Biol. Pharm. Bull. 29(11) 2295—2300 (2006)

which is frequently isolated from clinical specimens obtained from burns, surface wounds, the urinary tract, ear and eye infections, and the lungs of patients with cystic fibrosis. P. aeruginosa secretes many extracellular proteins, and of these, several proteases including alkaline protease (AprA) and elastase (LasB) are known to play important roles in the pathogenesis of human infections caused by P. aeruginosa. In response to iron deprivation, P. aeruginosa produces two unrelated siderophores, pyoverdin and pyochelin. Both of these siderophores promote the growth of P. aeruginosa when added to media containing transferrins as an iron source. Pyoverdin is considered to be the essential or more effective siderophore because of its higher iron-binding constant (pyoverdin 10 and pyochelin 10). Moreover, a pyochelin-deficient mutant was able to grow in human sera, whereas a pyoverdin-deficient mutant exhibited severely retarded growth, and the addition of pyoverdin was found to restore the retarded growth of pyoverdin-deficient mutants in media containing transferrins. P. aeruginosa produces several extracellular proteases. Of these, AprA and LasB have been extensively studied. These two proteases are produced more profoundly in irondeficient conditions than in iron-sufficient conditions, and are able to destroy transferrin, which is a major iron-withholding protein in human body fluids. Because of the high affinity of transferrin for iron, transferrin-bound iron is not freely-available for P. aeruginosa growth. Accordingly, the proteolytic cleavage of transferrin can allow free iron to be released from transferrin, and thus facilitate the pyoverdin-mediated iron-uptake and growth of P. aeruginosa. However, this hypothesis still remains unresolved despite considerable efforts. Some researchers have reported that LasB facilitates pyoverdin-mediated iron-uptake via the proteolytic cleavage of transferrins, but others found that LasB had no effect on the iron-uptake from transferrins because no difference was observed between the growths of a LasB-deficient mutant and its wild type strain and LasB was produced after the onset of stationary growth phase in media containing transferrin as an iron source. Similarly, Döring et al. found that AprA had no effect on pyoverdin-mediated iron-uptake from transferrins, but Shigematsu et al. more recently reported that AprA can facilitate siderophore-mediated iron-uptake via the proteolytic cleavage of transferrins. Although Shigematsu et al. observed that the growth of an aprA-insertion mutant was suppressed in media containing transferrins versus the wild type strain, they did not directly observe whether the AprA produced during culture could destroy transferrins. Rather, they exogenously added purified AprA to culture media in order to induce the proteolytic cleavage of transferrins at culture start. As the aprA-insertional mutation may also cause unexpected changes in the phenotypes of P. aeruginosa, the role of AprA should be verified in accordance with the molecular version of Koch’s postulates. Moreover, the exogenous addition of purified AprA can not accurately simulate the AprA production occurring during batch culture. Moreover, the proteolytic cleavage of transferrins alone can not prove that AprA facilitates siderophore-mediated iron-uptake from transferrins and promotes the growth of P. aeruginosa on transferrins. In order that AprA can facilitate siderophore-mediated iron-uptake via the cleavage of transferrins, first of all, sufficient AprA must be produced to destruct transferrins concomitantly with siderophore production during the exponential growth phase when P. aeruginosa is actively growing and consuming most iron in a medium. We previously reported that Vibro vulnificus metalloprotease VvpE had no direct effect on iron-acquisition from transferrins, because it was produced after the onset of the stationary growth phase, by which time V. vulnificus had already consumed most of the iron in medium for growth, although it was the sole protease capable of destroying transferrins. In contrast, we also reported that Bacillus subtilis proteases can facilitate siderophore-mediated iron-uptake via the proteNovember 2006 2295