Pseudomonas Aeruginosa Elastase Disrupts the Cortisol-Binding Activity of Corticosteroid-Binding Globulin

Abstract

The serine protease inhibitor (SERPIN) family member corticosteroid-binding globulin (CBG) is the main carrier of glucocorticoids in plasma. Human CBG mediates the targeted release of cortisol at sites of inflammation through cleavage of its reactive center loop (RCL) by neutrophil elastase. The RCLs of SERPIN family members are targeted by diverse endogenous and exogenous proteases, including several bacterial proteases. We tested different bacteria for their ability to secrete proteases that disrupt CBG cortisol-binding activity, and characterized the responsible protease and site of CBG cleavage. Serum CBG integrity was assessed by Western blotting and cortisol-binding capacity assay. Effects of time, pH, temperature, and protease inhibitors were tested. Proteolytically active proteins from bacterial media were purified by fast protein liquid chromatography, and the active protease and CBG cleavage sites were identified by mass spectrometry. Among the bacteria tested, medium from Pseudomonas aeruginosa actively disrupted the cortisol-binding activity of CBG. This proteolytic activity was inhibited by zinc chelators and occurred most efficiently at pH 7 and elevated physiological temperature (ie, 41°C). Mass spectrometric analysis of a semi-purified fraction of P. aeruginosa media identified the virulence factor LasB as the responsible protease, and this was confirmed by assaying media from LasB-deficient P. aeruginosa. This metalloprotease cleaves the CBG RCL at a major site, distinct from that targeted by neutrophil elastase. Our results suggest that humoral responses to P. aeruginosa infection are influenced by this pathogen's ability to secrete a protease that promotes the release of the anti-inflammatory steroid, cortisol, from its plasma transport protein.

Asn347 Glycosylation of Corticosteroid-binding Globulin Fine-tunes the Host Immune Response by Modulating Proteolysis by Pseudomonas aeruginosa and Neutrophil Elastase

“…Importantly, other pathogens (i.e. Burkholderia cenocepacia and Staphylococcus aureus) did not produce RCL-cleaving proteases, suggesting that this ability may be unique to P. aeruginosa (33). Supporting these observations, the RCL of CBG was here shown to be a proteolytic target of PAE; two equally preferred cleavage sites (Thr 345 -Leu 346 and Asn 347 -Leu 348 ) were identified.…”

“…Cell colonies were transferred to 5 ml of LB medium and incubated overnight at 37°C, and the culture media containing PAE were collected. The culture media were centrifuged at 5,000 ϫ g, and the abundantly expressed PAE was crudely separated from other more acidic molecular components in the supernatant fraction as described previously (33). In brief, the supernatant was mixed with strong anion exchange resin (Q-Sepharose, GE Healthcare) equilibrated in 0.02 M sodium phosphate, pH 7.7.…”

“…Finally, P. aeruginosa elastase (PAE), the major virulence factor of this opportunistic Gram-negative pathogen and a zinc metalloprotease that is structurally unrelated to the serine proteases NE and chymotrypsin, was shown to cleave the RCL of CBG primarily between Asn 347 and Leu 348 , leading to reduced cortisol affinity and release of the hormone from CBG (33). It (33).…”

“…Finally, P. aeruginosa elastase (PAE), the major virulence factor of this opportunistic Gram-negative pathogen and a zinc metalloprotease that is structurally unrelated to the serine proteases NE and chymotrypsin, was shown to cleave the RCL of CBG primarily between Asn 347 and Leu 348 , leading to reduced cortisol affinity and release of the hormone from CBG (33). It (33). Interestingly, PAE and NE are known to co-exist in the inflamed and bacteria-infected respiratory tract of individuals with cystic fibrosis and chronic obstructive pulmonary disease (34).…”

“…PAE produced by virulent P. aeruginosa was shown to cleave the RCL of CBG, causing a diminished cortisol binding capacity (33). Importantly, other pathogens (i.e.…”