Pseudomonas aeruginosa elastase disrupts the cortisol-binding activity of corticosteroid-binding globulin.
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.
Department of Cellular and Physiological Sciences (M.S., L.A.H., C.M.U., G.L.H.), University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada, V6T 1Z3; Department of Pathology and Laboratory Medicine (B.O.K.), University of British Columbia, Child and Family Research Institute, 950 W 28th Ave, Vancouver, British Columbia, Canada, V5Z 4H4; Department of Microbiology and Immunology (I.V.), University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada, V6T 1Z3; and Department of Microbiology and Immunology (R.E.W.H.), University of British Columbia, Centre for Microbial Diseases and Immunity Research, 2259 Lower Mall Research Station, Vancouver, British Columbia, Canada, V6T 1Z4.
Marc
M
Simard
Lesley A
LA
Hill
Caroline M
CM
Underhill
Bernd O
BO
Keller
Ivan
I
Villanueva
Robert E W
RE
Hancock
Geoffrey L
GL
Hammond
D001426:Bacterial Proteins / Q000502:physiology / Q000633:toxicity*; D017077:Culture Media, Conditioned; D006801:Humans; D006854:Hydrocortisone / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D019272:Leukocyte Elastase / Q000502:physiology; D008666:Metalloendopeptidases / Q000502:physiology / Q000633:toxicity*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000472:pathogenicity; D013696:Temperature; D014107:Tosyllysine Chloromethyl Ketone; D014156:Transcortin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D037521:Virulence Factors / Q000633:toxicity; D015032:Zinc
D001426:Bacterial Proteins; D017077:Culture Media, Conditioned; D037521:Virulence Factors; D014107:Tosyllysine Chloromethyl Ketone; D014156:Transcortin; D019272:Leukocyte Elastase; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa; D015032:Zinc; D006854:Hydrocortisone