Elastase activity of Vibrio vulnificus was highly dependent on growth phase, reached a maximum during the stationary phase, and was regulated at the level of transcription. The stationary phase production of elastase in crp or rpoS mutants, which were constructed by allelic exchanges, decreased about 3-and 10-fold, respectively. However, the promoter activity of vvpE encoding elastase was unaffected by those mutations in the log phase when analyzed using a vvpE-lux fusion. A primer extension analysis revealed that the transcription of vvpE begins at two different sites, consisting of putative promoter L (PL) and promoter S (PS). The PL activity was constitutive through the log and stationary phases, lower than the PS activity, and unaffected by the crp or rpoS mutations. The transcription of PS, induced only in the stationary phase, was dependent on RpoS. The mutation in crp reduced the activity of PS; however, the additional inactivation of crp did not influence the PS activity in the rpoS mutant, indicating that CRP exerted its effects through PS requiring RpoS. These results demonstrate that vvpE expression is differentially directed by PL and PS depending on the growth phase and elevated by RpoS and CRP in the stationary phase.The pathogenic marine bacterium Vibrio vulnificus is the causative agent of food-borne diseases, such as life-threatening septicemia and possibly gastroenteritis, in individuals with underlying predisposed conditions. The mortality from septicemia is very high (Ͼ50%), and death may occur within 1-2 days after the first signs of illness (1). Several potential virulence factors including an endotoxin, a polysaccharide capsule, ironsequestering systems, a cytolytic hemolysin, an elastase, a phospholipase A2, and other exotoxins have been identified for V. vulnificus (for a recent review, see Ref. 2).Among the putative virulence factors is an elastolytic metalloprotease. Elastase, with a broad substrate specificity including biologically important host molecules, has been suggested to be an important virulence factor of various human pathogenic bacteria (3, 4). The characteristics of the elastase of V. vulnificus as a potential virulence factor have been studied primarily using the purified protein in animal models (5-7). Injection of purified elastase reproduced many of the observed aspects of disease caused by V. vulnificus, including dermonecrosis, destruction of tissues, edema, and ulceration. However, when the isogenic mutant deficient in the elastase was compared with the parental strain for virulence, it appeared that elastase is less important in the pathogenesis of V. vulnificus than would have been predicted from examining the effects of administering purified proteins to animals (4, 8). One possible explanation for this contradiction is that the expression of vvpE encoding elastase may not be sufficient at least under the conditions used; hence the effects of the inactivation of the vvpE on the virulence of the pathogen were not apparent. This possibility strongly underscores th...

To assess the role of the flagellum which was detected by immunoscreening of surface proteins of Vibrio vulnificus, an flgE-deleted mutant was constructed and tested for its pathogenicity. The ability of this nonmotile mutant to adhere to INT-407 cells and its role in biofilm were decreased, as was its lethality to mice.Vibrio vulnificus is a gram-negative bacterium that causes gastroenteritis and primary septicemia, especially in immunocompromised humans (11). Several virulence factors have been discovered in V. vulnificus, including the expression of lipopolysaccharide (1), capsular polysaccharide (21), elastase (6), and a phospholipase A 2 (20), as well as iron availability (22). Motility could be proposed to be another virulence determinant in addition to the aforementioned factors (3). V. vulnificus is a highly motile organism by virtue of a polar flagellum, as are the closely related vibrios. In V. cholerae, nonmotile mutants have been shown to accumulate less fluid in rabbit ligated ileal loops (17). Recently, a V. vulnificus mutant showing a decreased cytotoxicity to HeLa cells was found to have a transposon insertion at the flgC gene encoding a flagellar basal body (8).We performed an experiment to identify bacterial surface molecules, which are required for the initiation of pathogenic interactions of V. vulnificus with a host. From an extensive screening process, a clone containing the flgDEF operon which encodes the components of the flagellum was obtained. Having constructed a knockout mutant of the flgE gene, we made a flagellum-deficient V. vulnificus mutant, and we then went on to investigate the role of flagellum-derived motility in the virulence of this pathogen to host cells.Isolation of the flgDEF clone from immunoscreening of surface proteins of V. vulnificus. The strains and plasmids used in this study are listed in Table 1. To prepare whole-cell lysate, exponential-phase V. vulnificus ATCC 29307 was resuspended in 10 mM Tris-HCl (pH 7.4) and disrupted with an ultrasonic liquid processor (model XL2020 sonicator; Misonix). After ultracentrifugation (100,000 ϫ g) for 1 h at 4°C, the pellet was added to 0.1% sodium lauryl sarkosinate in 7 mM EDTA. A sarkosyl-insoluble fraction (140 g) was used for three consecutive immunizations of a rabbit. Ten days after the last injection, the blood of the immunized rabbit was collected and used for immunoscreening of the ZAPII-based expression library of V. vulnificus.Approximately 20,000 plaques from the expression library were screened for clones interacting with the anti-surface protein serum described above. Five of the 11 candidate plaques showed reproducible immune reactions with the serum during further purification steps. The plasmids derived from the excision of these five clones, pBKH1 to pBKH5, were found to contain the identical DNA fragment; therefore, pBKH1 was used for further studies.Restriction analysis of pBKH1 by using BamHI showed that it had an insert of 2.7 kb (Fig. 1A). The DNA insert of pBKH1 was found to contain a partial sequence ...

A gene homologous to rpoS was cloned from a fatal human pathogen, Vibrio vulnificus. The functional role of rpoS in V. vulnificus was accessed by using an rpoS knockout mutant strain. This mutant was impaired in terms of the ability to survive under oxidative stress, nutrient starvation, UV irradiation, or acidic conditions. The increased susceptibility of the V. vulnificus mutant in the exponential phase to H 2 O 2 was attributed to the reduced activity of hydroperoxidase I (HPI). Although S synthesis was induced and HPI activity reached the maximal level in the stationary phase, the mutant in the stationary phase showed the same susceptibility to H 2 O 2 as the wild-type strain in the stationary phase. In addition, HPII activity, which is known to be controlled by S in Escherichia coli, was not detectable in V. vulnificus strains under the conditions tested. The mutant in the exponential phase complemented with multiple copies of either the rpoS or katG gene of V. vulnificus recovered both resistance to H 2 O 2 and HPI activity compared with the control strain. Expression of the katG gene encoding HPI in V. vulnificus was monitored by using a katG::luxAB transcriptional fusion. The expression of this gene was significantly reduced by deletion of S in both the early exponential and late stationary phases. Thus, S is necessary for increased synthesis and activity of HPI, and S is required for exponentially growing V. vulnificus to develop the ability to survive in the presence of H 2 O 2 .

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ABSTRACT. Chronic wasting disease (CWD) is a naturally occurring prion disease in North American deer (Odocoileus species), Rocky mountain elk (Cervus elaphus nelsoni) and moose (Alces alces). The disease was first confirmed in the Republic of Korea in 2001, and subsequent cases were diagnosed in 2004, 2005 and 2010. The experimental host range of CWD includes ferrets, several species of voles, white-footed mice, deer mice and Syrian golden hamsters. In addition, CWD was transmitted to the transgenic mouse over-expressing elk or deer prion protein efficiently, but not to wild type mouse. Here, we report the experimental transmission of elk CWD to conventional VM/ Dk mice reaching 100% attack rate after second passage. The CWD-prion-affected wild type mice will be a useful model for future CWD studies.