Degradation of protease inhibitors, immunoglobulins, and other serum proteins by Serratia protease and its toxicity to fibroblast in culture

Abstract

We investigated the effect of the extracellular protease of Serratia marcescens on human serum constituents such as immunoglobulins, fibronectin, alpha 1-protease inhibitor, alpha 2-macroglobulin, lysozyme, and transferrin. At a very low concentration of Serratia 56-kilodalton protease (56K protease), purified human plasma fibronectin was degraded rapidly into three structural domains or small fragments. Immunoglobulin G3 (IgG3) and IgA1 were also degraded within 30 min with 1 microgram of this protease per ml, more rapidly than their other subclass of IgG or IgA. alpha 1-Protease inhibitor, which did not inhibit the 56K protease, was degraded similarly by the protease. These events were demonstrated by fluorescence polarization and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protease was considerably inhibited by human alpha 2-macroglobulin and chicken ovomacroglobulin. However, when there was a 2 M excess of ovomacroglobulin or a 4 M excess of alpha 2-macroglobulin over the 56K protease, about 25 or 40% proteolytic activity remained, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the protease degraded the alpha 2-macroglobulin extensively during prolonged incubation, which paralleled with regeneration of the protease activity. The protease also cleaved human lysozyme, although moderately. Human serum transferrin was degraded slightly, and human serum albumin was almost resistant to the 56K protease. The enzyme seemed to have no effect on reconstituted collagen, but it degraded rat tropocollagen and yielded fragments of beta and gamma chains by cleaving the intramolecular cross-links. Most of the above proteolysis by the 56K protease appears to result in a limited type of substrate specificity. Thus, the present study demonstrates that the protease is capable of degrading defense-oriented humoral proteins and tissue constituents. Furthermore, it is toxic to fibroblasts. These findings also clarified the possible role of Serratia protease as a virulence factor in the pathogenesis of serratial infections. We recently demonstrated this notion in vivo with rabbit cornea (R. Kamata et al., Ophthalmology 92:1452-1459, 1985).

Role of Microbial Proteases in Pathogenesis

“…Furthermore, clinical flu symptoms caused by rhinovirus or influenza virus infection are now known as a consequence of kinin-generation (65 Many bacterial proteases can destroy defense-oriented proteins in vivo. In addition to the antibodies and complement described in preceding sections, lysozyme and transferrin were found to be degraded by serratial protease (49), and the same is true for other bacterial proteases. Both transferrin and lactoferrin are important iron carriers in the blood and milk of mammals, and they exhibit a bacteriostatic effect by removing free iron from the medium.…”

“…The first example was elucidated for Pseudomonas alkaline protease by Morihara et al (56). Table 1 represents examples obtained by the experiments using Serratia 56K metalloprotease, serralysin (37,49,53) and acid protease of Candida albicans (27). a1-protease inhibitor, the most abundant inhibitor in blood plasma which inhibits neutrophil elastase and thus exerts an anti-inflammatory effect, was inactivated very rapidly by serralysin even at a 200 molar excess of al-protease inhibitor over the protease (27,33).…”

Yeasts, enteric rods and pseudomonads in the subgingival flora of severe adult periodontitis

“…Among enterics and pseudomonads, some species (especially P. aeruginosa) have been shown to be tissue invasive (9), elaborate extracellular leukotoxins (5,7,52) and hemolysins (10,50), suppress lymphocyte proliferation (32), inactivate complement components (62), inactivate plasma alpha-1 -proteinase inhibitor (43,47), degrade immunoglobulins TgG and TgA (17,43), kill fibroblasts (43), inhibit mammalian cell protein synthesis (31), degrade basement membrane laminin (25), release potent enterotoxins and endotoxins (9), and elaborate coUagenolytic (26,43) and other proteolytic enzymes (10,43,44).…”

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.…”