Bacterial Pathogenesis. Brenda A. Wilson

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an opsonin. As will be seen in the next chapter, antibodies can also perform this function. Without opsonization, phagocytes have difficulty ingesting a bacterium unless it is trapped in a small space. Because bacteria do not stick well to the phagocyte surface, the action of pseudopod encirclement can actually propel the bacterium away from the phagocytes—analogous to the way a fish can slip from your hands as you try to grab it. Phagocytes have C3b receptors on their surfaces that specifically bind C3b (Figure 3-12). Thus, by binding to the bacterial surface, complement component C3b gives the phagocyte something to grab on to as it tries to engulf the bacteria. Antibodies bound to bacterial surfaces can also act as opsonins, because a conserved portion of the heavy chain of the antibody, the Fc region, is recognized by Fc receptors on the phagocyte. The difference between these two processes is that antibodies bind to specific molecules on the surface of a bacterium, whereas C3b binds nonspecifically to glycosyl groups commonly found on bacterial surfaces. The combined effect of C3b and Fc receptors binding to their respective ligands on the bacterial surface works synergistically to enhance phagocytosis.

      Figure 3-12. Opsonization of a bacterium by activated complement component C3b and antibodies. Combined opsonization by both C3b and antibodies considerably enhances the uptake of the bacterium by phagocytes. IgG, immunoglobulin G.

      Activated complement components can also lead to direct killing of bacteria. Activated component C5b binds to the O-antigen of LPS of Gram-negative bacteria and recruits sequentially C6, C7, C8, and multiple C9 proteins to form a MAC in the bacterial membranes (Figure 3-13). Formation of the MAC kills bacteria by creating holes in their membranes. Bacteria that can be lysed by MAC are said to be serum sensitive, i.e., the addition of serum-containing complement components to the bacteria will kill the bacteria. Some Gram-negative bacteria have altered LPS O-antigens that do not bind C5b and therefore cannot form a MAC on their surfaces, while others have extra-long LPS O-antigens that can still bind C5b but cannot form a MAC in the bacterial membrane. Gram-negative bacteria that cannot be lysed by MAC are said to be serum resistant, i.e., they are resistant to killing by serum complement via MAC formation. Gram-positive bacteria are inherently serum resistant, because their thick cell walls prevent MAC assembly at the cytoplasmic membrane.

      Figure 3-13. Formation of the membrane attack complex (MAC). (A) C5-convertase assembled on the bacterial cell surface cleaves C5 to generate C5a, which is a chemokine for phagocytes, and C5b, which binds to the O-antigen of LPS on Gram-negative bacteria and recruits C6 through C8 to initiate MAC formation and insertion into the membrane. Up to fifteen C9 proteins then oligomerize into the complex. (B) MAC pores in the membrane that then kills the bacteria. Reproduced from Janeway C, Travers P, Walport M, Shlomchik M. 2004. Immunobiology, 6/e. Garland Science, New York, NY, with permission.

      The steps in complement activation by each of the three pathways are shown in more detail in Figure 3-14. The classical complement pathway (called that because it was the first to be discovered) is initiated when the Fc regions of several antigen-bound IgG molecules or one pentameric IgM molecule bind to the surface of a bacterium and are subsequently cross-linked by C1, a multi-protein complex of hexameric C1q, two C1r and two C1s zymogens (C1qr2s2) (Figure 3-14A). C1q binds directly to the heavy chain of antibodies bound to the bacterial surface and activates the two C1r serine proteases, which then in turn cleave and activate the two C1s serine proteases. Activated C1s cleaves C4 into C4a and C4b. C4b, like C3b, has a reactive thioester group that enables it to covalently attach to the bacterial surface at a site near the C1qr2s2 complex. C1s also cleaves C2 into C2a and C2b. C4b binds to C2a to form the C3 convertase (C2aC4b). C3 convertase then cleaves C3 to C3a, which diffuses away from the site, and C3b, which covalently binds to the bacterial surface.

      Figure 3-14. Activation of complement by the classical, lectin, and alternative pathways. (A) Classical pathway: Two IgG molecules or one IgM molecule attached to the surface of a bacterium bind complement component C1, causing an autoproteolytic event that activates it. C1, C4b, and C2a bind to each other and to the bacterium’s surface to form C3 convertase. The addition of C3b produces C5 convertase that triggers assembly of the MAC. (B) Lectin pathway: The mannose-binding lectins (MBLs) activate the complement pathway similarly to antibodies, except that they interact with C4 and C2 rather than C1. After that point, this pathway is the same as the classical pathway. (C) Alternative pathway: C3-H2O, an activated form of C3 that resembles C3b in conformation, is normally produced at low levels. If it binds a host cell surface through serum factor H, then the H-bound C3-H2O complex is targeted for destruction by serum protein I. If C3-H2O binds to the surface of a bacterium, it can form a complex with factor B, which is targeted for cleavage by factor D to generate Ba and Bb. The C3-H2O complexed with Bb then cleaves more C3 into C3a and C3b. The C3b then covalently binds to the bacterial surface, where it binds factor B, which gets cleaved by factor D to form C3bBb (C3 convertase) on the bacteria surface. Addition of more C3b produces C5 convertase. C5 convertase triggers assembly of the MAC.

      The lectin pathway converges with the classical pathway in that it also stimulates cleavage of C4 and C2, with generation of C4b and C2a that form the C3 convertase complex (Figure 3-14B). The collectin MBL circulates in the blood as a multimeric complex with one or more serine proteases, called MBL-associated serine protease (MASP) proteins and small MASP-associated protein (sMAP). MASPs can also bind to ficolins (FCNs). There are three known MASPs: MASP-1, MASP-2, and MASP-3. MASP-1 and MASP-2 resemble C1r and C1s, respectively. When MBL binds mannose groups of glycoproteins found on the surface of many pathogens, the MASP-2 protein cleaves C4 and C2 and initiates formation of the C3 convertase as in the classical pathway. MASP-1 can also cleave C3 directly, whereas MASP-3 appears to associate primarily with FCN but does not appear to activate complement, and its function remains unclear.

      Activation via the alternative pathway (named because it was discovered after the “classical” pathway) bypasses the need for C1, C2, C4, antibodies, or MBL/MASP, and relies instead on C3b as the initiating component. As C3 circulates in blood and tissue, it is occasionally activated into a water-bound form (C3-H2O) that can interact with the serum proteins, factor B or factor H. Tissues of the body are coated with sialic acid residues, that can bind to one end of factor H. In the absence of a bacterial surface, C3-H2O binds to the other end of factor H to form a complex that produces C3b (Figure 3-14C). This binding of C3b to factor H changes the conformation of C3b and targets it for proteolytic cleavage into iC3b by the serum protease factor I. In the absence of a bacterial surface, the iC3b bound to factor H is targeted for further destruction by factor I. However, iC3b can still covalently bind to a bacterial surface via its internal thioester group and as such can serve as an opsonin, even though iC3b can no longer form active C3 convertase.

      If C3-H2O instead binds to serum protein factor B, then another serum protein factor D cleaves that B to Ba and Bb. The resulting complex of C3-H2O bound to Bb cleaves another C3 molecule to produce C3a and C3b, that then covalently binds to the bacterial surface. This membrane-bound C3b binds another factor B, and the complex C3b-B is then targeted for cleavage by factor D to form the C3 convertase C3bBb, which in turn continues to cleave more C3 to make more C3a and C3b. C3bBb, which normally has a relatively short half-life of about 90 minutes, is further stabilized by binding of the protein properdin (P), which increases the half-life of the resulting

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