Acquisition of Complement Factor H Is Important for Pathogenesis ofStreptococcus pyogenesInfections: Evidence from Bacterial In Vitro Survival and Human Genetic Association

Abstract

Streptococcus pyogenes (or group A streptococcus [GAS]) is a major human pathogen causing infections, such as tonsillitis, erysipelas, and sepsis. Several GAS strains bind host complement regulator factor H (CFH) via its domain 7 and, thereby, evade complement attack and C3b-mediated opsonophagocytosis. Importance of CFH binding for survival of GAS has been poorly studied because removal of CFH from plasma or blood causes vigorous complement activation, and specific inhibitors of the interaction have not been available. In this study, we found that activation of human complement by different GAS strains (n = 38) correlated negatively with binding of CFH via its domains 5–7. The importance of acquisition of host CFH for survival of GAS in vitro was studied next by blocking the binding with recombinant CFH5–7 lacking the regulatory domains 1–4. Using this fragment in full human blood resulted in death or radically reduced multiplication of all of the studied CFH-binding GAS strains. To study the importance of CFH binding in vivo (i.e., for pathogenesis of streptococcal infections), we used our recent finding that GAS binding to CFH is diminished in vitro by polymorphism 402H, which is also associated with age-related macular degeneration. We showed that allele 402H is suggested to be associated with protection from erysipelas (n = 278) and streptococcal tonsillitis (n = 209) compared with controls (n = 455) (p < 0.05). Taken together, the bacterial in vitro survival data and human genetic association revealed that binding of CFH is important for pathogenesis of GAS infections and suggested that inhibition of CFH binding can be a novel therapeutic approach in GAS infections.

Complement Factor H Binds to Human Serum Apolipoprotein E and Mediates Complement Regulation on High Density Lipoprotein Particles

“…and emm8 was from ATCC (ATCC catalogue number 12349). These strains were suitable controls for bacterial whole-blood multiplication and FH binding assays as shown earlier (18). The Staphylococcus aureus 2151 strain is a blood isolate collected from a blood culture of a septic patient with the permission of the Ethical Review board of the Hospital District of Helsinki and Uusimaa (448/13/03/00/09).…”

“…200 -1000 cfu in 100 l of PBS or PBS with 20 g of FH5-7 were added in 1.2 ml of 40% (N. meningitidis) or 100% (other bacte-ria) human blood supplemented with lepirudin (Refludan 50 g/ml). The concentration of FH5-7 in the assay was chosen according to the previous study where the final concentration of FH5-7 was 0.7 mM, and the FH5-7/FH ratio was ϳ1:3 (18).The samples were incubated at 35°C or 37°C for 60 (S. aureus, S. pyogenes), 180 (S. aureus), or 90 min (N. meningitidis). At the 0 and 60-, 90-, 120-, and 180-min time points dilutions of the samples were cultured on agar plates for 17 h. The cfu were counted, and the multiplication factor (MF) at the final time point was determined by counting the ratio between final cfu present at each time point and the original cfu present in the inoculum as described previously (18).…”

“…Binding-We have previously shown that ex vivo survival of a bacterium that uses FH binding for immune evasion is reduced in the presence of FH5-7 lacking the active domains 1-4 of FH (18). To detect which of the main human pathogens has impaired survival in the presence of FH5-7 fragment, we also studied N. meningitidis and S. aureus.…”

“…The formation of soluble C5b-9 was analyzed by ELISA as described earlier (18). The 96-well plates (Maxisorp, Nunc) were coated with 2 g/ml anti-C9 neoepitope antibody WU13-15 (Hycult Biotech) in 100 l of 0.2 M Na 2 CO 3 , pH 10.6.…”

“…Several other microbial pathogens have also been shown to use FH for immune evasion via two regions, FH6 -7 and FH19 -20 (17). At least Neisseria meningitidis and most of the pathogenic S. pyogenes strains interact only with the FH5-7 region on FH, thereby increasing their immune evasion and survival in serum (18). The reasons why several microbes acquire FH particularly via FH5-7 or why mutations or polymorphisms in this region lead to complement related inflammatory diseases are not fully understood.…”