The hyphal and yeast forms of Candida albicans bind the complement regulator C4b-binding protein

Candida albicans, an important pathogenic yeast, activates all three pathways of the complement system. To understand how this yeast evades the effects of the activated system, we have analyzed the binding of the classical pathway inhibitor C4b-binding protein (C4BP) by C. albicans. Purified native as well as recombinant C4BP bound dose dependently to the yeast and hyphal forms, as shown by multiple methods, such as confocal microscopy, flow cytometry, a novel enzyme-linked immunosorbent assay, absorption from human serum, and direct binding assays with purified proteins. A prominent binding site was identified at the tip of the germ tube, a structure that is considered important for tissue penetration and pathogenesis. The binding site in C4BP was localized to the two N-terminal complement control protein domains by using recombinant deletion constructs and site-specific monoclonal antibodies. As the alternative pathway inhibitors factor H and FHL-1 also bind to C. albicans, the binding of all three plasma proteins was compared. Simultaneous binding of the classical regulator C4BP and the alternative pathway regulator factor H was demonstrated by confocal microscopy. In addition, FHL-1 competed for binding with C4BP, suggesting that these two related complement regulators bind to the same structures on the yeast surface. The surface-attached C4BP maintains its complement regulatory activities and inactivates C4b. The surface-attached human C4BP serves multiple functions relevant for immune evasion and likely pathogenicity. It inhibits complement activation at the yeast surface and, in addition, mediates adhesion of C. albicans to host endothelial cells.     

The opportunistic human pathogenic fungus Aspergillus fumigatus evades the host complement system

The opportunistic human pathogenic fungus Aspergillus fumigatus causes severe systemic infections and is a major cause of fungal infections in immunocompromised patients. A. fumigatus conidia activate the alternative pathway of the complement system. In order to assess the mechanisms by which A. fumigatus evades the activated complement system, we analyzed the binding of host complement regulators to A. fumigatus. The binding of factor H and factor H-like protein 1 (FHL-1) from human sera to A. fumigatus conidia was shown by adsorption assays and immunostaining. In addition, factor H-related protein 1 (FHR-1) bound to conidia. Adsorption assays with recombinant factor H mutants were used to localize the binding domains. One binding region was identified within N-terminal short consensus repeats (SCRs) 1 to 7 and a second one within C-terminal SCR 20. Plasminogen was identified as the fourth host regulatory molecule that binds to A. fumigatus conidia. In contrast to conidia, other developmental stages of A. fumigatus, like swollen conidia or hyphae, did not bind to factor H, FHR-1, FHL-1, and plasminogen, thus indicating the developmentally regulated expression of A. fumigatus surface ligands. Both factor H and plasminogen maintained regulating activity when they were bound to the conidial surface. Bound factor H acted as a cofactor to the factor I-mediated cleavage of C3b. Plasminogen showed proteolytic activity when activated to plasmin by urokinase-type plasminogen activator. These data show that A. fumigatus conidia bind to complement regulators, and these bound host regulators may contribute to evasion of a host complement attack.     

Immune evasion of Borrelia burgdorferi by acquisition of human complement regulators FHL-1/reconectin and Factor H

To understand immune evasion mechanisms of Borrelia burgdorferi we compared serum-resistant B. afzelii and serum-sensitive B. garinii isolates for their capacity toacquire human complement regulators. Here we demonstrate that the two borrelial genospecies show different binding of the two important human complement regulators, FHL-1/reconectin and Factor H. All serum-resistant B. afzelii isolates bound FHL-1/reconectin and also Factor H, and all analyzed serum-sensitive B. garinii isolates showed no or a significantly lower binding activity. Using recombinant deletion mutants, the binding domains were localized to the C terminus of FHL-1/reconectin to short consensus repeats 5-7. The borrelial binding proteins were located in the surface of the bacteria as demonstrated by immunofluorescence staining of intact, serum-exposed bacteria and by enrichment of outer membrane proteins. The surface-attached complement regulators maintained complement regulatory activity as demonstrated in a cofactor assay. By ligand blotting two different borrelial binding proteins were identified that were responsible for the surface attachment of FHL-1/reconectin and Factor H. These borrelial complement regulators acquiring surface proteins (CRASP) were further characterized as either CRASP-1, a 27.5-kDa molecule which preferentially binds FHL-1/reconectin and which was present in all serum-resistant borreliae, or CRASP-2, a 20/21-kDa protein which interacts preferentially with Factor H and the expression of which was more restricted, being detected in four of the six isolates analyzed. In summary, we describe a new immune evasion mechanism of B. burgdorferi, as these bacteria acquire human complement regulators to control complement activation on their surface and to prevent formation of toxic activation products.     

Immune evasion by acquisition of complement inhibitors: the mould Aspergillus binds both factor H and C4b binding protein

Pathogenic fungi represent a major threat particularly to immunocompromised hosts, leading to severe, and often lethal, systemic opportunistic infections. Although the impaired immune status of the host is clearly the most important factor leading to disease, virulence factors of the fungus also play a role. Factor H (FH) and its splice product FHL-1 represent the major fluid phase inhibitors of the alternative pathway of complement, whereas C4b-binding protein (C4bp) is the main fluid phase inhibitor of the classical and lectin pathways. Both proteins can bind to the surface of various human pathogens conveying resistance to complement destruction and thus contribute to their pathogenic potential. We have recently shown that Candida albicans evades complement by binding both Factor H and C4bp. Here we show that moulds such as Aspergillus spp. bind Factor H, the splicing variant FHL-1 and also C4bp. Immunofluorescence and flow cytometry studies show that the binding of Factor H and C4bp to Aspergillus spp. appears to be even stronger than to Candida spp. and that different, albeit possibly nearby, binding moieties mediate this surface attachment.     

Assessment of the interaction of human complement regulatory proteins with group A Streptococcus. Identification of a high-affinity group A Streptococcus binding site in FHL-1

Group A Streptococcus (GAS), the most frequent bacterial cause of suppurative infections in humans, expresses on the cell surface M proteins with capacity to bind factor H, FHL-1 and C4b binding protein (C4BP). This has been interpreted as a mechanism developed by this pathogen to decrease phagocytosis by macrophages and polymorphonuclear cells. We report the analysis of the capacity to bind factor H, FHL-1 and C4BP of 69 clinical isolates from 19 different serotypes. We show that strains binding complement regulators (30/69) belong to specific M serotypes. Of these, M18 strains are relatively frequent and interact with all three complement regulators simultaneously. However, the most virulent M1 and M3 strains did not bind complement regulators in our assays. The relevance of the interaction between complement regulators and S. pyogenes was analyzed using different approaches with the conclusion that under physiological conditions only FHL-1 and C4BP bind to streptococci. We show that FHL-1 presents a higher binding affinity for S. pyogenes than factor H because it carries a hydrophobic, high-affinity, GAS binding site in addition to the heparin binding site in SCR7. Using synthetic peptides we provide evidence that the high-affinity GAS binding site in FHL-1 involves the hydrophobic tail (Ser-Phe-Thr-Leu) that distinguishes FHL-1 from factor H.     

Identification and characterization of the factor H and FHL-1 binding complement regulator-acquiring surface protein 1 of the Lyme disease spirochete Borrelia spielmanii sp. nov

Borrelia spielmanii, one of the etiological agents of Lyme disease found in Europe, evades host complement-mediated killing by recruitment of the immune regulators factor H and FHL-1 from human serum. Serum-resistant and intermediate serum-resistant isolates express up to 3 distinct complement regulator-acquiring surface proteins (CRASPs) that bind factor H and/or FHL-1. The present study describes identification and functional characterization of BsCRASP-1 as the dominant factor H and FHL-1 binding protein of B. spielmanii. BsCRASP-1 is a 27.7kDa outer surface lipoprotein, which after processing has a predicted mass of 24.9kDa. BsCRASP-1 is encoded by a single copy gene, cspA, that maps to a linear plasmid of approximately 55kb. Ligand affinity blot techniques revealed that both native and recombinant BsCRASP-1 from different isolates can strongly bind FHL-1, but only weakly factor H. Deletion mutants of recombinant BsCRASP-1 were generated and a high-affinity binding site for factor H and FHL-1 was mapped to its carboxy-terminal 10-amino-acid residue domain. Similarly, the dominant binding site of factor H and FHL-1 was localized to short consensus repeats (SCRs) 5-7. Factor H and FHL-1 maintained cofactor activity for factor I-mediated C3b inactivation when bound to full-length BsCRASP-1 but not to a deletion mutant lacking the carboxy-terminal 10-amino-acid residue domain. In conclusion, BsCRASP-1 binds the host immune regulators factor H and FHL-1, and is suggested to represent a key molecule of B. spielmanii for complement resistance. Thus, BsCRASP-1 most likely contributes to persistence of B. spielmanii and to pathogenesis of Lyme disease.     

Demonstration of factor H-like protein 1 binding to Treponema denticola, a pathogen associated with periodontal disease in humans

Treponema denticola is an important contributor to periodontal disease. In this study we investigated the ability of T. denticola to bind the complement regulatory proteins factor H and factor H-like protein 1 (FHL-1). The binding of these proteins has been demonstrated to facilitate evasion of the alternative complement cascade and/or to play a role in adherence and invasion. Here we demonstrate that T. denticola specifically binds FHL-1 via a 14-kDa, surface-exposed protein that we designated FhbB. Consistent with its FHL-1 binding specificity, FhbB binds only to factor H recombinant fragments spanning short consensus repeats (SCRs) 1 to 7 (H7 construct) and not to SCR constructs spanning SCRs 8 to 15 and 16 to 20. Binding of H7 to FhbB was inhibited by heparin. The specific involvement of SCR 7 in the interaction was demonstrated using an H7 mutant (H7AB) in which specific charged residues in SCR 7 were replaced by alanine. This construct lost FhbB binding ability. Analyses of the ability of FHL-1 bound to the surface of T. denticola to serve as a cofactor for factor I-mediated cleavage of C3b revealed that C3b is cleaved in an FHL-1/factor I-independent manner, perhaps by an unidentified protease. Based on the data presented here, we hypothesize that the primary function of FHL-1 binding by T. denticola might be to facilitate adherence to FHL-1 present on anchorage-dependent cells and in the extracellular matrix.     

Human pathogenic Borrelia spielmanii sp. nov. resists complement-mediated killing by direct binding of immune regulators factor H and factor H-like protein 1

Borrelia spielmanii sp. nov. has recently been shown to be a novel human pathogenic genospecies that causes Lyme disease in Europe. In order to elucidate the immune evasion mechanisms of B. spielmanii, we compared the abilities of isolates obtained from Lyme disease patients and tick isolate PC-Eq17 to escape from complement-mediated bacteriolysis. Using a growth inhibition assay, we show that four B. spielmanii isolates, including PC-Eq17, are serum resistant, whereas a single isolate, PMew, was more sensitive to complement-mediated lysis. All isolates activated complement in vitro, as demonstrated by covalent attachment of C3 fragments; however, deposition of the later activation products C6 and C5b-9 was restricted to the moderately serum-resistant isolate PMew and the serum-sensitive B. garinii isolate G1. Furthermore, serum adsorption experiments revealed that all B. spielmanii isolates acquired the host alternative pathway regulators factor H and factor H-like protein (FHL-1) from human serum. Both complement regulators retained their factor I-mediated C3b inactivation activities when bound to spirochetes. In addition, two distinct factor H and FHL-1 binding proteins, BsCRASP-1 and BsCRASP-2, were identified, which we estimated to be approximately 23 to 25 kDa in mass. A further factor H binding protein, BsCRASP-3, was found exclusively in the tick isolate, PC-Eq17. This is the first report describing an immune evasion mechanism utilized by B. spielmanii sp. nov., and it demonstrates the capture of human immune regulators to resist complement-mediated killing.     

Complement escape of human pathogenic bacteria by acquisition of complement regulators

Pathogenic micro-organisms employ a broad range of strategies to survive in and to persistently infect the human host. Far from being completely understood by which highly sophisticated means invading pathogens overcome the host's destructive immune defence, there is a growing body of evidence on particular mechanisms which play a pivotal role for immune evasion. This review focuses on evasion of medically and scientifically important bacteria by acquisition of host derived fluid-phase complement regulatory proteins, in particular factor H, FHL-1, and C4b binding protein. Expression of microbial surface molecules binding to human complement regulators and thus fixing them in a functionally active state allows pathogens to inhibit and finely regulate complement activation directly on their surface. Further studies on the utilization of host complement regulatory proteins will likely have a marked impact on a more efficient and specific clinical treatment.     

Acquisition of regulators of complement activation by Streptococcus pyogenes serotype M1

Opsonization of bacteria by complement proteins is an important component of the immune response. The pathogenic bacterium Streptococcus pyogenes has evolved multiple mechanisms for the evasion of complement-mediated opsonization. One mechanism involves the binding of human regulators of complement activation such as factor H (FH) and FH-like protein 1 (FHL-1). Acquisition of these regulatory proteins can limit deposition of the opsonin C3b on bacteria, thus decreasing the pathogen's susceptibility to phagocytosis. Binding of complement regulatory proteins by S. pyogenes has previously been attributed to the streptococcal M and M-like proteins. Here, we report that the S. pyogenes cell surface protein Fba can mediate binding of FH and FHL-1. We constructed mutant derivatives of S. pyogenes that lack Fba, M1 protein, or both proteins and assayed the strains for FH binding, susceptibility to phagocytosis, and C3 deposition. Fba expression was found to be sufficient for binding of purified FH as well as for binding of FH and FHL-1 from human plasma. Plasma adsorption experiments also revealed that M1(+) Fba(+) streptococci preferentially bind FHL-1, whereas M1(-) Fba(+) streptococci have similar affinities for FH and FHL-1. Fba was found to contribute to the survival of streptococci incubated with human blood and to inhibit C3 deposition on bacterial cells. Streptococci harvested from log-phase cultures readily bound FH, but binding was greatly reduced for bacteria obtained from stationary-phase cultures. Bacteria cultured in the presence of the protease inhibitor E64 maintained FH binding activity in stationary phase, suggesting that Fba is removed from the cell surface via proteolysis. Western analyses confirmed that E64 stabilizes cell surface expression of Fba. These data indicate that Fba is an antiopsonic, antiphagocytic protein that may be regulated by cell surface proteolysis.     

Molecular analyses of the interaction of Borrelia hermsii FhbA with the complement regulatory proteins factor H and factor H-like protein 1

Borrelia hermsii, the primary etiological agent of tick-borne relapsing fever in North America, binds the complement regulatory protein factor H (FH) as a means of evading opsonophagocytosis and the alternative complement pathway. The ability of FH-binding protein A (FhbA) to bind FH-like protein 1 (FHL-1) has not been assessed previously. In this study, using a whole-cell absorption assay, we demonstrated that B. hermsii absorbs both FH and FHL-1 from human serum. Consistent with this, affinity ligand binding immunoblot analyses revealed that FH constructs spanning short consensus repeats 1 to 7 and 16 to 20 bind to FhbA. To investigate the molecular basis of the interaction of FhbA with FH/FHL-1, recombinant FhbA truncated proteins were generated and tested for FH/FHL-1 binding. Binding required determinants located in both the N- and C-terminal domains of FhbA, suggesting that long-range intramolecular interactions are involved in the formation and presentation of the FH/FHL-1-binding pocket. To identify specific FhbA residues involved in binding, random mutagenesis was performed. These analyses identified a loop region of FhbA that may serve as a contact point for FH/FHL-1. The data presented here expand our understanding of the pathogenic mechanisms of the relapsing fever spirochetes and of the molecular nature of the interaction between FH/FHL-1 and FhbA.     

Assessment of the regions within complement regulator-acquiring surface protein (CRASP)-2 of Borrelia burgdorferi required for interaction with host immune regulators FHL-1 and factor H

The ability of Borrelia burgdorferi sensu lato to survive and persist in a variety of vertebrate hosts is a multifactorial process. Several potential mechanisms of immune evasion have been identified. We have shown that some Borrelia species bind host-derived fluid-phase immune regulators FHL-1 and factor H to their surface via complement regulator-acquiring surface proteins (CRASPs). Factor H and FHL-1 serve as cofactors for factor I, a serine protease that cleaves C3b directly on the cell surface and thereby confers resistance of spirochetes to complement-mediated lysis. Among the CRASP molecules produced by B. burgdorferi, BbCRASP-2 represents a novel FHL-1 and factor H binding protein that is distinct from other borrelial CRASP molecules and is predominantly expressed by serum-resistant Borrelia strains. The aim of this study was to identify BbCRASP-2 determinants required for FHL-1 and factor H binding. A number of recombinant BbCRASP-2 mutants were generated by in vitro mutagenesis and tested for factor H and FHL-1 binding employing ELISA. Up to 8 amino acid substitutions in the proposed binding regions 2 and 3 of BbCRASP-2 resulted in reduced or complete loss of FHL-1 and/or factor H binding. These results suggest that the factor H/FHL-1 binding regions are discontinuous and long distance interaction is involved in binding of both immune regulators. Furthermore, putative coiled-coil structural elements as recently discussed to be important in the interaction of BbCRASP-1 with factor H seem to play a subordinate role for binding of BbCRASP-2 to FHL-1 and factor H. The elucidation of host–pathogen interactions will help to develop novel therapeutic strategies against Lyme disease/borreliosis.     

Recruitment of complement factor H-like protein 1 promotes intracellular invasion by group A streptococci

Numerous microbial pathogens exploit complement regulatory proteins such as factor H (FH) and factor H-like protein 1 (FHL-1) for immune evasion. Fba is an FHL-1 and FH binding protein expressed on the surface of the human pathogenic bacterium, Streptococcus pyogenes, a common agent of pharyngeal, skin, and soft-tissue infections. In the present study, we demonstrate that Fba and FHL-1 work in concert to promote invasion of epithelial cells by S. pyogenes. Fba fragments were expressed as recombinant proteins and assayed for binding of FHL-1 and FH by Western blotting, enzyme-linked immunosorbent assay, and surface plasmon resonance. A binding site for FHL-1 and FH was localized to the N-terminal half of Fba, a region predicted to contain a coiled-coil domain. Deletion of this coiled-coil domain greatly reduced FHL-1 and FH binding. PepSpot analyses identified a 16-amino-acid segment of Fba which overlaps the coiled-coil domain that binds both FHL-1 and FH. To localize the Fba binding site in FHL-1 and FH, surface plasmon resonance was used to assess the interactions between the streptococcal protein and a series of recombinant FH deletion constructs. The Fba binding site was localized to short consensus repeat 7 (SCR 7), a domain common to FHL-1 and FH. SCR 7 contains a heparin binding site, and heparin was found to inhibit FHL-1 binding to Fba. FHL-1 promoted entry of Fba(+) group A streptococci into epithelial cells in a dose-dependent manner but did not affect invasion by an isogenic fba mutant. To our knowledge, this is the first report of a bacterial pathogen exploiting a soluble complement regulatory protein for entry into host cells.     

Impact of the SpeB protease on binding of the complement regulatory proteins factor H and factor H-like protein 1 by Streptococcus pyogenes

Microbial pathogens often exploit human complement regulatory proteins such as factor H (FH) and factor H-like protein 1 (FHL-1) for immune evasion. Fba is an FH and FHL-1 binding protein expressed on the surface of the human pathogenic bacterium Streptococcus pyogenes, a common agent of pharyngeal, skin, and soft-tissue infections. Fba has been shown to contribute to phagocytosis resistance, intracellular invasion, and virulence in mice. Here, we look at the role of Fba in recruitment of FH and FHL-1 by five serotype M1 isolates of streptococci. Inactivation of fba greatly inhibited binding of FH and FHL-1 by all isolates, indicating that Fba is a major FH and FHL-1 binding factor of serotype M1 streptococci. For three isolates, FH binding was significantly reduced in stationary-phase cultures and correlated with high levels of protease activity and SpeB (an extracellular cysteine protease) protein in culture supernatants. Analysis of a speB mutant confirmed that SpeB accounts for the loss of Fba from the cell surface, suggesting that the protease may modulate FH and FHL-1 recruitment during infection. Comparisons of fba DNA sequences revealed that the FH and FHL-1 binding site in Fba is conserved among the M1 isolates. Although the ligand binding site is not strictly conserved in Fba from a serotype M49 isolate, the M49 Fba protein was found to bind both FH and FHL-1. Collectively, these data indicate that binding of FH and FHL-1 is a conserved function of Fba while modulation of Fba function by SpeB is variable.     

Borrelia burgdorferi regulates expression of complement regulator-acquiring surface protein 1 during the mammal-tick infection cycle

During the natural mammal-tick infection cycle, the Lyme disease spirochete Borrelia burgdorferi comes into contact with components of the alternative complement pathway. B. burgdorferi, like many other human pathogens, has evolved the immune evasion strategy of binding two host-derived fluid-phase regulators of complement, factor H and factor H-like protein 1 (FHL-1). The borrelial complement regulator-acquiring surface protein 1 (CRASP-1) is a surface-exposed lipoprotein that binds both factor H and FHL-1. Analysis of CRASP-1 expression during the mammal-tick infectious cycle indicated that B. burgdorferi expresses this protein during mammalian infection, supporting the hypothesized role for CRASP-1 in immune evasion. However, CRASP-1 synthesis was repressed in bacteria during colonization of vector ticks. Analysis of cultured bacteria indicated that CRASP-1 is differentially expressed in response to changes in pH. Comparisons of CRASP-1 expression patterns with those of other infection-associated B. burgdorferi proteins, including the OspC, OspA, and Erp proteins, indicated that each protein is regulated through a unique mechanism.     

FHR-1, an additional human plasma protein,binds to complement regulator-acquiring surface proteins of Borrelia burgdorferi

The Borrelia burgdorferi strain LW2, a causative agent of Lyme disease, expresses up to five distinct complement regulator-acquiring surface proteins (CRASPs) that bind the human immune regulators factor H and/or FHL-1. In the present study, we identify FHR-1, a member of the human factor H protein family, as an additional ligand for CRASP-3, CRASP-4, and CRASP-5 but not for CRASP-1 and CRASP-2. A comparative analysis of the binding characteristics revealed unique and distinct binding profiles of the three host immune regulators to CRASPs. FHR-1 binds to CRASP-3, CRASP-4, and CRASP-5; factor H binds to all five CRASPs, and FHL-1 binds preferentially to CRASP-1 and CRASP-2. On the pathogen site, CRASP-3 interacts predominantly with factor H; CRASP-4 shows a preference for FHR-1, and CRASP-5 binds strongly and with equal intensity FHR-1 and factor H. Thus, expression of several CRASPs with distinct binding properties for host proteins allows the pathogen to attach functionally distinct host proteins to its surface.     

The human complement regulator factor H binds pneumococcal surface protein PspC via short consensus repeats 13 to 15

The innate ability of Streptococcus pneumoniae to resist complement activation and complement-mediated phagocytosis may be a direct consequence of the ability of the bacteria to bind components of the complement regulatory system. One such component, factor H (fH), is a crucial fluid-phase negative regulator of the alternative pathway of complement and is utilized by a number of pathogenic organisms to resist complement attack. The pneumococcal surface protein C (PspC [also known as CbpA] and SpsA) has been shown to bind fH, although the exact binding site within one or more of the 20 short consensus repeats (SCRs) of the molecule is not known. The purpose of the current study was to map specific SCRs on fH responsible for this binding. Initial experiments utilizing type 2 pneumococcal strain D39 and its isogenic PspC-negative derivative (D39/pspC mutant) showed that fH binding was PspC dependent. A purified recombinant protein derivative of PspC that lacked the proline-rich region (PspCDeltaPro) had a reduced binding efficiency for fH, thereby directly showing the importance of this region for the fH interaction. We have specifically shown by inhibition experiments that SCRs responsible for heparin and C3b binding of fH are not involved in binding PspC and the interaction between fH and PspC is largely hydrophobic, since no inhibition was observed in the presence of high concentrations of NaCl. Construction of SCR proteins encompassing the whole fH molecule showed that SCRs 8 to 15 (SCR 8-15) mediated binding to PspC. Further localization experiments revealed that SCR 13 and SCR 15 were required for full binding, although partial binding was retained when either SCR was removed.     

Tuf of Streptococcus pneumoniae is a surface displayed human complement regulator binding protein

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.