TY - JOUR
T1 - Bacterial killing by complement requires membrane attack complex formation via surface-bound C5 convertases
AU - Heesterbeek, Dani A.C.
AU - Bardoel, Bart W.
AU - Parsons, Edward S.
AU - Bennett, Isabel
AU - Ruyken, Maartje
AU - Doorduijn, Dennis J.
AU - Gorham, Ronald D.
AU - Berends, Evelien T.M.
AU - Pyne, Alice L.B.
AU - Hoogenboom, Bart W.
AU - Rooijakkers, Suzan H.M.
N1 - Funding Information:
We would like to acknowledge Piet Gros, Paul Parren, and Jos van Strijp for proofreading the manuscript and Zvi Fishelshon for fruitful discussions; Jannik Luebke and Richard Thorogate for assistance with AFM experiments; Richard Wubbolts for assistance with microscopy, U-Protein Express BV for protein expression facilities. We thank Ilse Jongerius and Diane Wouters (Sanquin Research, Department of Immunopathology, Amsterdam, the Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands) for providing ∆CD46/∆CD55/∆CD59 HAP1 cells; John Lambris (University of Philadelphia) for providing compstatin and Frank Beurs-kens (Genmab, Utrecht) for providing Eculizumab. The work was funded by: an ERC Starting grant (639209-ComBact, to S.H.M.R); the EMBO Young Investigator Program (3418 to S.H.M.R); UK RCUK | Biotechnology and Biological Sciences Research Council (BBSRC) and MRC project grants (BB/N015487/1 and MR/R000328/1, to B.W.H.); UK RCUK | Engineering and Physical Sciences Research Council (EPSRC) and MRC fellowships (EP/M507970/1 to E.S.P.; EP/ M506448/1 and MR/R024871/1 to A.L.B.P.); UCL Impact Award (EP/M506448/1 to I. B.); and UK EPSRC investments in AFM equipment (EP/K031953/1 via the IRC in Early-Warning Sensing Systems for Infectious Diseases; and EP/ M028100/1).
Funding Information:
We would like to acknowledge Piet Gros, Paul Parren, and Jos van Strijp for proofreading the manuscript and Zvi Fishelshon for fruitful discussions; Jannik Luebke and Richard Thorogate for assistance with AFM experiments; Richard Wubbolts for assistance with microscopy, U-Protein Express BV for protein expression facilities. We thank Ilse Jongerius and Diane Wouters (Sanquin Research, Department of Immunopathology, Amsterdam, the Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands) for providing ?CD46/?CD55/?CD59 HAP1 cells; John Lambris (University of Philadelphia) for providing compstatin and Frank Beurskens (Genmab, Utrecht) for providing Eculizumab. The work was funded by: an ERC Starting grant (639209-ComBact, to S.H.M.R); the EMBO Young Investigator Program (3418 to S.H.M.R); UK RCUK | Biotechnology and Biological Sciences Research Council (BBSRC) and MRC project grants (BB/N015487/1 and MR/R000328/1, to B.W.H.); UK RCUK | Engineering and Physical Sciences Research Council (EPSRC) and MRC fellowships (EP/M507970/1 to E.S.P.; EP/M506448/1 and MR/R024871/1 to A.L.B.P.); UCL Impact Award (EP/M506448/1 to I. B.); and UK EPSRC investments in AFM equipment (EP/K031953/1 via the IRC in Early-Warning Sensing Systems for Infectious Diseases; and EP/M028100/1).
Publisher Copyright:
© 2019 The Authors. Published under the terms of the CC BY 4.0 license
PY - 2019/2/15
Y1 - 2019/2/15
N2 - 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.
AB - 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.
KW - atomic force microscopy
KW - complement; convertase
KW - Gram-negative bacteria
KW - membrane attack complex
UR - http://www.scopus.com/inward/record.url?scp=85059950173&partnerID=8YFLogxK
U2 - 10.15252/embj.201899852
DO - 10.15252/embj.201899852
M3 - Article
C2 - 30643019
AN - SCOPUS:85059950173
SN - 0261-4189
VL - 38
JO - EMBO Journal
JF - EMBO Journal
IS - 4
M1 - e99852
ER -