TY - JOUR
T1 - Contribution of the complement Membrane Attack Complex to the bactericidal activity of human serum
AU - Berends, Evelien T M
AU - Mohan, Sarbani
AU - Miellet, Willem R.
AU - Ruyken, Maartje
AU - Rooijakkers, Suzan H. M.
PY - 2015/6/1
Y1 - 2015/6/1
N2 - Direct killing of Gram-negative bacteria by serum is usually attributed to the Membrane Attack Complex (MAC) that is assembled upon activation of the complement system. In serum bactericidal assays, the activity of the MAC is usually blocked by a relatively unspecific method in which certain heat-labile complement components are inactivated at 56. °C. The goal of this study was to re-evaluate MAC-driven lysis towards various Gram-negative bacteria. Instead of using heat-treatment, we included the highly specific C5 cleavage inhibitor OmCI to specifically block the formation of the MAC. Using a C5 conversion analysis tool, we monitored the efficacy of the inhibitor during the incubations. Our findings indicate that 'serum-sensitive' bacteria are not necessarily killed by the MAC. Other heat-labile serum factors can contribute to serum bactericidal activity. These unidentified factors are most potent at serum concentrations of 10% and higher. Furthermore, we also find that some bacteria can be killed by the MAC at a slower rate. Our data demonstrate the requirement for the use of specific inhibitors in serum bactericidal assays and revealed that the classification of serum-sensitive and resistant strains needs re-evaluation. Moreover, it is important to determine bacterial viability at multiple time intervals to differentiate serum susceptibility between bacterial species. In conclusion, these data provide new insights into bacterial killing by the humoral immune system and may guide future vaccine development studies for the treatment of pathogenic serum-resistant bacteria.
AB - Direct killing of Gram-negative bacteria by serum is usually attributed to the Membrane Attack Complex (MAC) that is assembled upon activation of the complement system. In serum bactericidal assays, the activity of the MAC is usually blocked by a relatively unspecific method in which certain heat-labile complement components are inactivated at 56. °C. The goal of this study was to re-evaluate MAC-driven lysis towards various Gram-negative bacteria. Instead of using heat-treatment, we included the highly specific C5 cleavage inhibitor OmCI to specifically block the formation of the MAC. Using a C5 conversion analysis tool, we monitored the efficacy of the inhibitor during the incubations. Our findings indicate that 'serum-sensitive' bacteria are not necessarily killed by the MAC. Other heat-labile serum factors can contribute to serum bactericidal activity. These unidentified factors are most potent at serum concentrations of 10% and higher. Furthermore, we also find that some bacteria can be killed by the MAC at a slower rate. Our data demonstrate the requirement for the use of specific inhibitors in serum bactericidal assays and revealed that the classification of serum-sensitive and resistant strains needs re-evaluation. Moreover, it is important to determine bacterial viability at multiple time intervals to differentiate serum susceptibility between bacterial species. In conclusion, these data provide new insights into bacterial killing by the humoral immune system and may guide future vaccine development studies for the treatment of pathogenic serum-resistant bacteria.
KW - Bactericidal assay
KW - Complement
KW - Gram-negative bacteria
KW - Membrane Attack Complex
KW - Serum
UR - http://www.scopus.com/inward/record.url?scp=84925640200&partnerID=8YFLogxK
U2 - 10.1016/j.molimm.2015.01.020
DO - 10.1016/j.molimm.2015.01.020
M3 - Article
C2 - 25725315
AN - SCOPUS:84925640200
SN - 0161-5890
VL - 65
SP - 328
EP - 335
JO - Molecular Immunology
JF - Molecular Immunology
IS - 2
ER -