Contribution of the Infection-Associated Complement Regulator-Acquiring Surface Protein 4 (ErpC) to Complement Resistance of \u3cem\u3eBorrelia Burgdorferi\u3c/em\u3e

Borrelia burgdorferi evades complement-mediated killing by interacting with complement regulators through distinct complement regulator-acquiring surface proteins (CRASPs). Here, we extend our analyses to the contribution of CRASP-4 in mediating complement resistance of B. burgdorferi and its interaction with human complement regulators. CRASP-4 (also known as ErpC) was immobilized onto magnetic beads and used to capture proteins from human serum. Following Western blotting, factor H (CFH), CFH-related protein 1 (CFHR1), CFHR2, and CFHR5 were identified as ligands of CRASP-4. To analyze the impact of native CRASP-4 on mediating survival of serum-sensitive cells in human serum, a B. garinii strain was generated that ectopically expresses CRASP-4. CRASP-4-producing bacteria bound CFHR1, CFHR2, and CFHR5 but not CFH. In addition, transformed spirochetes deposited significant amounts of lethal complement components on their surface and were susceptible to human serum, thus indicating that CRASP-4 plays a subordinate role in complement resistance of B. burgdorferi

Contribution of the Infection-Associated Complement Regulator-Acquiring Surface Protein 4 (ErpC) to Complement Resistance of Borrelia burgdorferi

Borrelia burgdorferi evades complement-mediated killing by interacting with complement regulators through distinct complement regulator-acquiring surface proteins (CRASPs). Here, we extend our analyses to the contribution of CRASP-4 in mediating complement resistance of B. burgdorferi and its interaction with human complement regulators. CRASP-4 (also known as ErpC) was immobilized onto magnetic beads and used to capture proteins from human serum. Following Western blotting, factor H (CFH), CFH-related protein 1 (CFHR1), CFHR2, and CFHR5 were identified as ligands of CRASP-4. To analyze the impact of native CRASP-4 on mediating survival of serum-sensitive cells in human serum, a B. garinii strain was generated that ectopically expresses CRASP-4. CRASP-4-producing bacteria bound CFHR1, CFHR2, and CFHR5 but not CFH. In addition, transformed spirochetes deposited significant amounts of lethal complement components on their surface and were susceptible to human serum, thus indicating that CRASP-4 plays a subordinate role in complement resistance of B. burgdorferi

Complement Factor H-Related Proteins CFHR2 and CFHR5 Represent Novel Ligands for the Infection-Associated CRASP Proteins of Borrelia burgdorferi

Background: One virulence property of Borrelia burgdorferi is its resistance to innate immunity, in particular to complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASP) which interact with complement regulator factor H (CFH) and factor H-like protein 1 (FHL1) or factor H-related protein 1 (CFHR1). In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi. Methodology/Principal Findings: To elucidate whether CRASP-5 and CRASP-3 interact with various human proteins, both borrelial proteins were immobilized on magnetic beads. Following incubation with human serum, bound proteins were eluted and separated by Glycine-SDS-PAGE. In addition to CFH and CFHR1, complement regulators CFHR2 and CFHR5 were identified as novel ligands for both borrelial proteins by employing MALDI-TOF. To further assess the contributions of CRASP-3 and CRASP-5 to complement resistance, a serum-sensitive B. garinii strain G1 which lacks all CFH-binding proteins was used as a valuable model for functional analyses. Both CRASPs expressed on the B. garinii outer surface bound CFH as well as CFHR1 and CFHR2 in ELISA. In contrast, live B. garinii bound CFHR1, CFHR2, and CFHR5 and only miniscute amounts of CFH as demonstrated by serum adsorption assays and FACS analyses. Further functional analysis revealed that upon NHS incubation, CRASP-3 or CRASP-5 expressing borreliae were killed by complement. Conclusions/Significance: In the absence of CFH and the presence of CFHR1, CFHR2 and CFHR5, assembly and integration of the membrane attack complex was not efficiently inhibited indicating that CFH in co-operation with CFHR1, CFHR2 and CFHR5 supports complement evasion of B. burgdorferi

Survival strategies of the human respiratory tract pathogen Haemophilus influenzae

Haemophilus influenzae is an important respiratory tract pathogen responsible for a variety of infections in humans. Encapsulated H. influenzae belongs to one of six serotypes (a-f), of which type b is the most virulent one causing serious and sometimes life-threatening diseases (e.g., epiglottitis, septicaemia and meningitis). In contrast, non-typeable H. influenzae (NTHi) accounts for the majority of local and upper and lower respiratory tract infections. The pathogenesis of many microorganisms relies on the capacity of pathogens to avoid, resist or neutralise the host defence including the complement system. We demonstrate that H. influenzae interferes with both the classical/lectin and alternative pathways of the complement system. NTHi binds C4BP, the inhibitor of the classical pathway, and the majority of the H. influenzae tested bound factor H, the inhibitor of the alternative pathway. Importantly, the capacity to bind C4BP and factor H appears to render the bacteria more resistant to serum mediated killing. Furthermore, both C4BP and factor H bound to the surface of H. influenzae retains its cofactor activity as determined by analysis of C4b and/or C3b degradation. In addition to interacting with the classical/lectin and alternative pathways, we demonstrate that Haemophilus surface fibrils (Hsf), which is expressed by encapsulated H. influenzae, binds vitronectin, a regulator of the terminal pathway of the complement system. Mapping of the membrane bound Hsf with gold-labelled specific antibodies in transmission electron microscopy (TEM) revealed a double-folded 100 nm long fibrillar structure. Using a series of mutants, we showed that when the C-terminal translocator domain was inactivated, Hsf was not translocated to the bacterial surface. Interestingly, we also show that outer membrane vesicles (OMV) secreted by the bacteria carry Hsf, and that Hsf is secreted into the extracellular milieu. IgD-binding is another important feature of encapsulated H. influenzae type b. By using a series of different IgD chimeric proteins, the site on the IgD molecule responsible for the interaction with H. influenzae was characterised. The binding site was localised to the CH1 region of IgD. In summary, H. influenzae binds C4BP, factor H and vitronectin, which are regulators of the complement system. The interaction between H. influenzae and these regulators protects the bacteria and makes them more resistant to the bactericidal activity of human serum. Finally, H. influenzae type b binds human IgD

Haemophilus influenzae and the complement system.

The respiratory tract pathogen Haemophilus influenzae is responsible for a variety of infections in humans including septicemia, bronchitis, pneumonia, and acute otitis media. The pathogenesis of H. influenzae relies on its capacity to resist human host defenses including the complement system, and thus H. influenzae has developed several efficient strategies to circumvent complement attack. In addition to attracting specific host complement regulators directly to the bacterial surface, the capsule, lipooligosaccharides, and several outer membrane proteins contribute to resistance against complement-mediated attacks and hence increased bacterial survival. Insights into the mechanisms of complement evasion by H. influenzae are important for understanding pathogenesis and for developing vaccines and new therapies aimed at patients with, for example, chronic obstructive pulmonary disease. Here we overview current knowledge on the different mechanisms by which H. influenzae evades attack by the host complement system

Complement evasion strategies of pathogens-Acquisition of inhibitors and beyond.

Activation of the complement system and resulting opsonisation with C3b are key events of the innate immune defense against infections. However, a wide variety of bacterial pathogens subvert complement attack by binding host complement inhibitors such as C4b-binding protein, factor H and vitronectin, which results in diminished opsonophagocytosis and killing of bacteria by lysis. Another widely used strategy is production of proteases, which can effectively degrade crucial complement components. Furthermore, bacterial pathogens such as Moraxella catarrhalis and Staphylococcus aureus capture and incapacitate the key complement component C3. The current review describes examples of these three strategies. Targeting binding sites for complement inhibitors on bacterial surfaces and complement-degrading proteases with vaccine-induced antibodies may be used to enhance a common vaccine design strategy that depends on the generation of complement-dependent bactericidal and opsonophagocytic antibody activities

Characterization of the IgD binding site of encapsulated Haemophilus influenzae serotype b.

Encapsulated Haemophilus influenzae is a causative agent of invasive disease, such as meningitis and septicemia. Several interactions exist between H. influenzae and the human host. H. influenzae has been reported to bind IgD in a nonimmune manner, but the responsible protein has not yet been identified. To define the binding site on IgD for H. influenzae, full-length IgD and four chimeric IgDs with interspersed TgG sequences and Ag specificity for dansyl chloride were expressed in stably transfected Chinese hamster ovary cells. The binding of recombinant IgD to a panel of encapsulated H. influenzae serotype b (Hib) and nontypeable strains were investigated using a whole cell ELISA and flow cytometry. IgD binding was detected in 50% of the encapsulated Hib strains examined, whereas nontypeable H. influenzae did not interact with IgD. Finally, mapping experiments using the chimeric IgD/IgG indicated that IgD C(H)1 aa 198-224 were involved in the interaction between IgD and H. influenzae. Thus, by using recombinant IgD and chimeras with defined Ag specificity, we have confirmed that Hib specifically binds IgD, and that this binding involves the IgD CHI region

Binding of complement regulators to invasive nontypeable Haemophilus influenzae is not increased compared to nasopharyngeal isolates, but serum resistance is linked to disease severity.

The aim of the present study was to analyse the importance for non-typeable Haemophilus influenzae (NTHi) isolated from patients with sepsis (invasive isolates) compared to nasopharyngeal isolates from patients with upper respiratory tract infection to resist the complement-mediated attack in human serum and to correlate this to disease severity. We in detail studied and characterized cases of invasive NTHi disease. All patients with invasive NTHi isolates were adults and 35 % had a clinical presentation of severe sepsis according to the ACCP/SCCM classification of sepsis grading. Moreover, 41 % of the cases had evidence of immune deficiency. The different isolates were analyzed for survival in human serum, for binding of [(125)I]-labeled purified human complement inhibitors C4b-binding protein (C4BP), Factor H and vitronectin in addition to binding of regulators directly from serum. No significant differences were found when blood and nasopharyngeal isolates were compared, suggesting that interactions with the complement system are equally important for NTHi strains irrespectively of isolation site. Interestingly, a correlation between serum resistance and invasive disease severity was found. The ability to resist the attack of the complement system seems to be important for NTHi strains infecting the respiratory tract as well as the blood stream

Interaction with C4b-binding protein contributes to nontypeable Haemophilus influenzae serum resistance.

Complement evasion by various mechanisms is important for microbial virulence and survival in the host. One strategy used by some pathogenic bacteria is to bind the complement inhibitor of the classical pathway, C4b-binding protein (C4BP). In this study, we have identified a novel interaction between nontypeable Haemophilus influenzae (NTHi) and C4BP, whereas the majority of the typeable H. influenzae (a-f) tested showed no binding. One of the clinical isolates, NTHi 506, displayed a particularly high binding of C4BP and was used for detailed analysis of the interaction. Importantly, a low C4BP-binding isolate (NTHi 69) showed an increased deposition of C3b followed by reduced survival as compared with NTHi 506 when exposed to normal human serum. The main isoform of C4BP contains seven identical a-chains and one beta-chain linked together with disulfide bridges. Each a-chain is composed of eight complement control protein (CCP) modules and we have found that the NTHi 506 strain did not interact with rC4BP lacking CCP2 or CCP7 showing that these two CCPs are important for the binding. Importantly, C4BP bound to the surface of H. influenzae retained its cofactor activity as determined by analysis of C3b and C4b degradation. Taken together, NTHi interferes with the classical complement activation pathway by binding to C4BP