Immune Evasion of Enterococcus faecalis by an Extracellular Gelatinase That Cleaves C3 and iC3b This information is current as Shin Yong Park, Yong Pyo Shin, Chong Han Kim, Ho Jin of October 1, 2021. Park, Yeon Sun Seong, Byung Sam Kim, Sook Jae Seo and In Hee Lee J Immunol 2008; 181:6328-6336; ; doi: 10.4049/jimmunol.181.9.6328 http://www.jimmunol.org/content/181/9/6328 Downloaded from References This article cites 44 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/181/9/6328.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Immune Evasion of Enterococcus faecalis by an Extracellular Gelatinase That Cleaves C3 and iC3b1 Shin Yong Park,* Yong Pyo Shin,* Chong Han Kim,* Ho Jin Park,* Yeon Sun Seong,† Byung Sam Kim,‡ Sook Jae Seo,§ and In Hee Lee2* Enterococcus faecalis (Ef) accounts for most cases of enterococcal bacteremia, which is one of the principal causes of nosocomial bloodstream infections (BSI). Among several virulence factors associated with the pathogenesis of Ef, an extracellular gelatinase (GelE) has been known to be the most common factor, although its virulence mechanisms, especially in association with human BSI, have yet to be demonstrated. In this study, we describe the complement resistance mechanism of Ef mediated by GelE. Using purified GelE, we determined that it cleaved the C3 occurring in human serum into a C3b-like molecule, which was inactivated rapidly via reaction with water. This C3 convertase-like activity of GelE was shown to result in a consumption of C3 and thus inhibited the activation of the complement system. Also, GelE was confirmed to degrade an iC3b that was deposited on the Ag surfaces without affecting the bound Downloaded from C3b. This proteolytic effect of GelE against the major complement opsonin resulted in a substantial reduction in Ef phagocytosis by human polymorphonuclear leukocytes. In addition, we verified that the action of GelE against C3, which is a central component of the complement cascade, was human specific. Taken together, it was suggested that GelE may represent a promising molecule for targeting human BSI associated with Ef. The Journal of Immunology, 2008, 181: 6328–6336. he complement system is an essential part of the innate sumption of C3 induced by the excessive assembly of C3 conver- immune system, and performs crucial functions in the rec- tase on microbial surfaces (3). As the major complement opsonin http://www.jimmunol.org/ T ognition and elimination of invading microorganisms in for phagocytosis by polymorphonuclear leukocytes (PMN), iC3b the tissue fluid, as well as the blood of mammals. It is activated by is generated by protease factor (f) I with the aid of its cofactor, three distinct pathways (the alternative, classical, and lectin path- which are fH in serum (4), complement receptor (CR) 1 or mem- ways), which differ in terms of their modes of activation, but com- brane cofactor protein (5). CR3 on PMN recognizes iC3b as a monly result in the deposition of iC3b (inactive form of C3b) and ligand displayed on the surfaces of microorganisms (6), and this 3 the formation of the membrane attack complex (C5b-9) (MAC) receptor-ligand interaction is a prerequisite for the complement- on microbial surfaces via the activity of C3 convertase (C4b2a or mediated clearance of circulating pathogens. C3bBb) (1, 2). In the activation of three complement pathways, Pathogenic microorganisms have developed a variety of strategies by guest on October 1, 2021 C3b is a central component, as it behaves as a recognition protein to overcome or evade the complement system. Previous studies con- that attaches covalently to microbial surfaces via its reactive thio- cerning complement evasion by microbial virulence factors will have ester. Bound C3b provides a platform for the generation of more a marked impact on the search for more efficient and specific clinical C3 convertase (C3bBb) in the alternative pathway, and C5 con- treatment for infectious disease. In recent years, several complement vertase (C4b2a3b or C3bBb3b) in all three pathways. The majority evasion strategies employed by pathogenic microbes have been elu- of bound C3b is changed to iC3b to prevent the unnecessary con- cidated, and many pathogen-encoded proteins that contribute to those strategies have also been identified as novel virulence factors (1, 7, 8). Microbial pathogens utilize two types of strategies for complement *Department of Biotechnology, Hoseo University, Asan City, Chungnam, South Ko- system inhibition. One is to resist the deposition of C3b on their sur- rea; †Department of Biochemistry, College of Medicine, Dankook University, Cheo- nan, South Korea; ‡Immunomodulation Research Center, University of Ulsan, Ulsan, faces. To do this, 1) they derange the initial recognition steps for South Korea; and §Division of Life Science, College of Natural Sciences, Gyeong- complement activation (9, 10), 2) inactivate C3 convertase, thereby sang National University, Chinju, South Korea preventing further C3b deposition on their surfaces (11), 3) modulate Received for publication March 31, 2008. Accepted for publication August 21, 2008. C3 and its split products (C3b and iC3b) (12, 13). The other strategy The costs of publication of this article were defrayed in part by the payment of page involves the blockage of the terminal step in the complement cascade, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. namely the assembly of cytolytic molecules (C5b-9) on the microbial 1 This work was supported by a grant (2005-015-C00447) from Korea Research surfaces (14, 15). Foundation (KRF). S.Y.P. and C.H.K. received a scholarship from the World-Class Enterococci are the third most common pathogens isolated from 2030 Project of Hoseo University. S.J.S. and B.S.K. were supported by a scholarship human bloodstream infections (BSIs) (16, 17). It has been previ- (or grant) from the BK21 Program, the Ministry of Education and Human Resources Development, Korea. ously reported that up to 90% of enterococcal infections in human 2 Address correspondence and reprint requests to Dr. In Hee Lee, Department of are caused by Enterococcus faecalis (Ef) (18). Whereas the mech- Biotechnology, Hoseo University, 165 Sechuli, Baebangmyun, Asan City, Chung- anisms of their antibiotic resistance and spread have been exten- nam, South Korea. E-mail address: [email protected] sively studied, virulence mechanisms for enterococcal infections 3 Abbreviations used in this paper: MAC, membrane attack complex; PMN, poly- remain largely unknown. In our previous study (19), we purified an morphonuclear leukocyte; f, factor; CR, complement receptor; BSI, bloodstream in- fection; Ef, Enterococcus faecalis; GelE, gelatinase; NHS, normal human serum; extracellular gelatinase (GelE) (GenBank EF105504) of Ef GM C3-def HS, C3-deficient human serum; O-Ef, pre-opsonized Ef; CVF, cobra venom (GenBank EF120452), which has been described previously as an factor. important virulence factor of Ef. In addition, we demonstrated that the Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 GelE of Ef GM evidenced an identity of 99% at the nucleotide and www.jimmunol.org The Journal of Immunology 6329 Downloaded from FIGURE 1. Proteolytic effects of GelE on complement components in the human serum. A, Each reaction mixture was subjected to SDS-PAGE (upper panel) and immunoblotting analysis (lower panel). B, Proteolytic activity of GelE to each purified complement factor. One microgram of each complement factor was treated with GelE at the indicated concentrations for 30 min at room temperature and then subjected to SDS-PAGE. C, The assembly of fluid-phase C3 convertase with fB, fD, and C3bЈ generated via GelE action. Validity of C3bЈ as a component of C3 convertase was monitored by the appearance of Bb after the incubation of the mixture. Lane 1, human C3b; lane 2, human C3 treated with GelE; lane 3, mixture of C3b, fB, and fD; lane 4, mixture of C3, GelE, fB, and fD. D, Effects of fH and fI on assembled C3 convertase. Lane 1, C3 treated with assembled C3 convertase for control; lane 2, C3 treated with assembled C3 convertase in the presence of fH and fI; lane 3, mixture of C3, GelE, fB, fD, fH, and fI. E, Native SDS-PAGE analysis http://www.jimmunol.org/ to examine the fD cofactor activity of C3b and C3bЈ. Of note, whereas the band for C3 was not changed upon incubation with fB (third lane), those for C3b and C3bЈ were moved upward in the presence of fB. In A and E, symbols ϩ and Ϫ indicate addition and no addition of corresponding protein into the sample, respectively. amino acid sequence levels with the GelE of Ef V583. It was also sence of GelE. The reaction volume was 10 ␮l of PBS and the quantity of shown that proteolytic activity of GelE affected the complement sys- each protein used in these tests was adjusted to 1 ␮g.