Bacterial killing by complement requires membrane attack complex formation via surface‐bound C5 convertases

Abstract

The immune system kills bacteria by the formation of lytic membrane attack complexes (MACs), triggered when complement enzymes cleave C5. At present, it is not understood how the MAC perturbs the composite cell envelope of Gram‐negative bacteria. Here, we show that the role of C5 convertase enzymes in MAC assembly extends beyond the cleavage of C5 into the MAC precursor C5b. Although purified MAC complexes generated from preassembled C5b6 perforate artificial lipid membranes and mammalian cells, these components lack bactericidal activity. In order to permeabilize both the bacterial outer and inner membrane and thus kill a bacterium, MACs need to be assembled locally by the C5 convertase enzymes. Our data indicate that C5b6 rapidly loses the capacity to form bactericidal pores; therefore, bacterial killing requires both in situ conversion of C5 and immediate insertion of C5b67 into the membrane. Using flow cytometry and atomic force microscopy, we show that local assembly of C5b6 at the bacterial surface is required for the efficient insertion of MAC pores into bacterial membranes. These studies provide basic molecular insights into MAC assembly and bacterial killing by the immune system.

How the Membrane Attack Complex Damages the Bacterial Cell Envelope and Kills Gram‐Negative Bacteria

“…[36] In general, monomers of the MACPF/CDC protein family first bind to a specific target on the target surface via a membrane-binding domain. Our recent report, describing how surface-bound C5 convertases are essential to properly insert MAC pores into target bacteria, [22] supports this hypothesis. [37] After binding to the membrane, these monomers homo-oligomerize and form a "prepore" that does not yet permeate the membrane.…”

“…These C5 convertases, which are multimeric, enzymatic complexes that cleave C5 into C5a and C5b, are covalently linked to the bacterial surface when the complement system is activated. [22,29,49] In contrast to convertase-generated MAC pores, pores that were generated from preassembled C5b6 were sensitive to trypsin and not visible by AFM, suggesting that these pores were not stably inserted into the bacterial OM. Local MAC assembly by C5 convertases is crucial to efficiently insert MAC pores into the bacterial OM.…”

“…Since this composite cell envelope of Gram-negative bacteria spans over 30 nm (as shown in Figure 2A), [19] it seems physically impossible for the MAC (that has a transmembrane domain of ≈5 nm [29] ) to damage the full cell envelope. B) Schematic overview of how local MAC assembly damages the cell envelope of Gramnegative bacteria, explained using the recently developed assay for measuring cell envelope integrity in Heesterbeek et al [22] When the MAC is locally generated by surface-bound C5 convertases, these pores are stably inserted into the bacterial OM and permeate the OM leading to leakage of periplasmic proteins (as depicted by the absence of red periplasmic content). Moreover, as Gram-negative bacteria are metabolically active, an active cellular process could also be involved in bacterial killing by MAC pores.…”

“…[22] In order to efficiently kill a bacterium, the MAC needs to be generated by surface-bound C5 convertases. [22] In order to efficiently kill a bacterium, the MAC needs to be generated by surface-bound C5 convertases.…”

“…[16][17][18] However, the exact molecular mechanism by which MAC pores kill Gram-negative bacteria remains largely unknown. [22] This review provides an overview of these recent structural findings on how MAC pores assemble and summarizes these recent functional studies on bacteria. [19] Several hypotheses on how MAC pores could permeate the complex cell envelope of Gram-negative bacteria in order to kill the bacterium have previously been reviewed.…”