Metalloproteases, containing zinc as an essential metal ion for enzyme catalysis, are elaborated by many bacteria and are classified into 30 major families, which include the thermolysin-like, elastase-like, Serratia protease-like metalloproteases, and the neurotoxins of Clostridium tetani and Clostridium botulinum type B (211). V. vulnificus expresses a thermolysin-like metalloprotease (175,212214), which was originally described by Kreger and Lockwood (174). It was later isolated and characterized by Kothary and Kreger (214), who assigned it a molecular weight of 50.5 kDa and found that it had both caseinolytic and elastinolytic activity. Miyoshi et al. (213) also showed that the metalloprotease underwent autoproteolytic conversion to a 35 KDa active form, losing both a 186-amino-acid N-terminal propeptide and a 10 kDa C-terminal peptide in the process. Furthermore, it has been shown by a number of investigators that the metalloprotease possessed similar biological and immunological properties with the common zinc-metalloproteases expressed by V. cholerae (211), V. mimicus (175,176,214,215), V. tubiashii (216), V. anguillarum, Vibrio proteolyticus, and Pseudomonas aeruginosa (211).
The extracellular metalloprotease is thought to be important in aiding invasion of the bacterium into tissues by degrading elastin and collagen and may be responsible, at least in part, for the extensive tissue necrosis observed clinically during infections (217). Other studies by Miyoshi et al. (218) demonstrated that the metalloprotease proteolytically activates the Hageman factor (factor XII), generating bradykinin, a 9-amino-acid inflammatory polypeptide (219,220). The kinins bradykinin and lysylbradykinin are important mediators of inflammatory responses and are potent vasoactive peptides with wide-ranging properties, including the ability to (a) increase vascular permeability, (b) cause vasodilation, pain, and smooth muscle contraction, and (c) stimulate arachidonic acid metabolism. Activation occurs at sites of negatively charged molecules, such as collagen fibers associated with the interstitial tissue space (221). High molecular weight kininogen (HMWK) and prekallikrein (circulating as a 1: 1 stoichiometric complex, together with the Hageman factor) binds to collagen, and the metalloprotease then activates the Hageman factor to convert prekallikren to kallikren, which then digests HMWK to release lysylbradyknin, which is then converted to brady-kinin. More research is needed to determine if the metalloprotease acts alone or in combination with other V. vulnificus bacterial products in conditioning the Hageman factor-collagen activation site. However, it is likely that the protease activation of the kinin system is involved in the intravascu-lar dissemination. Miyoshi et al. (222) have carried these experiments a step further by injecting the metalloprotease intradermally into dorsal skin of a rabbit and then following the level of in vivo hemorrhagic activity. The results showed that the metalloprotease was found to be significantly correlative with that of the in vitro proteolytic activity for the reconstituted basement membrane gel, which consists primarily of laminin and type IV collagen. Only type IV collagen was easily digested by the metalloprotease. Additionally, type IV collagen antibodies, but not antibodies against laminin, showed sufficient protection against the hemorrhagic reaction. Capillary vessels are known to be stabilized by binding of the basal surface of vascular endothelial cells to the basement membrane. Therefore, specific degradation of type IV collagen may cause destruction of the basement membrane and subsequent breakdown of capillary vessels, resulting in leakage of blood components including erythrocytes into the affected area. Alternatively, this may explain why the organism targets itself to the skin and why a hemorrhagic rash, the most notable symptom observed during infection, occurs. a2-Macroglobulin, a plasma protein, has been shown to be a potent inhibitor of the metalloprotease (223). Because of this liability, Narukawa et al. (224) developed an a2-macroglobulin-resistant derivative of the metalloprotese, which was also stable in vivo. The metalloprotease was modified with activated monomethoxy polyethylene glycol. The modified protease retained full activity to a peptide substrate and 10-20% activity to protein substrates and was resistant to entrapment by a2-macroglobulin.
Another role that the metalloprotease may play is in iron metabolism. It is thought that V. vulnificus obtains iron from a variety of heme proteins through proteolytic action of the metalloprotease (225,226). Nishina et al. (227) showed that both clinical and environmental isolates of V. vulnificus could grow in a synthetic medium supplemented with heme protein as the iron source. In support of this, Fouz et al. (228,229) demonstrated that proteolysis by an outer membrane preparation, which was enriched for a 36 kDa protein (presumably the metalloprotease) expressed by V. vulnificus biotype 2 cells, caused the release of iron from native hemoglobin and hemin. Similarly, Miyoshi et al. (226) showed that the purified metalloprotease could liberate protoheme (iron-proto-porphyrin IX) through proteolytic digestion of these heme proteins; the iron itself or in association with protoporphyrin IX is then transported into the cell. Further observations by Miyoshi et al. (230) also provide additional support that V. vulnificus can obtain iron from hemoglobin bound to haptoglobin types 1 and 2 and synthetic iron sources, such as Fe-alpha, beta, gamma, and delta-tetraphenylporphine tetrasulfonic acid, but not that bound to haptoglobin type 2-1. Results from a study by Nishina et al. (227) confirmed that metalloprotease was needed for heme liberation. The protease digested all of the heme proteins tested and elicited the liberation of heme from the proteins. Lastly, an in vivo mouse peritonitis model employed by Helms et al. (231) demonstrated that heme-containing molecules enhanced the lethality of infections by V. vulnificus. The lethality of inocula of the bacteria injected intraperitoneally (ip) was increased by concurrent injections (ip) of hemoglobin, methemoglobin, or hematin, but not by myoglobin. These investigators obtained similar results in mice with phenylhydrazine-induced hemoglobinemia.
Several investigators have shown that the protease can cause a variety of eyrthrocytes from vertebrate species (rabbit, sheep) to agglutinate (232-234). The C-terminal portion of the protease is thought to be essential for hemagglutination, binding to the erythrocyte membrane, and for proteolysis of membrane proteins (213). Together, these results suggest that V. vulnificus is capable of extracting iron from hemoglobin in vivo for use as a nutrilite and that protease contributes to the efficient utilization of heme and iron metabolism by V. vulnificus.
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