Cloning and Characterization ofvuuA, a Gene Encoding theVibrio vulnificusFerric Vulnibactin Receptor

Abstract

The ability of Vibrio vulnificus to acquire iron from the host has been shown to correlate with virulence. Many iron transport genes are regulated by iron, and in V. vulnificus, transcriptional regulation by iron depends on the fur gene. The N-terminal amino acid sequence of a 72-kDa iron-regulated outer membrane protein purified from a V. vulnificus fur mutant had 53% homology with the first 15 amino acids of the mature protein of the Vibrio cholerae vibriobactin receptor, ViuA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for VuuA, the vulnibactin receptor of V. Vibrio vulnificus is a halophilic marine bacterium that has been associated with primary septicemia and serious wound infections (7,39,40,42). Primary septicemia is often acquired by eating raw oysters or shellfish, and wound infections are associated with exposure of wounds to seawater (30, 62). Primary septicemia is often associated with patients who have diseases predisposing them to iron overload, such as cirrhosis, hemochromatosis, and alcoholism, or who are immunocompromised (28).Iron is an essential element for the growth of most bacteria. In the mammalian host, most intracellular iron is found as heme, ferritin, hemoglobin, and hemosiderin. The concentration of available iron in the extracellular environment is extremely low because of the binding of iron to host high-affinity iron-binding proteins, such as transferrin and lactoferrin (3). Bacteria have evolved various mechanisms for the acquisition of iron from the host, including specific uptake of iron-chelating siderophores or the use of host iron compounds directly. Production of these iron uptake systems is repressed in the presence of iron by an iron-binding repressor protein called Fur (for ferric uptake regulation) (2).Iron appears to be particularly important in the pathogenesis of V. vulnificus infections. Stelma et al. (60) found that iron overload was a more significant risk factor for infection than impaired immune function. Virulent isolates were resistant to inactivation by serum complement, produced a phenolate (catechol) siderophore, had high titers of hemolysin, and utilized transferrin-bound iron. Avirulent isolates in that study were unable either to utilize transferrin-bound iron or to produce significant amounts of catechol siderophore. The results suggest that the phenolate (catechol) siderophore enables the virulent isolates to acquire iron from highly saturated transferrin. Morris et al. (41) also found a significant association between virulence and the utilization of transferrin as an iron source. The structure of the phenolate siderophore of V. vulnificus, named vulnibactin, has been characterized (47). We recently isolated a V. vulnificus mutant that was unable to produce catechol siderophores or to acquire iron from transferrin (34). This mutant showed reduced virulence in an infant mouse model.A V. vulnificus fur deletion mutant overexpresses at least two normally iron-regul...

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

“…Although the methods and conditions used were different from those of the present study, Litwin and co-workers also demonstrated that both a venBmutant and a vuuA-deficient mutant were unable to grow on transferrin-bound iron. [11][12][13] The above results indicate that vulnibactin is essentially required for iron-uptake from transferrins, and that VvpE has no direct effect on facilitating vulnibactin-mediated iron-uptake from transferrins both in vitro and in a human ex vivo experimental system.…”

“…In addition, they showed that a vuuA mutant incapable of expressing vulnibactin receptor could also neither acquire iron from transferrins nor grow on the agars containing them. 11,13) However, they did not rule out the effect of VvpE on the vulnibactin-mediated iron-uptake from transferrin.…”

“…vulnificus vulnibactin is known to play important roles in the iron-uptake from transferrins and the expression of virulence. [10][11][12][13][14] Litwin and co-workers reported that a venB mutant incapable of producing vulnibactin could not acquire iron from transferrins, and thus could not grow on agars containing transferrins. In addition, they showed that a vuuA mutant incapable of expressing vulnibactin receptor could also neither acquire iron from transferrins nor grow on the agars containing them.…”

“…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).…”