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Identification of Staphylococcus aureus surface protein SdrE as a complement factor H-binding molecule
Abstracts / Molecular Immunology 48 (2011) 1666–1733
Methods and results: In order to better characterize this activity, the poison was further purified and after three steps of chromatography, five subfractions were obtained (S1–S5). The subfractions S2 and S5 induced reduction of the hemolytic activity of the classical pathway. This effect occurred by activation of the CS as showed by complement-dependent neutrophil chemotaxis (migration distances of 66.25 m for S2 and 55.47 m for S5 versus 37.5 m for the negative control, p < 0.05). S2 and S5 preincubated with human normal serum were able to produce C3 fragments as showed by bidimensional immunoelectrophoresis. S2 also was able to generate SC5b-9 soluble complexes as measured by an ELISA assay (S2 versus negative control, p < 0.01). Conclusions: In this work, we isolated two subfractions able to induce the activation of the CS. This study allowed a better evaluation of some components present in the complex mixture which is the poison of Rhinella schneideri, that in the future can generate important pharmacological tools for the study of diverse pathologies related to the complement system. FAPESP and CAPES. doi:10.1016/j.molimm.2011.06.309 P90 Complement activation in Alzheimer’s disease W.H. Rutjes-van den Hurk a,∗ , C. Theunissen b , R. Veerhuis b , L-.O. Wahlund c , H. VanderStichele d , M. Tsolaki e , R. VandenBerghe f , P. Johannsen g , A. Lleo h , L. Fröhlich i , P.J. Visser j a
Hycult Biotech, Uden, Netherlands VU Medical Center, Amsterdam, Netherlands c Karolinska Institutet, Stockholm, Sweden d Innogenetics, Zijnaarde, Belgium e Aristotle University of Thessaloniki, Thessalonike, Greece f KU Leuven, Leuven, Belgium g Rigshospitalet, Copenhagen, Denmark h Universitat de Barcelona, Barcelona, Spain i Forschungszentrum Jülich, Jülich, Germany j University of Maastricht, Maastricht, Netherlands b
Introduction: Inflammation is an early event in neurodegeneration. The role of the innate immune system in Alzheimer’s disease (AD) was investigated by measuring complement factors in cerebrospinal fluid (CSF) of subjects with mild cognitive impairment (MCI), AD and other dementias (oDem). Methods: We selected healthy controls (n = 19, age = 63, MMSE = 29.1), subjects with MCI (n = 56, age = 70, MMSE = 26.9), AD (n = 45, age = 72, MMSE = 22.9) and oDem (n = 31, age = 69 MMSE = 24.1) from the EDAR study (www.edarstudy.eu). We measured C3a, C5a, and the soluble C5b-9 terminal complement complex (TCC), A1-42, total tau, and serum amyloid P. We compared groups and correlated complement markers with other biomarkers, neuropsychological markers, and markers of functional impairment using non-parametric tests. Results: C3a was decreased by 50–60% in subjects with MCI, AD, and oDem relative to controls (p < 0.003 all comparisons). C5a was increased 3–6-fold in AD relative to controls (p = 0.077), MCI (p = 0.016), and oDem (p = 0.07). TCC did not differ between the groups. In the combined sample of subjects with MCI, AD, and oDem, higher levels of C3a correlated with higher levels of TCC (r = 0.51, p < 0.001), SAP (r = 0.40, p < 0.001), and tau (r = 0.18, p = 0.048). Higher levels of C5a correlated with more impairment on memory, executive functioning, and the MMSE (r = 0.22-0.30, p = 0.05-0.002). Higher levels of TCC correlated with higher SAP levels (r = 0.41, p < 0.001) and more functional impairment (r = 0.25, p = 0.018).
1695
Conclusion: The innate immune system is actively involved in neurodegeneration and may provide diagnostic and prognostic information for AD. doi:10.1016/j.molimm.2011.06.310 P91 Identification of Staphylococcus aureus surface protein SdrE as a complement factor H-binding molecule K.M. Cunnion a,∗ , J.A. Sharp a , P.S. Hair a , C.G. Echague a , M.D. Ward a , T.J. Foster b , J.O. Nyalwidhe a a b
Eastern Virginia Medical School, Norfolk, United States Trinity College, Dublin, Ireland
Background and rationale: S. aureus utilizes a repertoire of immune-evasive tactics to circumvent host defenses including the complement system, a crucial component of our innate immune response that is central for controlling S. aureus infections. We have previously shown that S. aureus acquires the complement regulatory protein factor H (fH) to its surface as a means of evading complement attack. Identifying the S. aureus surface protein that binds fH may provide a therapeutic target for blocking this interaction. Methods: To identify S. aureus cell-wall proteins that bind fH, cross-linking of purified fH to the bacterial surface as well as farwestern analysis of cell-wall preparations were performed. Bands evident for fH binding were analyzed via ESI-LC–MS/MS. Putative targets were expressed in recombinant form and assessed for fH binding via dot-blot and modified ELISA using purified fH and heat-inactivated serum. Recombinant proteins were assayed for functionality by capturing factor H and measuring factor I cleavage of C3b to iC3b. Results: SdrE was identified as a putative factor H-binding protein by both overlay and cross-linking methods. Recombinant SdrE bound both purified fH and serum fH efficiently. Increasing amounts of purified fH bound to immobilized rSdrE positively correlated with iC3b generation. Conclusions: These studies provide the first identification of an S. aureus cell-wall protein, SdrE, which binds factor H. Furthermore, rSdrE-bound fH retains cofactor activity for factor I-mediated cleavage of C3b. doi:10.1016/j.molimm.2011.06.311 P92 Hyperglycemic conditions inhibit C3-mediated host defenses against bacterial pathogens and change the tertiary structure of C3 K.M. Cunnion ∗ , P.S. Hair, J.O. Nyalwidhe Eastern Virginia Medical School, Norfolk, United States In diabetics, Staphylococcus aureus is the most common cause of foot infections and polymicrobial limb infections frequently lead to amputation. These studies provide new insight into hyperglycemia altering the interaction between the host defense complement system, S. aureus, and Gram-negative bacteria. Elevated glucose concentrations were found to increase C3 binding to S. aureus 6fold. However, minimal conversion of C3 to the functional forms C3b and iC3b was found to occur on the S. aureus surface. Because the C3 was not covalently bound, the glucose-mediated C3 binding to S. aureus was reversible upon return to normal glucose concentrations. Elevated glucose dramatically increased C3 binding to all clinical S. aureus isolates tested and increased C3 binding
Methods and results: In order to better characterize this activity, the poison was further purified and after three steps of chromatography, five subfractions were obtained (S1–S5). The subfractions S2 and S5 induced reduction of the hemolytic activity of the classical pathway. This effect occurred by activation of the CS as showed by complement-dependent neutrophil chemotaxis (migration distances of 66.25 m for S2 and 55.47 m for S5 versus 37.5 m for the negative control, p < 0.05). S2 and S5 preincubated with human normal serum were able to produce C3 fragments as showed by bidimensional immunoelectrophoresis. S2 also was able to generate SC5b-9 soluble complexes as measured by an ELISA assay (S2 versus negative control, p < 0.01). Conclusions: In this work, we isolated two subfractions able to induce the activation of the CS. This study allowed a better evaluation of some components present in the complex mixture which is the poison of Rhinella schneideri, that in the future can generate important pharmacological tools for the study of diverse pathologies related to the complement system. FAPESP and CAPES. doi:10.1016/j.molimm.2011.06.309 P90 Complement activation in Alzheimer’s disease W.H. Rutjes-van den Hurk a,∗ , C. Theunissen b , R. Veerhuis b , L-.O. Wahlund c , H. VanderStichele d , M. Tsolaki e , R. VandenBerghe f , P. Johannsen g , A. Lleo h , L. Fröhlich i , P.J. Visser j a
Hycult Biotech, Uden, Netherlands VU Medical Center, Amsterdam, Netherlands c Karolinska Institutet, Stockholm, Sweden d Innogenetics, Zijnaarde, Belgium e Aristotle University of Thessaloniki, Thessalonike, Greece f KU Leuven, Leuven, Belgium g Rigshospitalet, Copenhagen, Denmark h Universitat de Barcelona, Barcelona, Spain i Forschungszentrum Jülich, Jülich, Germany j University of Maastricht, Maastricht, Netherlands b
Introduction: Inflammation is an early event in neurodegeneration. The role of the innate immune system in Alzheimer’s disease (AD) was investigated by measuring complement factors in cerebrospinal fluid (CSF) of subjects with mild cognitive impairment (MCI), AD and other dementias (oDem). Methods: We selected healthy controls (n = 19, age = 63, MMSE = 29.1), subjects with MCI (n = 56, age = 70, MMSE = 26.9), AD (n = 45, age = 72, MMSE = 22.9) and oDem (n = 31, age = 69 MMSE = 24.1) from the EDAR study (www.edarstudy.eu). We measured C3a, C5a, and the soluble C5b-9 terminal complement complex (TCC), A1-42, total tau, and serum amyloid P. We compared groups and correlated complement markers with other biomarkers, neuropsychological markers, and markers of functional impairment using non-parametric tests. Results: C3a was decreased by 50–60% in subjects with MCI, AD, and oDem relative to controls (p < 0.003 all comparisons). C5a was increased 3–6-fold in AD relative to controls (p = 0.077), MCI (p = 0.016), and oDem (p = 0.07). TCC did not differ between the groups. In the combined sample of subjects with MCI, AD, and oDem, higher levels of C3a correlated with higher levels of TCC (r = 0.51, p < 0.001), SAP (r = 0.40, p < 0.001), and tau (r = 0.18, p = 0.048). Higher levels of C5a correlated with more impairment on memory, executive functioning, and the MMSE (r = 0.22-0.30, p = 0.05-0.002). Higher levels of TCC correlated with higher SAP levels (r = 0.41, p < 0.001) and more functional impairment (r = 0.25, p = 0.018).
1695
Conclusion: The innate immune system is actively involved in neurodegeneration and may provide diagnostic and prognostic information for AD. doi:10.1016/j.molimm.2011.06.310 P91 Identification of Staphylococcus aureus surface protein SdrE as a complement factor H-binding molecule K.M. Cunnion a,∗ , J.A. Sharp a , P.S. Hair a , C.G. Echague a , M.D. Ward a , T.J. Foster b , J.O. Nyalwidhe a a b
Eastern Virginia Medical School, Norfolk, United States Trinity College, Dublin, Ireland
Background and rationale: S. aureus utilizes a repertoire of immune-evasive tactics to circumvent host defenses including the complement system, a crucial component of our innate immune response that is central for controlling S. aureus infections. We have previously shown that S. aureus acquires the complement regulatory protein factor H (fH) to its surface as a means of evading complement attack. Identifying the S. aureus surface protein that binds fH may provide a therapeutic target for blocking this interaction. Methods: To identify S. aureus cell-wall proteins that bind fH, cross-linking of purified fH to the bacterial surface as well as farwestern analysis of cell-wall preparations were performed. Bands evident for fH binding were analyzed via ESI-LC–MS/MS. Putative targets were expressed in recombinant form and assessed for fH binding via dot-blot and modified ELISA using purified fH and heat-inactivated serum. Recombinant proteins were assayed for functionality by capturing factor H and measuring factor I cleavage of C3b to iC3b. Results: SdrE was identified as a putative factor H-binding protein by both overlay and cross-linking methods. Recombinant SdrE bound both purified fH and serum fH efficiently. Increasing amounts of purified fH bound to immobilized rSdrE positively correlated with iC3b generation. Conclusions: These studies provide the first identification of an S. aureus cell-wall protein, SdrE, which binds factor H. Furthermore, rSdrE-bound fH retains cofactor activity for factor I-mediated cleavage of C3b. doi:10.1016/j.molimm.2011.06.311 P92 Hyperglycemic conditions inhibit C3-mediated host defenses against bacterial pathogens and change the tertiary structure of C3 K.M. Cunnion ∗ , P.S. Hair, J.O. Nyalwidhe Eastern Virginia Medical School, Norfolk, United States In diabetics, Staphylococcus aureus is the most common cause of foot infections and polymicrobial limb infections frequently lead to amputation. These studies provide new insight into hyperglycemia altering the interaction between the host defense complement system, S. aureus, and Gram-negative bacteria. Elevated glucose concentrations were found to increase C3 binding to S. aureus 6fold. However, minimal conversion of C3 to the functional forms C3b and iC3b was found to occur on the S. aureus surface. Because the C3 was not covalently bound, the glucose-mediated C3 binding to S. aureus was reversible upon return to normal glucose concentrations. Elevated glucose dramatically increased C3 binding to all clinical S. aureus isolates tested and increased C3 binding