Cloning and genetic analysis of the Vibrio vulnificus fur gene and construction of a fur mutant by in vivo marker exchange

Abstract

Vibrio vulnificus is a halophilic, marine pathogen that has been associated with septicemia and serious wound infections in immunocompromised individuals and patients who have hemochromatosis, cirrhosis, or alcoholism (3,41,42). Many of the V. vulnificus infections occur in patients with iron overload. Septicemia is often acquired by eating shellfish, and the mortality rates of patients with septicemia often exceed 50% (26).A number of factors have been proposed as possible virulence determinants for V. vulnificus, including an extracellular cytolysin (20,21,30), an elastolytic protease (27,28), siderophores (54), a phospholipase (61), the presence of a polysaccharide capsule (29,31,56,64,65), resistance to the bactericidal effects of sera (25,64,65), resistance to phagocytosis (25,29,65), and the ability to acquire iron from transferrin (44, 55, 56). Wright et al. directly correlated virulence of V. vulnificus with the availability of iron (63).

Molecular characterization of the ferric-uptake regulator, Fur, from Staphylococcus aureus The GenBank accession numbers for the S. aureus fur gene and fhu operon reported in this paper are AF118839 and AF132117, respectively.

“…As shown in Fig. 1(b), S. aureus Fur appeared to be closely related to the Fur of B. subtilis (73 % identity) and more distantly related to the Fur of Gramnegative bacteria ["30 % identity with Fur of Vibrio vulnificus (Litwin & Calderwood, 1993b) and of Pseudomonas aeruginosa (Prince et al, 1993)]. An additional Fur homologue has been described in the literature in S. epidermidis (Heidrich et al, 1996).…”

Iron and Quorum Sensing Coordinately Regulate the Expression of Vulnibactin Biosynthesis in Vibrio vulnificus

“…When iron is abundant, the Fur-iron complex binds to a consensus sequence called the Fur box (5Ј-GATAATGATA-ATCATTATC-3Ј) in the region upstream of target genes to block transcription (10). In V. vulnificus, Fur is a 149-amino acid protein, showing 70% homology to Escherichia coli Fur, and 93% homology to V. cholerae Fur (11). The mechanism of how iron-siderophores are imported into Gram-negative bacteria is well established (3), but the factors involved in the regulation of siderophore synthesis have yet to be fully elucidated.…”

Vibrio vulnificus Vulnibactin, But Not Metalloprotease VvpE, Is Essentially Required for Iron-Uptake from Human Holotransferrin

“…However, the roles of vulnibactin and VvpE in V. vulnificus ironuptake from transferrins have been determined using different methods under different experimental conditions. [11][12][13]20) Therefore, we considered that a definitive comparison and reevaluation of the roles of VvpE and vulnibactin is required, i.e., using the same methods and experimental conditions, in vitro and in a human ex vivo experimental system, and in accordance with the molecular version of Koch's postulates. 1) To achieve these ends, we constructed a V. vulnificus vvpEdeletion mutant and its vvpE-complemented strain, and a furdeletion mutant exhibiting enhanced vulnibactin productivity and a vis-insertion mutant incapable of producing vulnibactin.…”

“…10) Of these, the catechol-siderophore (called vulnibactin) is known to play the more important role in iron-uptake from transferrins and the virulence of V. vulnificus. [10][11][12][13][14] Expression of the vulnibactin-mediated iron-uptake system is regulated by Fur, a repressor regulating iron uptake. Both the mutation of venB gene encoding an enzyme required for vulnibactin synthesis and the mutation of vuuA gene encoding vulnibactin receptor protein can abolish the ability of V. vulnificus to assimilate iron from transferrins and to grow on holotransferrin (HT).…”