Distinct localization of the complement C5b-9 complex on Gram-positive bacteria

Abstract

SummaryThe plasma proteins of the complement system fulfil important immune defence functions, including opsonization of bacteria for phagocytosis, generation of chemo-attractants and direct bacterial killing via the Membrane Attack Complex (MAC or C5b-9). The MAC is comprised of C5b, C6, C7, C8, and multiple copies of C9 that generate lytic pores in cellular membranes. Gram-positive bacteria are protected from MAC-dependent lysis by their thick peptidoglycan layer. Paradoxically, several Grampositive pathogens secrete small proteins that inhibit C5b-9 formation. In this study, we found that complement activation on Gram-positive bacteria in serum results in specific surface deposition of C5b-9 complexes. Immunoblotting revealed that C9 occurs in both monomeric and polymeric (SDS-stable) forms, indicating the presence of ringstructured C5b-9. Surprisingly, confocal microscopy demonstrated that C5b-9 deposition occurs at specialized regions on the bacterial cell. On Streptococcus pyogenes, C5b-9 deposits near the division septum whereas on Bacillus subtilis the complex is located at the poles. This is in contrast to C3b deposition, which occurs randomly on the bacterial surface. Altogether, these results show a previously unrecognized interaction between the C5b-9 complex and Gram-positive bacteria, which might ultimately lead to a new model of MAC assembly and functioning.

Contribution of the complement Membrane Attack Complex to the bactericidal activity of human serum

“…Human serum was derived from 20 healthy volunteers, pooled, and prepared as described (Berends et al, 2013). Heat inactivated serum (30 min incubation in 56 • C water bath) or serum containing 20 g/ml OmCI were used where indicated.…”

“…Supernatants of the bactericidal assay were isolated after centrifugation and analyzed for the presence of C5a by a calcium mobilization assay as described previously (Berends et al, 2013). In short, U937 cells transfected with the C5a receptor (U937-C5aR) (Kew et al, 1997), were labeled with 2 M Fluo-3-AM (Life technologies).…”

Complement and Bacterial Infections: From Molecular Mechanisms to Therapeutic Applications

“…1a); this specifically kills Gram-negative bacteria. Gram-positive bacteria are protected from MAC-dependent killing, likely because their thick peptidoglycan outer layer prevents insertion of the MAC into the cell membrane [16].…”

Catch Me if You Can: <b><i>Streptococcus pyogenes</i></b> Complement Evasion Strategies

“…Although the MAC can deposit on the surface of S. pyogenes in distinct locations [58], the role this has in mediating an immune response is not understood and clearly calls for further investigation to elucidate if the MAC affects bacteria physiology or somehow serves as a signalling molecule for the cells of the immune system.…”