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Borrelia (B.) bavariensis exhibits a marked tropism for nervous tissues and frequently causes neurological manifestations in humans. The molecular mechanism by which B. bavariensis overcomes innate immunity, in particular, complement remains elusive. In contrast to other serum-resistant spirochetes, none of the B. bavariensis isolates investigated bound complement regulators of the alternative (AP) and classical pathway (CP) or proteolytically inactivated complement components. Focusing on outer surface proteins BGA66 and BGA71, we demonstrated that both molecules either inhibit AP, CP and terminal pathway (TP) activation, or block activation of the CP and TP respectively. Both molecules bind complement components C7, C8 and C9, and thereby prevent assembly of the terminal complement complex. This inhibitory activity was confirmed by the introduction of the BGA66 and BGA71 encoding genes into a serum-sensitive B. garinii strain. Transformed spirochetes producing either BGA66 or BGA71 overcome complement-mediated killing, thus indicating that both proteins independently facilitate serum resistance of B. bavariensis. The generation of C-terminally truncated proteins as well as a chimeric BGA71 protein lead to the localization of the complement-interacting binding site within the N-terminus. Collectively, our data reveal a novel immune evasion strategy of B. bavariensis that is directed against the activation of the TP.

Haemophilus influenzae is a Gram-negative human pathogen that resides in the upper respiratory tract. Encapsulated H. influenzae type b (Hib) and type f (Hif) are the most common serotypes associated with invasive disease. H. influenzae displays various strategies to circumvent the host innate immune response, including the bactericidal effect of the complement system. In this study, we identified an H. influenzae lipoprotein having the ability to bind factor H (FH), the major regulator of the alternative pathway of complement activation. This protein, named protein H (PH), was surface exposed and was found in all clinical Hib and Hif isolates tested. Deletion of the gene encoding for PH (lph) in Hib and Hif significantly reduced the interaction between bacteria and FH. When Hib and Hif PH variants were separately expressed in nontypeable (unencapsulated) H. influenzae, which did not bind FH, an increased FH affinity was observed. We recombinantly expressed the two PH variants in Escherichia coli, and despite sharing only 56% identical amino acids, both FH-binding Haemophilus proteins similarly interacted with the complement regulator FH short consensus repeats 7 and 18–20. Importantly, Hib and Hif resistance against the bactericidal effect of human serum was significantly reduced when bacterial mutants devoid of PH were tested. In conclusion, we have characterized a hitherto unknown bacterial protein that is crucial for mediating an interaction between the human pathogen H. influenzae and FH. This novel interaction is important for H. influenzae resistance against complement activation and will consequently promote bacterial pathogenesis.

Haemophilus influenzae type b (Hib) escapes the host immune system by recruitment of the complement regulator vitronectin, which inhibits the formation of the membrane attack complex (MAC) by inhibiting C5b-C7 complex formation and C9 polymerization. We reported previously that Hib acquires vitronectin at the surface by using Haemophilus surface fibrils (Hsf). Here we studied in detail the interaction between Hsf and vitronectin and its role in the inhibition of MAC formation and the invasion of lung epithelial cells. The vitronectin-binding region of Hsf was defined at the N-terminal region comprising Hsf amino acids 429 to 652. Moreover, the Hsf recognition site on vitronectin consisted of the C-terminal amino acids 352 to 374. H. influenzae was killed more rapidly in vitronectin-depleted serum than in normal human serum (NHS), and increased MAC deposition was observed at the surface of an Hsf-deficient H. influenzae mutant. In parallel, Hsf-expressing Escherichia coli selectively acquired vitronectin from serum, resulting in significant inhibition of the MAC. Moreover, when vitronectin was bound to Hsf, increased bacterial adherence and internalization into epithelial cells were observed. Taking our findings together, we have defined a fine-tuned protein-protein interaction between Hsf and vitronectin that may contribute to increased Hib virulence.

CspA of the Lyme disease spirochete Borrelia burgdorferi represents a key molecule in immune evasion, protecting borrelial cells from complement-mediated killing. As previous studies focused almost exclusively on CspA of B. burgdorferi , here we investigate the different binding capacities of CspA orthologs of Borrelia burgdorferi , B. afzelii , and B. spielmanii for complement regulator factor H and plasminogen and their ability to inhibit complement activation by either binding these host-derived plasma proteins or independently by direct interaction with components involved in formation of the lethal, pore-like terminal complement complex. To further examine their function in serum resistance in vivo , a serum-sensitive B. garinii strain was used to generate spirochetes, ectopically producing functional CspA orthologs. Irrespective of their species origin, all three CspA orthologs impart resistance to complement-mediated killing when produced in a serum-sensitive B. garinii surrogate strain. To analyze the inhibitory effect on complement activation and to assess the potential to inactivate C3b by binding of factor H and plasminogen, recombinant CspA orthologs were also investigated. All three CspA orthologs simultaneously bound factor H and plasminogen but differed in regard to their capacity to inactivate C3b via bound plasmin(ogen) and inhibit formation of the terminal complement complex. CspA of B. afzelii binds plasmin(ogen) and inhibits the terminal complement complex more efficiently than CspA of B. burgdorferi and B. spielmanii . Taken together, CspA orthologs of serum-resistant Lyme disease spirochetes act as multifunctional evasion molecules that inhibit complement on two central activation levels, C3b generation and assembly of the terminal complement complex.

Complement is a central homeotic system of mammals and represents the first defense line of innate immunity. The human complement system is aimed to maintain homeostasis by recognizing and removing damaged or modified self material, as well as infectious foreign microbes. However, pathogenic microbes also control and escape the host complement and immune attack. The increasing resistance of microbial pathogens to either antibiotics or antifungal drugs is a major health problem and is of global interest. Therefore the topic how pathogenic microbes escape human complement and immune control is of high and of central interest. Identifying and defining the action of proteins involved in this intense immune interaction and understanding how these proteins interact is of relevance to design new control strategies. In this review we summarize the complement system of the human host and how this cascade drives effector functions. In addition, we summarize how diverse pathogenic microbes control, modulate and block the complement response of their host. The characterization of pathogen derived virulence factors and complement escape proteins reveals patterns of multiplicity, diversity and redundancy among pathogen encoded proteins. Sequence variability of immune and also complement escape proteins is largely driven by antigenic diversity and adaptive immunity. However common complement escape principles are, emerging in terms of conserved binding repertoire for host regulators and evasion among the large variety of infectious microbes. These conserved and common escape features are relevant and they provide challenging options for new therapeutic approaches. (C) 2013 Elsevier Ltd. All rights reserved.

Complement resistance is an important virulence trait of Yersinia enterocolitica (Ye). The predominant virulence factor expressed by Ye is Yersinia adhesin A (YadA), which enables bacterial attachment to host cells and extracellular matrix and additionally allows the acquisition of soluble serum factors. The serum glycoprotein vitronectin (Vn) acts as an inhibitory regulator of the terminal complement complex by inhibiting the lytic pore formation. Here, we show YadA-mediated direct interaction of Ye with Vn and investigated the role of this Vn binding during mouse infection in vivo. Using different Yersinia strains, we identified a short stretch in the YadA head domain of Ye O:9 E40, similar to the 'uptake region' of Y. pseudotuberculosis YPIII YadA, as crucial for efficient Vn binding. Using recombinant fragments of Vn, we found the C-terminal part of Vn, including heparin-binding domain 3, to be responsible for binding to YadA. Moreover, we found that Vn bound to the bacterial surface is still functionally active and thus inhibits C5b-9 formation. In a mouse infection model, we demonstrate that Vn reduces complement-mediated killing of Ye O:9 E40 and, thus, improved bacterial survival. Taken together, these findings show that YadA-mediated Vn binding influences Ye pathogenesis.

The human plasma protein β2-glycoprotein I (β2-GPI) is the major target of autoantibodies associated with antiphospholipid syndrome. However, the biologic function of this abundant protein is still unclear. Here we identify β2-GPI as a complement regulator. β2-GPI circulates in the plasma in an inactive circular form. On surface binding, such as to apoptotic cells, β2-GPI changes conformation to an elongated form that acquires C3/C3b binding activities. β2-GPI apparently changes conformation of C3, so that the regulator factor H attaches and induces subsequent degradation by the protease factor I. β2-GPI also mediates further cleavage of C3/C3b compared with factor H alone. Our data provide important insights into innate immune regulation by plasma protein β2-GPI, which may be exploited in the prevention and therapy of autoimmune disease antiphospholipid syndrome.

The complement system plays an important role in eliminating invading pathogens. Activation of complement results in C3b deposition (opsonization), phagocytosis, anaphylatoxin (C3a, C5a) release, and consequently cell lysis. Moraxella catarrhalis is a human respiratory pathogen commonly found in children with otitis media and in adults with chronic obstructive pulmonary disease. The species has evolved multiple complement evasion strategies, which among others involves the ubiquitous surface protein (Usp) family consisting of UspA1, A2, and A2 hybrid. In the present study, we found that the ability of M. catarrhalis to bind C3 correlated with UspA expression and that C3 binding contributed to serum resistance in a large number of clinical isolates. Recombinantly expressed UspA1 and A2 inhibit both the alternative and classical pathways, C3b deposition, and C3a generation when bound to the C3 molecule. We also revealed that the M. catarrhalis UspA-binding domain on C3b was located to C3d and that the major bacterial C3d-binding domains were within UspA1299–452 and UspA2165–318. The interaction with C3 was not species specific since UspA-expressing M. catarrhalis also bound mouse C3 that resulted in inhibition of the alternative pathway of mouse complement. Taken together, the binding of C3 to UspAs is an efficient strategy of Moraxella to block the activation of complement and to inhibit C3a-mediated inflammation.

Nontypeable Haemophilus influenzae (NTHi) commonly causes local disease in the upper and lower respiratory tract and has recently been shown to interfere with both the classical and alternative pathways of complement activation. The terminal pathway of the complement system is regulated by vitronectin that is a component of both plasma and the extracellular matrix. In this study, we identify protein E (PE; 16 kDa), which is a recently characterized ubiquitous outer membrane protein, as a vitronectin-binding protein of NTHi. A PE-deficient NTHi mutant had a markedly reduced survival in serum compared with the PE-expressing isogenic NTHi wild type. Moreover, the PE-deficient mutant showed a significantly decreased binding to both soluble and immobilized vitronectin. In parallel, PE-expressing Escherichia coli bound soluble vitronectin and adhered to immobilized vitronectin compared with controls. Surface plasmon resonance technology revealed a KD of 0.4 μΜ for the interaction between recombinant PE and immobilized vitronectin. Moreover, the PE-dependent vitronectin-binding site was located at the heparin-binding domains of vitronectin and the major vitronectin-binding domain was found in the central core of PE (aa 84–108). Importantly, vitronectin bound to the surface of NTHi 3655 reduced membrane attack complex-induced hemolysis. In contrast to incubation with normal human serum, NTHi 3655 showed a reduced survival in vitronectin-depleted human serum, thus demonstrating that vitronectin mediates a protective role at the bacterial surface. Our findings show that PE, by binding vitronectin, may play an important role in NTHi pathogenesis.

The immunoglobulin D (IgD)-binding protein MID/Hag of the human respiratory pathogen Moraxella catarrhalis is an outer membrane protein of approximately 200kDa belonging to the autotransporter family. MID also functions as an adhesin and hemagglutinin. In the present paper, the ultrastructure of MID was mapped. Using a series of Escherichia coli transformants, the last 210 aa of the C-terminal region were shown to translocate protein MID through the outer membrane suggesting that MID has a beta-barrel structure comprising of 10 transmembrane beta-sheets. Electron microscopy mapping with gold-labelled specific antibodies, and partial unravelling using guanidine hydrochloride showed that the rest of the MID protein forms an approximately 120nm long, fibrillar structure in which the individual monomers fold back on themselves to expose a globular distal domain at their tips comprising both the IgD-binding (MID962-1200) and adhesive (MID764-913) regions. This positions their N-termini close to the C-terminal membrane spanning domains. Mass measurements by scanning transmission electron microscopy (STEM) verified that the MID molecule is an oligomer.

Pathogenicity of many microbes relies on their capacity to resist innate immunity, and to survive and persist in an immunocompetent human host microbes have developed highly efficient and sophisticated complement evasion strategies. Here we show that different human pathogens including Gram-negative and Gram-positive bacteria, as well as the fungal pathogen Candida albicans, acquire the human terminal complement regulator vitronectin to their surface. By using truncated vitronectin fragments we found that all analyzed microbial pathogens (n = 13) bound human vitronectin via the same C-terminal heparin-binding domain (amino acids 352–374). This specific interaction leaves the terminal complement complex (TCC) regulatory region of vitronectin accessible, allowing inhibition of C5b-7 membrane insertion and C9 polymerization. Vitronectin complexed with the various microbes and corresponding proteins was thus functionally active and inhibited complement-mediated C5b-9 deposition. Taken together, diverse microbial pathogens expressing different structurally unrelated vitronectin-binding molecules interact with host vitronectin via the same conserved region to allow versatile control of the host innate immune response.

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 IgG 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 CH1 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 CH1 region.

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 α-chains and one β-chain linked together with disulfide bridges. Each α-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.

Borrelia (B.) bavariensis exhibits a marked tropism for nervous tissues and frequently causes neurological manifestations in humans. The molecular mechanism by which B. bavariensis overcomes innate immunity, in particular, complement remains elusive. In contrast to other serum-resistant spirochetes, none of the B. bavariensis isolates investigated bound complement regulators of the alternative (AP) and classical pathway (CP) or proteolytically inactivated complement components. Focusing on outer surface proteins BGA66 and BGA71, we demonstrated that both molecules either inhibit AP, CP and terminal pathway (TP) activation, or block activation of the CP and TP respectively. Both molecules bind complement components C7, C8 and C9, and thereby prevent assembly of the terminal complement complex. This inhibitory activity was confirmed by the introduction of the BGA66 and BGA71 encoding genes into a serum-sensitive B. garinii strain. Transformed spirochetes producing either BGA66 or BGA71 overcome complement-mediated killing, thus indicating that both proteins independently facilitate serum resistance of B. bavariensis. The generation of C-terminally truncated proteins as well as a chimeric BGA71 protein lead to the localization of the complement-interacting binding site within the N-terminus. Collectively, our data reveal a novel immune evasion strategy of B. bavariensis that is directed against the activation of the TP.

The opportunistic human pathogen Pseudomonas aeruginosa controls host innate immune and complement attack. Here we identify Dihydrolipoamide dehydrogenase (Lpd), a 57 kDa moonlighting protein, as the first P. aeruginosa protein that binds the two human terminal pathway inhibitors vitronectin and clusterin. Both human regulators when bound to the bacterium inhibited effector function of the terminal complement, blocked C5b-9 deposition and protected the bacterium from complement damage. P. aeruginosa when challenged with complement active human serum depleted from vitronectin was severely damaged and bacterial survival was reduced by over 50%. Similarly, when in human serum clusterin was blocked by a mAb, bacterial survival was reduced by 44%. Thus, demonstrating that Pseudomonas benefits from attachment of each human regulator and controls complement attack. The Lpd binding site in vitronectin was localized to the C-terminal region, i.e. to residues 354-363. Thus, Lpd of P. aeruginosa is a surface exposed moonlighting protein that binds two human terminal pathway inhibitors, vitronectin and clusterin and each human inhibitor when attached protected the bacterial pathogen from the action of the terminal complement pathway. Our results showed insights into the important function of Lpd as a complement regulator binding protein that might play an important role in virulence of P. aeruginosa.

Allergies are an increasing issue in human health and can, eventually, cause severe anaphylactic shock. Aspergillus fumigatus and Candida albicans are leading causes of life-threatening invasive fungal infections in immunocompromised patients, but can also cause severe allergic responses in otherwise healthy individuals. The glycolytic enzyme enolase is known as a major allergen despite its function in intracellular metabolism. Therefore, its presentation on surfaces of different fungal species was investigated by using antibodies raised against recombinant enolases from A. fumigatus and C. albicans. Examination of antibody specificity revealed cross-reactivity to cell-free extracts from Aspergillus terreus, Aspergillus flavus, Aspergillus nidulans and Candida glabrata, but not against any of the three human enolases. Antibody specificity was further confirmed by hybridization with other recombinant fungal enolases, where the antibodies recognized different subsets of fungal enolases. When surface presentation of enolase was tested on intact fungal cells, a positive staining was obtained with those antibodies that also recognized the enzyme from the respective cell-free extract. This implies a general surface presentation of this glycolytic enzyme among fungal species and provides hints for its predominant recognition as an allergen. Additionally, A. fumigatus and C. albicans enolase bound to human plasminogen, which remained accessible for the plasminogen activator uPA. This implies a potential role of enolase in the invasion and dissemination process during fungal infections.

SummaryStreptococcus pneumoniae serotype 3 strains are highly resistant to opsonophagocytosis due to recruitment of the complement inhibitor Factor H via Hic, a member of the pneumococcal surface protein C (PspC) family. In this study, we demonstrated that Hic also interacts with vitronectin, a fluid-phase regulator involved in haemostasis, angiogenesis, and the terminal complement cascade as well as a component of the extracellular matrix. Blocking of Hic by specific antiserum or genetic deletion significantly reduced pneumococcal binding to soluble and immobilised vitronectin and to Factor H, respectively. In parallel, ectopic expression of Hic on the surface of Lactococcus lactis conferred binding to soluble and immobilised vitronectin as well as Factor H. Molecular analyses with truncated Hic fragments narrowed down the vitronectin-binding site to the central core of Hic (aa 151–201). This vitronectin-binding region is separate from that of Factor H, which binds to the N-terminus of Hic (aa 38–92). Binding of pneumococcal Hic was localised to the C-terminal heparin-binding domain (HBD3) of vitronectin. However, an N-terminal region to HBD3 was further involved in Hic-binding to immobilised vitronectin. Finally, vitronectin bound to Hic was functionally active and inhibited formation of the terminal complement complex. In conclusion, Hic interacts with vitronectin and simultaneously with Factor H, and both human proteins may contribute to colonisation and invasive disease caused by serotype 3 pneumococci.

Streptococcus pneumoniae is a Gram-positive bacterium, causing acute sinusitis, otitis media, and severe diseases such as pneumonia, bacteraemia, meningitis and sepsis. Here we identify elongation factor Tu (Tuf) as a new Factor H binding protein of S. pneumoniae. The surface protein PspC which also binds a series of other human immune inhibitors, was the first identified pneumococcal Factor H binding protein of S. pneumoniae. Pneumococcal Tuf, a 55 kDa pneumococcal moonlighting protein which is displayed on the surface of pneumococci, is also located in the cytoplasm and is detected in the culture supernatant. Tuf binds the human complement inhibitors Factor H, FHL-1, CFHR1 and also the proenzyme plasminogen. Factor H and FHL-1 bound to Tuf, retain their complement regulatory activities. Similarly, plasminogen bound to Tuf was accessible for the activator uPA and activated plasmin cleaved the synthetic chromogenic substrate S-2251 as well as the natural substrates fibrinogen and the complement proteins C3 and C3b. Taken together, Tuf of S. pneumoniae is a new multi-functional bacterial virulence factor that helps the pathogen in complement escape and likely also in ECM degradation.

Adherence of Streptococcus pneumoniae is directly mediated by interactions of adhesins with eukaryotic cellular receptors or indirectly by exploiting matrix and serum proteins as molecular bridges. Pneumococci engage vitronectin, the human adhesive glycoprotein and complement inhibitor, to facilitate attachment to epithelial cells of the mucosal cavity, thereby modulating host cell signaling. In this study, we identified PspC as a vitronectin-binding protein interacting with the C-terminal heparin-binding domain of vitronectin. PspC is a multifunctional surface-exposed choline-binding protein displaying various adhesive properties. Vitronectin binding required the R domains in the mature PspC protein, which are also essential for the interaction with the ectodomain of the polymeric immunoglobulin receptor and secretory IgA. Consequently, secretory IgA competitively inhibited binding of vitronectin to purified PspC and to PspC-expressing pneumococci. In contrast, Factor H, which binds to the N-terminal part of mature PspC molecules, did not interfere with the PspC-vitronectin interaction. Using a series of vitronectin peptides, the C-terminal heparin-binding domain was shown to be essential for the interaction of soluble vitronectin with PspC. Binding experiments with immobilized vitronectin suggested a region N-terminal to the identified heparin-binding domain as an additional binding region for PspC, suggesting that soluble, immobilized, as well as cellularly bound vitronectin possesses different conformations. Finally, vitronectin bound to PspC was functionally active and inhibited the deposition of the terminal complement complex. In conclusion, this study identifies and characterizes (on the molecular level) the interaction between the pneumococcal adhesin PspC and the human glycoprotein vitronectin.

Mutations and deletions within the human CFHR gene cluster on chromosome 1 are associated with diseases, such as dense deposit disease, CFHR nephropathy or age-related macular degeneration. Resulting mutant CFHR proteins can affect complement regulation. Here we identify human CFHR2 as a novel alternative pathway complement regulator that inhibits the C3 alternative pathway convertase and terminal pathway assembly. CFHR2 is composed of four short consensus repeat domains (SCRs). Two CFHR2 molecules form a dimer through their N-terminal SCRs, and each of the two C-terminal ends can bind C3b. C3b bound CFHR2 still allows C3 convertase formation but the CFHR2 bound convertases do not cleave the substrate C3. Interestingly CFHR2 hardly competes off factor H from C3b. Thus CFHR2 likely acts in concert with factor H, as CFHR2 inhibits convertases while simultaneously allowing factor H assisted degradation by factor I.

In order to survive and persist in an immunocompetent human host, Borrelia burgdorferi controls the human immune attack and blocks the damaging effects of the activated complement system. These Gram-negative spirochetes use CspA (CRASP-1) and four additional immune evasion proteins to bind combinations of human plasma regulators, including factor H, factor H-like protein 1 (FHL-1), complement factor H-related protein 1 (CFHR1), CFHR2, CFHR5, and plasminogen. As many microbial immune evasion proteins have multiple functions, we hypothesized that CspA has additional roles in complement or immune control. Here, we identify CspA as a terminal complement inhibitor. Borrelial CspA binds the human terminal complement components C7 and C9 and blocks assembly and membrane insertion of the terminal complement complex (TCC). CspA inhibits TCC assembly at the level of C7, as revealed by hemolytic assays, and inhibits polymerization of C9. CspA, when ectopically expressed on the surface of serum-sensitive Borrelia garinii, blocks TCC assembly on the level of C7 and induces serum resistance in the transformed bacteria. This CspA-mediated serum resistance and terminal complement pathway inhibition allow B. burgdorferi to survive in the hostile environment of human plasma., IMPORTANCE The present study defines a new mechanism by which the pathogenic bacterium Borrelia burgdorferi controls the terminal complement pathway of the human host to survive in human serum. The borrelial CspA binds to terminal pathway proteins C7 and C9 and inhibits the terminal complement pathway at the step of C7 and thereby inhibits terminal complement complex (TCC) assembly and membrane insertion. CspA blocks TCC assembly and insertion when expressed at the bacterial surface. CspA is the first TCC inhibitor cloned and functionally characterized from a Gram-negative bacterium. This identification of a bacterial TCC inhibitor of pathogen origin expands our knowledge of complement evasion of pathogenic bacteria and shows that pathogenic bacteria target the terminal pathway of complement. Thus, CspA as a central microbial virulence factor can represent an interesting biomarker and a target to develop new therapeutics and vaccines against borreliae.

The opportunistic human pathogen Pseudomonas aeruginosa causes a wide range of diseases. To cross host innate immune barriers, P. aeruginosa has developed efficient strategies to escape host complement attack. In this study, we identify the 57-kDa dihydrolipoamide dehydrogenase (Lpd) as a surface-exposed protein of P. aeruginosa that binds the four human plasma proteins, Factor H, Factor H-like protein-1 (FHL-1), complement Factor H-related protein 1 (CFHR1), and plasminogen. Factor H contacts Lpd via short consensus repeats 7 and 18–20. Factor H, FHL-1, and plasminogen when bound to Lpd were functionally active. Factor H and FHL-1 displayed complement-regulatory activity, and bound plasminogen, when converted to the active protease plasmin, cleaved the chromogenic substrate S-2251 and the natural substrate fibrinogen. The lpd of P. aeruginosa is a rather conserved gene; a total of 22 synonymous and 3 nonsynonymous mutations was identified in the lpd gene of the 5 laboratory strains and 13 clinical isolates. Lpd is surface exposed and contributes to survival of P. aeruginosa in human serum. Bacterial survival was reduced when Lpd was blocked on the surface prior to challenge with human serum. Similarly, bacterial survival was reduced up to 84% when the bacteria was challenged with complement active serum depleted of Factor H, FHL-1, and CFHR1, demonstrating a protective role of the attached human regulators from complement attack. In summary, Lpd is a novel surface-exposed virulence factor of P. aeruginosa that binds Factor H, FHL-1, CFHR1, and plasminogen, and the Lpd-attached regulators are relevant for innate immune escape and most likely contribute to tissue invasion.

Yersinia adhesin A (YadA) is a major virulence factor of Yersinia enterocolitica. YadA mediates host cell binding and autoaggregation and protects the pathogen from killing by the complement system. Previous studies demonstrated that YadA is the most important single factor mediating serum resistance of Y. enterocolitica, presumably by binding C4b binding protein (C4BP) and factor H, which are both complement inhibitors. Factor H acts as a cofactor for factor I-mediated cleavage of C3b into the inactive form iC3b and thus prevents formation of inflammatory effector compounds and the terminal complement complex. In this study, we challenged the current direct binding model of factor H to YadA and show that Y. enterocolitica YadA recruits C3b and iC3b directly, without the need of an active complement cascade or additional serum factors. Enhanced binding of C3b does not decrease survival of YadA-expressing Yersiniae because C3b becomes readily inactivated by factor H and factor I. Binding of factor H to YadA is greatly reduced in the absence of C3. Experiments using Yersinia lacking YadA or expressing YadA with reduced trimeric stability clearly demonstrate that both the presence and full trimeric stability of YadA are essential for complement resistance. A novel mechanism of factor H binding is presented in which YadA exploits recruitment of C3b or iC3b to attract large amounts of factor H. As a consequence, formation of the terminal complement complex is limited and bacterial survival is enhanced. These findings add a new aspect of how Y. enterocolitica effectively evades the host complement system.

Borrelia burgdorferievades 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 ofB. burgdorferiand 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, aB. gariniistrain 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 ofB. burgdorferi.

Nontypeable Haemophilus influenzae (NTHi) causes otitis media and is commonly found in patients with chronic obstructive pulmonary disease (COPD). Adhesins are important for bacterial attachment and colonization. Protein E (PE) is a recently characterized ubiquitous 16 kDa adhesin with vitronectin-binding capacity that results in increased survival in serum. In addition to PE, NTHi utilizes Haemophilus adhesion protein (Hap) that binds to the basement-membrane glycoprotein laminin. We show that most clinical isolates bind laminin and that both Hap and PE are crucial for the NTHi-dependent interaction with laminin as revealed with different mutants. The laminin-binding region is located at the N-terminus of PE, and PE binds to the heparin-binding C-terminal globular domain of laminin. PE simultaneously attracts vitronectin and laminin at separate binding sites, proving the multifunctional nature of the adhesin. This previously unknown PE-dependent interaction with laminin may contribute to NTHi colonization, particularly in smokers with COPD.

Lyme disease-causing Borrelia burgdorferi spirochetes express up to 5 complement regulator-acquiring surface proteins (CRASPs). To better define how CRASP-1 contributes to infection, we aimed to identify novel CRASP-1-binding host proteins. Here, we identified a number of novel human CRASP-1-binding proteins, including bone morphogenic protein 2, collagen I, collagen III, collagen IV, fibronectin, laminin, and plasminogen. The plasminogen-binding regions were located in 2 separate regions of CRASP-1. Our results demonstrated that plasminogen-bound CRASP-1 can be converted to plasmin by the urokinase-type plasminogen activator and that proteolytically active plasmin cleaves the synthetic chromogenic substrate S-2251 and the natural substrate fibrinogen. In conclusion, CRASP-1 is a multifunctional protein of B. burgdorferi that binds to several human extracellular matrix proteins and plasminogen. These interactions may contribute to adhesion, bacterial colonization, and organ tropism and may allow dissemination of B. burgdorferi in the host.

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.

Yersinia adhesin A (YadA) is a trimeric autotransporter adhesin with multiple functions in host-pathogen interactions. The aim of this study was to dissect the virulence functions promoted by YadA in vitro and in vivo . To accomplish this, we generated Yersinia enterocolitica O:8 mutants expressing point mutations in YadA G389, a highly conserved residue in the membrane anchor of YadA, and analyzed their impact on YadA expression and virulence functions. We found that point mutations of YadA G389 led to impaired transport, stability, and surface display of YadA. YadA G389A and G389S mutants showed comparable YadA surface expression, autoagglutination, and adhesion to those of wild-type YadA but displayed reduced trimer stability and complement resistance in vitro and were 10- to 1,000-fold attenuated in experimental Y. enterocolitica infection in mice. The G389T, G389N, and G389H mutants lost trimer stability, exhibited strongly reduced surface display, autoagglutination, adhesion properties, and complement resistance, and were avirulent (>10,000-fold attenuation) in mice. Our data demonstrate that G389 is a critical residue of YadA, required for optimal trimer stability, transport, surface display, and serum resistance. We also show that stable trimeric YadA protein is essential for virulence of Y. enterocolitica .

The aim of the present study was to analyze the importance of nontypeable Haemophilus influenzae (NTHi) isolated from patients with sepsis (invasive isolates) compared to nasopharyngeal isolates from patients with upper respiratory tract infection for resistance to complement-mediated attack in human serum and to correlate this result with disease severity. We studied and characterized cases of invasive NTHi disease in detail. 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 patients had evidence of immune deficiency. The different isolates were analyzed for survival in human serum and 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-derived and nasopharyngeal isolates were compared, suggesting that interactions with the complement system are equally important for NTHi strains, irrespective 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 bloodstream.

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.

Factor H is the central regulator of the alternative complement pathway and controls early activation of the complement cascade at the level of the C3 convertase. Mutations in the Factor H gene are associated with severe and diverse diseases including the rare renal disorders hemolytic uremic syndrome (HUS) and membranoproliferative glomerulonephritis (MPGN) also termed dense deposit disease (DDD), as well as the more frequent retinal disease age related macular degeneration (AMD). In addition, pathogenic microbes utilize host complement Factor H for immune evasion and these pathogens express specific surface receptors which bind host innate immune regulators. Sequence variations or mutations of one single gene, coding for the host regulator Factor H, form the basis for multiple, different disorders such as human renal and retinal diseases as well as infections. This association of Factor H but also of additional related complement components and regulators with the same diseases demonstrate an important role of complement, particularly of the alternative pathway, for tissue homeostasis. Disturbances of this central immune surveillance system lead to damage of autologous tissues and surfaces and result in autoimmune diseases. Remarkably, pathogenic microbes copy this mechanism of immune surveillance: they mimic the composition of host cell's, bind Factor H to their surface and engage acquired host Factor H for immune disguise.

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.

Pathogenic microbes acquire human complement inhibitors to circumvent the innate immune system. In this study, we identify two novel host-pathogen interactions, factor H (FH) and factor H-like protein 1 (FHL-1), the inhibitors of the alternative pathway that binds to Hib. A collection of clinical Haemophilus influenzae isolates was tested and the majority of encapsulated and unencapsulated bound FH. The isolate Hib 541 with a particularly high FH-binding was selected for detailed analysis. An increased survival in normal human serum was observed with Hib 541 as compared with the low FH-binding Hib 568. Interestingly, two binding domains were identified within FH; one binding site common to both FH and FHL-1 was located in the N-terminal short consensus repeat domains 6–7, whereas the other, specific for FH, was located in the C-terminal short consensus repeat domains 18–20. Importantly, both FH and FHL-1, when bound to the surface of Hib 541, retained cofactor activity as determined by analysis of C3b degradation. Two H. influenzae outer membrane proteins of ∼32 and 40 kDa were detected with radiolabeled FH in Far Western blot. Taken together, in addition to interactions with the classical, lectin, and terminal pathways, H. influenzae interferes with the alternative complement activation pathway by binding FH and FHL-1, and thereby reducing the complement-mediated bactericidal activity resulting in an increased survival. In contrast to incubation with active complement, H. influenzae had a reduced survival in FH-depleted human serum, thus demonstrating that FH mediates a protective role at the bacterial surface.

Vitronectin inhibits the membrane attack complex of the complement system and is found both in plasma and the extracellular matrix. In this study, we have identified the outer membrane protein Haemophilus surface fibrils (Hsf) as the major vitronectin-binding protein in encapsulated H. influenzae type b. A H. influenzae mutant devoid of Hsf showed a significantly decreased binding to both soluble and immobilized vitronectin as compared with the wild-type counterpart. Moreover, Escherichia coli-expressing Hsf at the surface strongly adhered to immobilized vitronectin. Importantly, the H. influenzae Hsf mutant had a markedly reduced survival as compared with the wild-type bacterium when incubated with normal human serum. A series of truncated Hsf fragments were recombinantly manufactured in E. coli. The vitronectin binding regions were located within two separate binding domains. In conclusion, Hsf interacts with vitronectin and thereby inhibits the complement-mediated bactericidal activity, and thus is a major H. influenzae virulence factor.