophore involved in virulence of enteric bacteria, can only be brought into the cell using the TtpC2-TonB2 system, indicating an important evolutionary adaptation of TtpC2 and TonB2. Furthermore, in the absence of TonB1, TtpC2 is essential for a fully virulent phenotype as demonstrated using 50% lethal dose (LD(50)) experiments in mice.     

Siderophore-mediated iron acquisition mechanisms in Vibrio vulnificus biotype 2.

Vibrio vulnificus biotype 2 is a primary pathogen for eels and, as has recently been suggested, an opportunistic pathogen for humans. In this study we have investigated the ability of V. vulnificus biotype 2 to obtain iron by siderophore-mediated mechanisms and evaluated the importance of free iron in vibriosis. The virulence degree for eels was dependent on iron availability from host fluids, as was revealed by a reduction in the 50% lethal dose for iron-overloaded eels. This biotype produced both phenolate- and hydroxamate-type siderophores of an unknown nature and two new outer membrane proteins of around 84 and 72 kDa in response to iron starvation. No alterations in lipopolysaccharide patterns were detected in response to iron stress. Finally, our data suggest that V. vulnificus biotype 2 uses the hydroxamate-type siderophore for removal of iron from transferrin rather than relying on a receptor for this iron-binding protein.     

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.     

Virulence of Salmonella enteritidis phage type 4 is related to the possession of a 38 MDa plasmid

Nine strains of Salmonella enteritidis phage type 4 were examined for virulence in BALB/c mice. The possession of a 38 MDa plasmid was necessary for full virulence. Strains carrying this plasmid had LD50 values of less than 20 bacteria whilst plasmid-free strains had LD50 values of greater than 10(6) bacteria when challenged intraperitoneally. Pathogenesis of disease involved the widespread distribution of bacteria throughout the tissues. Possession of the 38 MDa plasmid could not be linked with the ability of strains to express novel outer membrane proteins, to produce toxins affecting Vero, Y1, HeLa, Henle or HEp-2 cells, or to invade HEp-2 cells. Furthermore, the 38 MDa plasmid did not encode an aerobactin-mediated iron uptake system or the production of a haemolysin. Strains of S. enteritidis PT4 isolated in 1967, 1978 or 1979 and possessing the 38 MDa plasmid showed the same virulence properties as the current plasmid-carrying strains. This suggests that the enhanced virulence of the current strains for poultry is unlikely to be the result of changes in the 38 MDa plasmid.     

Indole-positive Vibrio vulnificus isolated from disease outbreaks on a Danish eel farm

Vibrio vulnificus was isolated in 1996 from 2 disease outbreaks on a Danish eel farm which used brackish water. A characteristic clinical sign was extensive, deep muscle necrosis in the head region. V. vulnificus was isolated from kidney, mucus, spleen, gill and intestine of diseased eels. Thirty-two isolates were examined phenotypically and serologically for pathogenicity to eels and for correlation to ribotype and plasmid profile. Biochemically, the isolates showed properties similar to those described previously for eel-pathogenic strains of V. vulnificus, with the exception of indole production. Virulence was evaluated by LD50 (the 50% lethal dose), which ranged from < 9.4 x 10(3) to 2.3 x 10(5) CFU (colony-forming units) per fish. The isolates which were lethal for eels showed identical ribotypes and serotypes. A relationship between certain plasmids and virulence was not found. A serotyping system based on lipopolysaccharide (LPS)-associated O antigen type and on carbohydrate capsule antigens showed that the eel-virulent isolates shared a common LPS-based homogeneous O serogroup and a capsule antigen. V. vulnificus serovar O4 and capsule type 9 was identical serologically to the Japanese isolate ATCC 33149 and was the agent responsible for the disease outbreaks that occurred on the Danish eel farm. Despite absence of antibiotic resistance, treatment had little effect and disease reoccurred.     

铁摄取调节蛋白Fur功能和作用模式的研究进展

铁是大多数生物必需的微量元素,在健康和疾病,尤其是宿主-病原菌互作过程中发挥着至关重要的作用.细菌胞内铁离子浓度的高低不仅是调节自身高亲和力铁运输系统表达的信号,更是病原菌产生毒素和其他必要毒力因子的关键调控因素.而另一方面,超负荷的铁也会导致致命的细胞毒性.因此,生物体内铁稳态的维持受到严格控制,其中以铁摄取调节蛋白(ferric uptake regulator,Fur)的作用最为显著,其调控网络涵盖了细菌生命活动的各个方面.本综述将基于Fur的生物学功能,围绕其家族分类、结构特点和差异、调控网络和调控机制等方面进行总结和分析,以期为Fur和铁稳态调节等研究提供参考.     

Francisella novicida LPS has greater immunobiological activity in mice than F. tularensis LPS,and contributes to F. novicida murine pathogenesis

To further understand the role of LPS in the pathogenesis of Francisella infection, we characterized murine infection with F. novicida, and compared immunobiological activities of F. novicida LPS and the LPS from F. tularensis live vaccine strain (LVS). F. novicida had a lower intradermal LD(50) in BALB/cByJ mice than F. tularensis LVS, and mice given a lethal F. novicida dose intraperitoneally died faster than those given the same lethal F. tularensis LVS dose. However, the pattern of in vivo dissemination was similar, and in vitro growth of both bacteria in bone marrow-derived macrophages was comparable. F. novicida LPS stimulated very modest in vitro proliferation of mouse splenocytes at high doses, but F. tularensis LVS LPS did not. Murine bone marrow macrophages treated in vitro with F. novicida LPS produced IL12 and TNF-alpha, but did not produce detectable interferon-gamma, IL10, or nitric oxide; in contrast, murine macrophages treated with F. tularensis LVS LPS produced none of these mediators. In contrast to clear differences in stimulation of proliferation and especially cytokines, both types of purified LPS stimulated early protection against lethal challenge of mice with F. tularensis LVS, but not against lethal challenge with F. novicida. Thus, although LPS recognition may not be a major factor in engendering protection, the ability of F. novicida LPS to stimulate the production of proinflammatory cytokines including TNF-alpha likely contributes to the increased virulence for mice of F. novicida compared to F. tularensis LVS.     

Flagellar basal body flg operon as a virulence determinant of Vibrio vulnificus

Vibrio vulnificus, a halophilic estuarine bacterium causing a rapidly progressing fatal septicemia, is highly cytotoxic to eukaryotic cells. To identify new virulence factors associated with cytotoxicity, we constructed a mariner-based transposon (Tn Himar1) library of the highly virulent clinical isolate MO6-24/O having a double mutation in the hemolysin and protease genes. The Himar1 mutant library was extensively screened for the mutants showing decreased cytotoxicity to HeLa cells. We selected a cytotoxicity defective mutant having a Himar1 insertion in an open reading frame showing 96% identity to Vibrio parahaemolyticus FlgC, a flagella basal body rod protein. The Tn Himar1 insertion mutation also resulted in a significant decrease in motility, adhesion, cytotoxicity, and lethality to mice. This is the first report showing that flg genes, which are components of the flagellum biogenesis gene cluster, might play an important role in the virulence of V. vulnificus.     

Identification and functional analysis of vibrio vulnificus SmcR, a novel global regulator

Recently, quorum sensing has been implicated as an important global regulator controlling the production of numerous virulence factors such as capsular polysaccharides in bacterial pathogens. The nucleotide and deduced amino acid sequences of smcR, a homolog of V. harveyi luxR identified from V. vulnificus ATCC29307, were analyzed. The amino acid sequence of SmcR from V. vulnificus was 72 to 92% similar to those of LuxR homologs from Vibrio spp. Functions of SmcR were assessed by the construction of an isogenic mutant, whose smcR gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in vitro and in mice. The disruption of smcR resulted in a significant alteration in biofilm formation, in type of colony morphology, and in motility. When compared with the wild-type, the smcR mutant exhibited reduced survival under adverse conditions, such as acidic pH and hyperosmotic stress. The smcR mutant exhibited decreased cytotoxic activity toward INT 407 cells in vitro. Furthermore, the intraperitoneal LD50 of the smcR mutant was approximately 10(2) times higher than that of parental wild-type. Therefore, it appears that SmcR is a novel global regulator, controlling numerous genes contributing to the pathogenesis as well as survival of V. vulnificus.     

Cytotoxic mechanism of Vibrio vulnificus cytolysin in CPAE cells

Vibrio vulnificus is an estuarian bacterium that causes septicemia and serious wound infection. The cytolysin, one of the important virulence determinants in V. vulnificus infection, has been reported to have lethal activity primarily by increasing pulmonary vascular permeability. In the present study, we investigated the cytotoxic mechanism of V. vulnificus cytolysin in cultured pulmonary artery endothelial (CPAE) cells, which are possible target cells of cytolysin in vivo. V. vulnificus cytolysin caused the CPAE cell damages with elevation of the cytosolic free Ca2+, DNA fragmentation, and decrease of the cellular NAD+ and ATP level. These cytotoxic effects of V. vulnificus cytolysin were prevented by EGTA and aminobenzamide, but were not affected by verapamil or catalase. These results indicate that the elevation of cytosolic free Ca2+ induced by V. vulnificus cytolysin causes the increase of DNA fragmentation and the damaged DNA activates nuclear poly(ADP-ribose) synthetase, which depletes the cellular NAD+ and ATP, resulting in cell death.     

Expression of Vibrio vulnificus capsular polysaccharide inhibits biofilm formation

Vibrio vulnificus is a human pathogen that produces lethal septicemia in susceptible persons, and the primary virulence factor for this organism is capsular polysaccharide (CPS). The role of the capsule in V. vulnificus biofilms was examined under a variety of conditions, by using either defined CPS mutants or spontaneous CPS expression phase variants derived from multiple strains. CPS expression was shown to inhibit attachment and biofilm formation, which contrasted with other studies describing polysaccharides as integral to biofilms in related species.     

Virulence characteristics of clinical and environmental isolates of Vibrio vulnificus.

Twenty-four randomly selected clinical and environmental Vibrio vulnificus isolates were tested for virulence in iron-overloaded mice (250 mg of iron dextran per kg of body weight). The log10 50% lethal doses of 17 isolates were lower by greater than or equal to 3.5 log10 units in iron-overloaded mice than in control mice. These isolates were classified as virulent. The 50% lethal doses of these virulent isolates were also lower in mice that were immunosuppressed by treatment with cyclophosphamide (150 mg/kg). Four of the seven isolates initially classified as avirulent were virulent in mice that were simultaneously iron overloaded and immunosuppressed. These isolates were classified as moderately virulent. The remaining three isolates were avirulent under all conditions. The incidence of virulent strains among clinical and environmental isolates did not differ. The virulent isolates produced high titers of hemolysin, were resistant to inactivation by serum complement, produced phenolate siderophore, and utilized transferrin-bound iron. The moderately virulent isolates differed from the virulent isolates only in their increased sensitivity to inactivation by serum complement. The avirulent isolates differed from those of the other two classes in their inability to either produce significant amounts of phenolate siderophore or utilize transferrin-bound iron. A modified agar plate diffusion method for transferrin-bound iron utilization was developed to differentiate the two classes of virulent isolates from the avirulent isolates in vitro.     

Evidence for IFN-gamma as a mediator of the lethality of endotoxin and tumor necrosis factor-alpha.

Current evidence indicates that endogenously produced peptide cytokines, most notably TNF-alpha and IL-1, mediate the lethality of experimental endotoxemia. Because circulating serum levels of IFN-gamma can be detected soon after TNF-alpha and IL-1 in response to endotoxin, we investigated the role of IFN-gamma in endotoxin and TNF-alpha lethality. Specific neutralizing antibodies to murine TNF-alpha (anti-TNF-alpha Ab) or murine IFN gamma (anti-IFN-gamma Ab) produced in our laboratory protected mice against the lethality of Escherichia coli endotoxin (LPS) administered 6 h later. Serum IFN-gamma levels 2 h after i.v. LPS were lower in mice treated with anti-TNF-alpha Ab compared to mice that received nonimmune IgG (median less than 2.5 vs 3.0 U/ml, P2 less than 0.05). In contrast, serum TNF-alpha levels 1 h after i.v. LPS peaked more than fourfold higher in mice treated with anti-IFN-gamma Ab compared to controls (median greater than 6400 vs 1405 pg/ml, p2 less than 0.05). Doses of TNF-alpha (300 micrograms/kg) and IFN-gamma (50,000 U) which were well tolerated when given individually were synergistically lethal in combination (0% lethality vs 100% lethality, P2 less than 0.001), and were associated with higher serum levels of IL-6 than with either cytokine alone. Anti-IFN-gamma Ab provided complete protection against exogenous human rTNF-alpha at the LD100 dose (1400 micrograms/kg, p2 less than 0.001), and in fact prevented lethality at doses four- to fivefold greater than the LD100 human rTNF-alpha (up to 6000 micrograms/kg). We conclude that IFN-gamma is synergistic with TNF-alpha, is essential for the lethality of LPS and TNF-alpha, and may have modulating effects on the negative control of serum levels of TNF-alpha after LPS in mice.     

Studies on a murine model for evaluation of virulence of Streptococcus suis capsular type 2 isolates

Five different parameters, time of incubation of the culture, type of culture medium, inoculum, strain of inbred mice, and age of mice, were tested using the LD50 technique to standardize a murine model for the evaluation of the virulence of Streptococcus suis capsular type 2 isolates. A model using 28 day-old mice belonging to CF1 strain appeared to give the best results. The inoculum size was the parameter most influencing the 50% lethal dose obtained with mice. Inoculation with 1-ml volume of a bacterial suspension instead of 0.1 or 0.5 ml decreased the LD50. The standardized model was used to evaluate the virulence of some isolates of known pathogenicity for pigs. The minimum lethal dose was used in the model and it appeared that the virulence of Streptococcus suis capsular type 2 isolates can be measured from highly virulent to totally avirulent.     

Role of pili in the pathogenesis of Pseudomonas aeruginosa burn infection

The present study using three isogenic mutants (F+P-, F-P+, F-P-) of Pseudomonas aeruginosa indicates that the presence of pili enhances the virulence of the organisms in experimental P. aeruginosa burn infection of mice. The 50% lethal dose (LD50) value for burned mice inoculated with non-piliated (P-) mutant was at least ten times higher than those inoculated with piliated (P+) bacteria. Meanwhile the LD50 value for burned mice inoculated with non-flagellated (F-) mutant was at least 10(5) times higher than those inoculated with flagellated (F+) bacteria. At 24 hr after inoculation, the bacterial counts in burned skin of mice inoculated with P+ bacteria were ten times higher than those inoculated with P- bacteria; and at 48 hr the bacterial counts became a hundred times higher in the former mice than the latter. At 24 hr after inoculation, P+ bacteria were isolated from blood, liver (F+P+), lung (F+P+), and kidney, while P- bacteria were not present in these tissues. And at 48 hr after inoculation, P+ bacteria were isolated from all tissues, while P- bacteria were isolated from some sites only. These results suggested that pili and flagella each play an important role as virulence factors independently, and that pili-mediated enhancement of virulence of P. aeruginosa was attributed to pili-mediated enhanced colonization of the organisms at the burned skin surfaces.     

Protective mechanism of curcumin against Vibrio vulnificus infection

Curcumin, a natural polyphenolic flavonoid extracted from the rhizome of Curcuma longa L., has many beneficial biological activities. However, there are relatively few reports of the effects of curcumin on pathogen infections. This study examined the effect of curcumin on a Vibrio vulnificus infection. The cytotoxicity of V. vulnificus to HeLa cells was significantly inhibited by curcumin (at 10 or 30?μM). To further examine the inhibitory mechanism of curcumin against V. vulnificus-mediated cytotoxicity, the level of bacterial growth, bacterial motility, cell adhesion, RTX toxin expression and host cell reactions were evaluated. Curcumin inhibited V. vulnificus growth in HI broth. Curcumin inhibited both bacterial adhesion and RTX toxin binding to the host cells, which can be considered the major protective mechanisms for the decrease in V. vulnificus cytotoxicity. Curcumin also inhibited host cell rounding and actin aggregation, which are the early features of cell death caused by V. vulnificus. In addition, curcumin decreased the V. vulnificus-induced NF-κB translocation in HeLa cells. Finally, curcumin protected mice from V. vulnificus-induced septicemia. In conclusion, curcumin may be an alternative antimicrobial agent against fatal bacterial infections.