Pseudomonas aeruginosa Alkaline Protease Can Facilitate Siderophore-Mediated Iron-Uptake via the Proteolytic Cleavage of Transferrins

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen 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. 1) 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. 2,3)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 32 and pyochelin 10 5 ). 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. [4][5][6] P. aeruginosa produces several extracellular proteases. Of these, AprA and LasB have been extensively studied. [7][8][9] 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, 3,7) 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. 9) Similarly, Döring et al. 7) found that AprA had no effect on pyoverdin-mediated iron-uptake from transferrins, but Shigematsu et al.8) 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.8) 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 p...

Gene expression characteristics of a cystic fibrosis epidemic strain ofPseudomonas aeruginosaduring biofilm and planktonic growth

“…Planktonic AES‐2 showed enhanced expression of iron acquisition genes pyoverdine pvdA(PA2386 ) and pyoverdine synthetase pvdF(PA2396 ) (22.9 × and 5.4 × , respectively, Table 1) compared with infrequent clones; however, expression was significantly downregulated (−6.3 × and −8.8 × ) in AES‐2 biofilms, which showed expression similar to infrequent clone biofilms. Alkaline protease expression facilitates siderophore‐mediated iron uptake by P. aeruginosa (Kim et al , 2006); thus, enhanced expression of pyoverdine in planktonic culture may be due to the increased alkaline protease activity. It is possible that the infrequent clones utilize a different sequestration pathway to AES‐2, because they had significantly higher expression (8.3 × ) than AES‐2 of the B subunit of bacterioferritin, bfrB(PA3531) , another major iron‐storage protein in bacteria.…”

Cooperative pathogenicity in cystic fibrosis: Stenotrophomonas maltophilia modulates Pseudomonas aeruginosa virulence in mixed biofilm

“…P. aeruginosa causes severe tissue damage by the expression of the alkaline protease AprA, especially in CF patients where it has been associated with increased infectivity and virulence, suggesting a role in processes related to bacterial colonization and/or exacerbation in the CF lung (Burke et al, 1991 ; Kim et al, 2006 ). Our findings suggested that in mixed infections S. maltophilia might indirectly facilitate the onset of PEs in CF patients by inducing increased proteolytic activity in P. aeruginosa , probably via a Las/RhI independent pathway.…”

Iron acquisition in the cystic fibrosis lung and potential for novel therapeutic strategies

“…P. aeruginosa pyoverdine and pyochelin can displace iron from transferrin (Takase et al, 2000). P. aeruginosa also has the ability to release iron from transferrin using elastase (LasB; Wolz et al, 1994), the alkaline protease AprA (Kim et al, 2006) and the endoprotease PrpL (Wilderman et al, 2001). PrpL can also hydrolyse lactoferrin, in addition to other extracellular host proteins, leading to tissue damage and further contributing to the infection process (Wilderman et al, 2001).…”