Proteolytic inactivation of cytokines by Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa alkaline protease and elastase are thought to contribute to bacterial invasiveness, tissue damage, and immune suppression in animals and patients infected with the bacterium. This study examined the ability of the two proteases to inactivate a number of cytokines that mediate immune and inflammatory responses. Human recombinant gamma interferon (rIFN-y) and human recombinant tumor necrosis factor alpha were inactivated by both proteases. Murine rIFN-,y was relatively resistant to alkaline protease but was inactivated by elastase, and human recombinant interleukin-la and recombinant interleukin-lo were resistant to the effects of both proteases. Western immunoblots suggested that cytokine inactivation by these proteases, where it occurred, required only limited proteolysis of the polypeptides. The ability of different P. aeruginosa strains to inactivate IFN--y appeared to require the production of both proteases for optimum activity. These results indicate that in vitro cytokine inactivation by Pseudomonas proteases is selective, requires only limited proteolysis, and in certain instances reflects the cooperative effects of both proteases.Pseudomonas aeruginosa is an important pulmonary pathogen in conditions like cystic fibrosis (15,42). It has been suggested that the ability of P. aeruginosa to establish itself in the respiratory tract may be promoted by its suppressive effects on pulmonary immune responses (2,16,40). The mechanisms of this immunosuppression are not entirely understood, but proteolytic enzymes secreted by the bacterium have been shown to degrade surface receptors on hematogenous cells (36, 43) and inactivate interleukin-2 (IL-2) and gamma interferon (IFN--y) (16,17,41).In the case of human IFN--y, cytokine inactivation was caused by either Pseudomonas alkaline protease (AP) (17) or elastase (E) (16). Significant reductions in antiviral and immunomodulatory activities were associated with limited proteolysis of IFN--y. Of particular interest were the synergistic effects on IFN--y seen when both purified proteases were added to reaction mixtures (16). These results would predict that Pseudomonas strains that produce both enzymes should be particularly immunosuppressive, a property that may aid the bacterium in establishing initial colonization by significantly altering immune and inflammatory responses in infections like those seen in cystic fibrosis.This study was undertaken to address three questions relating to the effects of Pseudomonas protease on cytokines. First, what are the specificities of these proteases relative to the inactivation of cytokines that might be involved in Pseudomonas infections? Second, is limited proteolysis of the type seen with human IFN--y sufficient for the inactivation of other cytokines? Third, is the ability to inactivate cytokines a common property of Pseudomonas strains, and how does this property relate to their production of the two proteases? MATERIALS AND METHODSHuman subjects. The studies reported here were approved by the Human Subj...

Interferon‐gamma and resistance to bacterial infections

“…Structural components of the microorganism, such as Lipoarabinomannan of Mycobacteriae sp., can directly inhibit the responsiveness of macrophages to IFN-y (98). Substances produced by replication of bacteria, such as proteases produced by Pseudomonas aeruginosa, can destroy the activity of the IFN--y molecule through specific enzymatic degradation (86). In addition, infection with a pathogen such as Mycobacterium aviurn can increase the production of other cytokines (such as TGF-P) which in turn interfere with the antimicrobial activities of IFN-y (7).…”

“…However, no beneficial effect of IFN-y treatment was observed in burned mice infected with Pseudomonas aeruginosa, a common burn pathogen (43). Later studies by Parmely and associates have indicated that proteases, such as alkaline protease and elastase, produced during Pseudomonas infections specifically cleave IFN-y resulting in inactive forms of the cytokine (86). When a strain of Pseudomonas deficient in its ability to produce alkaline protease was used in the burn/infection model, IFN-y successfully protected mice from infection (90).…”

The detrimental impact of extracellular bacterial proteases on wound healing

“…P. aeruginosa proteases hinder a range of cytokine activities and are also able to induce degradation of cytokines (59). Examples include AprA degradation and inactivation of human interferon γ (INF-γ) (90), and inactivation of human tumour necrosis factor-α (TNF-α) by LasB (90,91). Both INF-γ and TNF-α play an important role in the host immune response, with a lack of INF-γ resulting in auto-inflammatory diseases (92,93) and TNF-α involved in systemic inflammation and apoptosis (77).…”

Actinobacillus pleuropneumoniae metalloprotease: cloning and in vivo expression

“…In addition to the direct action of microbial proteases, it is now evident that the activation of endogenous host protease zymogens such as clotting cascade and the inactivation of most of the plasma protease inhibitors contribute to damaging host tissues. Bacterial proteases may also potentate inflammatory processes, activate the bradykinin generating system, degrade immunoglobulins, and complement factors amongst other effects [22][23][24][25]. The precise role of the protease produced by A. pleuropneumoniae and its participation in porcine pleuropneumonia pathogenesis remains to be demonstrated.…”