The Research On Molecular Mechanism Of Interaction Between The Surface Protein Fhb Of Streptococcus Suis Serotype2and Complement System Components

Streptococcus suis serotype2(SS2) is an important emerging zoonotic pathogen inrecent years, SS2infection by wound is one of the major causes of meningitis,endocarditis, sepsis and streptococcal toxic shock syndrome(STSS) in humans.However, little is known about how S. suis2survives in the host blood, especially studyof SS2how to evade host complement-mediated phagocytosis remains very limited.Complement system, as an important component of the innate immune defense, is apowerful weapon protecting the host from invading pathogens by opsonizingmicroorganisms, activating the immune system, recruiting phagocytes and eventuallydirectly lysing certain pathogens. Complement system activation is a highly orderedcascade, the host complement system activation in three ways: the classical pathway;the mannose binding lectin pathway; the alternative pathway. All pathways lead to theformation of the C3convertases, with subsequently cleavage of C3to generate activatedcomplement component C3b, a large number of C3b molecules deposition on pathogenis an important event of complement activation. Most of pathogens causing invasivediseases would benefit from inhibiting deposition of C3b on their surface and inhibitingactivation of complement system, either by attracting host FH or by inactivating C3bdirectly.Previous research in our lab identified that SS2surface protein Fhb (factor H-bindingprotein), as a virulence factor, inhibits activation of the alternative pathway toincreasing resistance to phagocytosis by binding hFH. Through GST-pulldown, we identified that Fhb not only binding to hFH, but interacting with host complementprotein C3b and C3d. However, the molecular mechanism of these interactions betweenthem remains unclear.The first part of this study is mainly to verify the recombinant Fhb truncated fragmentFhb-Ⅲ*binding to the complement proteins hFH, C3b, C3d. Using gene engineeringrecombinant expression, based on the expression vector pET28a, we constructed sevenFhb truncated fragments(Fhb-Ⅰ45-344；Fhb-Ⅱ45-170；Fhb-Ⅲ171-344；Fhb-Ⅳ345-664；Fhb-Ⅴ345-483；Fhb-Ⅵ484-664；Fhb-Ⅲ*134-344) and four Fhb domain deletion proteins37(134-170aa)；3(171-344aa)；3N(171-232aa)；3C(233-344aa), andpurified them by nickel affinity chromatography. Through ELISA, ligand blotting andbiolayer interferometry (BLI) methods, we identified that Fhb can bind to thecomplement proteins hFH, C3b, C3d, and determined that the binding region is mainlylocated in Fhb-Ⅲ*(134-344aa). The affinity of interaction between hFH, C3b or C3dand Fhb-Ⅲ*was estimated using BLI. The obtained affinity constants wereKD=83.51nM for hFH-Fhb-Ⅲ*, KD=17.09nM for C3b-Fhb-Ⅲ*and KD=141.5nM forC3d-Fhb-Ⅲ*. In addition, we detected a decrease of hFH-Fhb-Ⅲ*binding whenincreasing NaCl and ethylene glycol concentrations, indicating that the interactionbetween hFH and Fhb-Ⅲ*is partially of ionic nature, more seems to depend onhydrophobic interaction as well as electrostatics.The content of the second part in this study is research on the role of Fhb-Ⅲ*domain ininhibiting of complement activation. First, we used the shuttle plasmid pAT18toconstruct the deletion complementary strain CΔSsfhb-3which knockout the Fhbfunctional domain Fhb-Ⅲ171-344. Using immunofluorescence-laser scanning confocalmicroscopy and immunoblotting to visualize the location of Fhb protein, it’s interestingto find that Fhb not only express on the bacterial surface, can also be detected in theculture supernatant. We next analyzed the impact of soluble Fhb in supernatant on complement activation by FACS, and found that the Fhb protein in the WT culturesupernatant can inhibit complement C3b/iC3b deposition on the surface of the mutantstrain ΔSsfhb, but not with the CΔSsfhb-3culture supernatant, suggesting that domainFhb-Ⅲ171-344play important role in inhibiting of complement activation by soluble Fhb.To conclude, in this study, we confirmed that Fhb can directly bind to the complementsystem component protein hFH, C3b and C3d through the functional domain Fhb-Ⅲ*134-344, and can play roles in immune evasion in the culture supernatant. The resultsindicated the probably dual functions of the Fhb protein:(1) The Fhb protein expressedon the bacterial surface can bind to the complement negative regulatory protein hFHand recruit C3b or C3d molecules to form a tripartite complex, degrading C3bmolecules and further inhibiting the C3convertase formation of the alternative pathway,thereby evade complement mediated killing.(2) The surface protein Fhb shed into theculture supernatant in some kind of force, exposes the active region of Fhb-Ⅲ*134-344and binds to C3b molecules in serum, competitively inhibiting C3convertase formationand deposition of C3b molecules, evading the complement mediated killing. Ourresearch disclosed the mechanism of SS2surface protein Fhb on antiphagocyticclearance and lays a foundation to further define the crystal structure of the Fhb incomplex with hFH/C3b/C3d, promotes understanding of the mechanism of S. suis inescaping the host innate immunity.