Human Pre-Elafin Inhibits a Pseudomonas aeruginosa -Secreted Peptidase and Prevents Its Proliferation in Complex Media

Abstract

Pseudomonas aeruginosa is a life-threatening opportunist human pathogen frequently associated with lung inflammatory diseases, namely, cystic fibrosis. Like other species, this gram-negative bacteria is increasingly drug resistant. During the past decade, intensive research efforts have been focused on the identification of natural innate defense molecules with broad antimicrobial activities, collectively known as antimicrobial peptides. Human pre-elafin, best characterized as a potent inhibitor of neutrophil elastase with anti-inflammatory properties, was also shown to possess antimicrobial activity against both gram-positive and gramnegative bacteria, including P. aeruginosa. Its mode of action was, however, not known. Using full-length pre-elafin, each domain separately, and mutated variants of pre-elafin with attenuated antipeptidase activity toward neutrophil elastase, we report here that both pre-elafin domains contribute, through distinct mechanisms, to its antibacterial activity against Pseudomonas aeruginosa. Most importantly, we demonstrate that the whey acidic protein (WAP) domain specifically inhibits a secreted peptidase with the characteristics of arginyl peptidase (protease IV). This is the first demonstration that a human WAP-motif protein inhibits a secreted peptidase to prevent bacterial growth in vitro. Since several WAP-motif proteins from various species demonstrate antimicrobial function with variable activities toward bacterial species, we suggest that this mechanism may be more common than initially anticipated.Pseudomonas aeruginosa is an opportunistic pathogen that is life-threatening for immunocompromised individuals and for patients suffering from chronic respiratory diseases such as cystic fibrosis (CF) (10, 27). Chronic lung P. aeruginosa infection is the primary cause of morbidity and mortality in CF patients, and its prevalence increases with age. P. aeruginosa is also the predominant bacteria associated with nosocomial infections, and acute P. aeruginosa infection may result in sepsis and death (26). P. aeruginosa is characterized by the expression of numerous virulence factors, reflected by the unusually large genome of this bacteria and has developed sophisticated mechanisms to escape the host immune defenses (10,15,27,33). In addition, due to low membrane permeability, active efflux of drugs, and the presence of ␤-lactamase activity, P. aeruginosa infections are becoming increasingly difficult to treat (17). Hence, the development of novel antimicrobial agents, preferably acting on targets exposed at the bacterial cell surface, is urgently needed.Among the P. aeruginosa virulence factors, the secreted peptidases are thought to play a critical role in tissue invasion, nutrient accessibility, and degradation of the innate host defense molecules and therefore constitute potential therapeutic targets (18)(19)(20). Because opportunistic pathogens are taking advantage of a weakened host protective shield, augmentation therapy with lung natural defense molecules also appears to be a ...

New Antimicrobial Strategies in Cystic Fibrosis

“…[33] Also, aerosol treatment with elastase inhibitor enhanced bacterial clearance in an in vitro model of chronic P. aeruginosa lung infection. [32] Clinical studies will be necessary to extend these observations to humans, and to determine whether protease inhibition can inhibit progression of CF lung disease in CF patients. [20,34] …”

“…Elafin is cleaved by its cognate enzyme neutrophil elastase, which is present in excessive concentrations in CF sputum, especially when the lung is colonized by P. aeruginosa. [31,32] Recombinant human elastase inhibitor was tested in a rat model of acute pulmonary damage, where it prevented injury caused by instillation of human secretions of CF sputum. [33] Also, aerosol treatment with elastase inhibitor enhanced bacterial clearance in an in vitro model of chronic P. aeruginosa lung infection.…”

Functional study of elafin cleaved by Pseudomonas aeruginosa metalloproteinases

“…The observed resistance of elafin to proteolysis mediated by Pseudomonas serine protease(s) suggests that it is not as sensitive as SLPI and AAT, or may be explained by the capacity of elafin to inhibit serine protease(s) secreted by the pathogenic microorganism. Indeed, a recent study suggests that elafin inhibits growth of P. aeruginosa in complex media via the inhibition of a serine protease produced by the pathogen, presumably the arginyl peptidase protease IV (Bellemare et al , 2008). Identification of the Pseudomonas metalloprotease(s) involved in the cleavage and inactivation of elafin was carried out using purified Pseudomonas metalloproteases: pseudolysin, staphylolysin (LasA) and aeruginolysin ( Pseudomonas alkaline protease, serralysin).…”

Functional Characterization of Polymorphisms in the Peptidase Inhibitor 3 (Elafin) Gene and Validation of Their Contribution to Risk of Acute Respiratory Distress Syndrome

“…Apart from its well known inhibitory properties, pre-elafin has been found to have broad antimicrobial properties in vitro and in vivo (14,(46)(47)(48). Although the biochemical mechanism responsible for the antimicrobial activities has not yet been elucidated, it is well known that the antimicrobial activities of pre-elafin are independent of their antiproteinase function (47)(48)(49).…”

“…Although the biochemical mechanism responsible for the antimicrobial activities has not yet been elucidated, it is well known that the antimicrobial activities of pre-elafin are independent of their antiproteinase function (47)(48)(49). Recent studies have demonstrated that the N terminus of pre-elafin, responsible for most of the antimicrobial activity of the protein, adopts an a-helical conformation in the presence of a membrane mimetic, which is typical of many antimicrobial peptides (49).…”