الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 238 |  |  |
| | | from | 238 | | |
Abstract
Infections caused by Enterococcus faecalis are considered a major public health concern worldwide. Complement system has a crucial role in the protection against different microbial pathogens including E. faecalis. However, there is limited information on the role of the classical pathway (CP) in protection against infections caused by E. faecalis. Thus, the present study aimed to evaluate the role of the CP of complement activation during E. faecalis infection. For that, we generated Fab fragments that can successfully block the CP via inhibition of a key enzyme named C1s-A. The anti-mC1s-A-SP Fab fragments were established in our study through three main steps. First, the mC1s-A-SP protein (the active moiety of the enzyme mC1s-A) was expressed via recombinant DNA technology in E. coli BL-21 (DE3) pLysS cells using pRSET-B as expression vector. Second, polyclonal anti-mC1s-A-SP antibodies were produced in rabbits, which were immunized subcutaneously with the purified mC1s-A-SP protein emulsified with Freund’s adjuvant. Third, the purified anti-mC1s-A-SP antibodies were digested via papain enzyme to produce the Fab fragments. The anti-mC1s-A-SP Fab fragments were purified via protein G Sepharose column and their binding capacity and functional activity were evaluated. Our results showed that anti-mC1s-A-SP Fab fragments block the CP mediated C3b and C4b deposition in vitro. Furthermore, administration of these Fab fragments significantly impairs the CP in vivo. The role of the CP in complement activation during E. faecalis infections was evaluated in vitro and in vivo using the prepared inhibitory Fab fragments (anti-mC1s-A-SP Fab fragments). Our results showed the importance of CP in recognition, opsonization, and clearance (phagocytosis) of E. faecalis. Moreover, our data depicted that treatment of infected mice with E. faecalis using anti-mC1s-A-SP Fab fragments significantly impairs bacterial clearance as determined from the viable bacterial counts recovered from blood, kidneys, spleens, livers, and lungs of infected mice. Over all, this study highlights the essential role of the CP pathway in host defense against E. faecalis.