- Email: [email protected]
The role of C5 convertases in membrane attack complex dependent killing of Gram-negative bacteria
154
Abstracts / Molecular Immunology 89 (2017) 152–160
076
077
MASP-3 specifically recognizes different Aspergillus species
The role of C5 convertases in membrane attack complex dependent killing of Gram-negative bacteria
Anne Rosbjerg 1,∗ , Mikkel Ole Skjoedt 1 , Reinhard Würzner 2 , Peter Garred 1 1
Laboratory of Molecular Medicine, Department of Clinical Immunology Section 7631, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 2 Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria Background: Complement is important in protection against Aspergillus fungal infections and both the classical and lectin pathway seem to be involved with a strong contribution from the alternative pathway amplification loop. A. fumigatus followed by A. flavus A. terreus, and A. niger are the most disease related Aspergillus species and elucidation of complement activation/evasion mechanisms is of interest to patients infected with drug resistant strains. MASP-3 is known to associate with lectin pathway pattern recognition molecules, but not in the role as a lectin pathway activator. Instead MASP-3 seems to work as an alternative pathway protease activator. The MASP-3 connection to both the lectin and alternative pathway led us to investigate the role of MASP-3 in the complement response to Aspergillus. Materials and methods: Clinical isolates of various fungi were cultured on agar plates and the conidia (spores) were harvested and heat-inactivated. Protein binding and complement deposition on the conidia were measured in flow cytometry and microscopy using recombinant proteins (rMASP-1, rMASP-3, rMAP-1 and rMASP-1/-3 heavy chain) and serum/plasma. Results: We found a clear direct binding of rMASP-3 to the four different Aspergillus species tested; fumigatus, terreus, flavus and niger. No binding was observed on Candida albicans or on three different Mucorales fungi tested (Lichtheimia corymbifera, Mucor circinelloides and Rhizopus arrhizus). rMASP-1 bound the four Aspergillus species, but to a lesser degree than rMASP-3. Only a minor or no binding of rMAP-1 or rMASP-1/-3 heavy chain was observed, suggesting that the serine protease domain of MASP-3 is involved. A. fumigatus conidia with pre-bound rMASP-3 appeared to convey lower levels of complement activation than rMASP-3-free conidia. Conclusion: MASP-3 binds specifically to Aspergillus species (fumigatus, terreus, flavus and niger) and not to C. albicans and various Mucorales fungi. Conceivably this demonstrates a novel evasion mechanism used by Aspergillus species to escape complement attack. http://dx.doi.org/10.1016/j.molimm.2017.06.111
D.A.C. Heesterbeek 1,∗ , B.W. Bardoel 1 , I. Bennett 2 , A. Pyne 2 , E.T.M. Berends 1 , M. Ruyken 1 , B. Hoogenboom 2 , S.H.M. Rooijakkers 1 1 Medical Microbiology, University Medical Center Utrecht, Utrecht, Netherlands 2 London Center for Nanotechnology, University College London, London, UK
Background: Gram-negative (GN) bacteria are effectively killed by human complement through the Membrane Attack Complex (MAC). The formation of MAC is driven by surface-bound C5 convertases that cleave C5 into C5b, which then interacts with components C6 to form C5b6. Subsequent binding of components C7, C8 and multiple copies of C9 results in formation of the MAC (C5b9). Recent structural studies showed that purified C5b-9 forms an asymmetrical pore that perturbs single-membrane liposomes. Here we study how the MAC can lyse GN bacteria that comprise two membranes with a peptidoglycan layer in between. Materials and methods: Dynamics of bacterial membrane disruption were studied using fluorescent DNA dyes in combination with flow cytometry and conventional plating assays. Bacteria were imaged using Scanning Electron Microscopy (SEM) and high resolution information of (pre)pores on bacterial membranes was gained using atomic force microscopy (AFM). Results and conclusions: In contrast to lysis of singlemembrane liposomes and erythrocytes, we observe that purified C5b-9 does not kill GN bacteria. Whereas human complement effectively kills ‘serum-sensitive’ Gram-negatives like Escherichia coli, Klebsiella pneumoniae and Salmonella, none of these bacteria were killed by C5b-9. However, purified MAC does kill bacteria that are labeled with C5 convertases. Using AFM, we find that C5 convertases are essential for stable insertion of pores into the bacterial membrane. Pore formation analyses in flow cytometry indicate that C5 convertases, bound to the bacterial outer membrane, are essential to form pores that (indirectly) damage the bacterial inner membrane. In conclusion, we show that C5 convertases are essential for the formation of bactericidal MAC. http://dx.doi.org/10.1016/j.molimm.2017.06.112 078 Defining the molecular interplay between antibodies and complement in bacterial infections Seline A. Zwarthoff 1,∗ , Maartje Ruyken 1 , Simone Magnoni 1 , Gestur Vidarsson 2 , Piet C. Aerts 1 , Carla J. de Haas 1 , Suzan H.M. Rooijakkers 1 1 Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands 2 Experimental Immunohematology, Sanquin Research, Amsterdam, The Netherlands
Background: Antibiotic resistance has become a serious problem world-wide and we need novel treatment strategies to control bacterial infections. Complement plays a crucial role in the elimination of bacteria from our body as it labels bacteria for phagocytosis and specifically kills Gram-negatives directly through formation of the membrane attack complex (MAC). Since complement can be specifically triggered via antibodies recognizing the bacterial
Abstracts / Molecular Immunology 89 (2017) 152–160
076
077
MASP-3 specifically recognizes different Aspergillus species
The role of C5 convertases in membrane attack complex dependent killing of Gram-negative bacteria
Anne Rosbjerg 1,∗ , Mikkel Ole Skjoedt 1 , Reinhard Würzner 2 , Peter Garred 1 1
Laboratory of Molecular Medicine, Department of Clinical Immunology Section 7631, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 2 Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria Background: Complement is important in protection against Aspergillus fungal infections and both the classical and lectin pathway seem to be involved with a strong contribution from the alternative pathway amplification loop. A. fumigatus followed by A. flavus A. terreus, and A. niger are the most disease related Aspergillus species and elucidation of complement activation/evasion mechanisms is of interest to patients infected with drug resistant strains. MASP-3 is known to associate with lectin pathway pattern recognition molecules, but not in the role as a lectin pathway activator. Instead MASP-3 seems to work as an alternative pathway protease activator. The MASP-3 connection to both the lectin and alternative pathway led us to investigate the role of MASP-3 in the complement response to Aspergillus. Materials and methods: Clinical isolates of various fungi were cultured on agar plates and the conidia (spores) were harvested and heat-inactivated. Protein binding and complement deposition on the conidia were measured in flow cytometry and microscopy using recombinant proteins (rMASP-1, rMASP-3, rMAP-1 and rMASP-1/-3 heavy chain) and serum/plasma. Results: We found a clear direct binding of rMASP-3 to the four different Aspergillus species tested; fumigatus, terreus, flavus and niger. No binding was observed on Candida albicans or on three different Mucorales fungi tested (Lichtheimia corymbifera, Mucor circinelloides and Rhizopus arrhizus). rMASP-1 bound the four Aspergillus species, but to a lesser degree than rMASP-3. Only a minor or no binding of rMAP-1 or rMASP-1/-3 heavy chain was observed, suggesting that the serine protease domain of MASP-3 is involved. A. fumigatus conidia with pre-bound rMASP-3 appeared to convey lower levels of complement activation than rMASP-3-free conidia. Conclusion: MASP-3 binds specifically to Aspergillus species (fumigatus, terreus, flavus and niger) and not to C. albicans and various Mucorales fungi. Conceivably this demonstrates a novel evasion mechanism used by Aspergillus species to escape complement attack. http://dx.doi.org/10.1016/j.molimm.2017.06.111
D.A.C. Heesterbeek 1,∗ , B.W. Bardoel 1 , I. Bennett 2 , A. Pyne 2 , E.T.M. Berends 1 , M. Ruyken 1 , B. Hoogenboom 2 , S.H.M. Rooijakkers 1 1 Medical Microbiology, University Medical Center Utrecht, Utrecht, Netherlands 2 London Center for Nanotechnology, University College London, London, UK
Background: Gram-negative (GN) bacteria are effectively killed by human complement through the Membrane Attack Complex (MAC). The formation of MAC is driven by surface-bound C5 convertases that cleave C5 into C5b, which then interacts with components C6 to form C5b6. Subsequent binding of components C7, C8 and multiple copies of C9 results in formation of the MAC (C5b9). Recent structural studies showed that purified C5b-9 forms an asymmetrical pore that perturbs single-membrane liposomes. Here we study how the MAC can lyse GN bacteria that comprise two membranes with a peptidoglycan layer in between. Materials and methods: Dynamics of bacterial membrane disruption were studied using fluorescent DNA dyes in combination with flow cytometry and conventional plating assays. Bacteria were imaged using Scanning Electron Microscopy (SEM) and high resolution information of (pre)pores on bacterial membranes was gained using atomic force microscopy (AFM). Results and conclusions: In contrast to lysis of singlemembrane liposomes and erythrocytes, we observe that purified C5b-9 does not kill GN bacteria. Whereas human complement effectively kills ‘serum-sensitive’ Gram-negatives like Escherichia coli, Klebsiella pneumoniae and Salmonella, none of these bacteria were killed by C5b-9. However, purified MAC does kill bacteria that are labeled with C5 convertases. Using AFM, we find that C5 convertases are essential for stable insertion of pores into the bacterial membrane. Pore formation analyses in flow cytometry indicate that C5 convertases, bound to the bacterial outer membrane, are essential to form pores that (indirectly) damage the bacterial inner membrane. In conclusion, we show that C5 convertases are essential for the formation of bactericidal MAC. http://dx.doi.org/10.1016/j.molimm.2017.06.112 078 Defining the molecular interplay between antibodies and complement in bacterial infections Seline A. Zwarthoff 1,∗ , Maartje Ruyken 1 , Simone Magnoni 1 , Gestur Vidarsson 2 , Piet C. Aerts 1 , Carla J. de Haas 1 , Suzan H.M. Rooijakkers 1 1 Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands 2 Experimental Immunohematology, Sanquin Research, Amsterdam, The Netherlands
Background: Antibiotic resistance has become a serious problem world-wide and we need novel treatment strategies to control bacterial infections. Complement plays a crucial role in the elimination of bacteria from our body as it labels bacteria for phagocytosis and specifically kills Gram-negatives directly through formation of the membrane attack complex (MAC). Since complement can be specifically triggered via antibodies recognizing the bacterial