Monoclonal antibodies against the complement control protein Factor H (β1 H)

Two mouse monoclonal antibodies against the human complement control protein, Factor H (beta 1H), are described. The antibodies are both IgG - gamma 1 - subclass and are directed against different epitopes on the human Factor H molecule. One of the antibodies, MRC OX 24, increases the cofactor activity of Factor H in Factor I-mediated cleavage of soluble C3b. The second antibody, MRC OX 23, which has no effect alone, reduces the increase in cofactor activity observed in the presence of the first antibody. However, MRC OX 24 inhibits the binding of 125I-labelled Factor H to surface-bound C3b (EAC3b). Again MRC OX 23 alone does not have an effect but decreases the inhibition in 125I-labelled Factor H binding to EAC3b observed with MRC OX 24. These studies show clearly that the interaction of Factor H with soluble C3b is different to its interaction with surface-bound C3b. In an indirect immunoprecipitation system using these monoclonal antibodies, single-chain molecules of 150 000 mol.wt. are specifically precipitated from human serum and also from the sera of other primates - rhesus monkey, cynomolgus monkey, and African green monkey. There was no precipitation from sera of cow, pig, sheep, chick, or rabbit. Using a radioimmunoassay with radiolabelled monoclonal MRC OX 23, the concentration of Factor H in human plasma was determined.     

Immune evasion of the human pathogen Pseudomonas aeruginosa: elongation factor Tuf is a factor H and plasminogen binding protein

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause a wide range of clinical symptoms and infections that are frequent in immunocompromised patients. In this study, we show that P. aeruginosa evades human complement attack by binding the human plasma regulators Factor H and Factor H-related protein-1 (FHR-1) to its surface. Factor H binds to intact bacteria via two sites that are located within short consensus repeat (SCR) domains 6-7 and 19-20, and FHR-1 binds within SCR domain 3-5. A P. aeruginosa Factor H binding protein was isolated using a Factor H affinity matrix, and was identified by mass spectrometry as the elongation factor Tuf. Factor H uses the same domains for binding to recombinant Tuf and to intact bacteria. Factor H bound to recombinant Tuf displayed cofactor activity for degradation of C3b. Similarly Factor H bound to intact P. aeruginosa showed complement regulatory activity and mediated C3b degradation. This acquired complement control was rather effective and acted in concert with endogenous proteases. Immunolocalization identified Tuf as a surface protein of P. aeruginosa. Tuf also bound plasminogen, and Tuf-bound plasminogen was converted by urokinase plasminogen activator to active plasmin. Thus, at the bacterial surface Tuf acts as a virulence factor and binds the human complement regulator Factor H and plasminogen. Acquisition of host effector proteins to the surface of the pathogen allows complement control and may facilitate tissue invasion.     

Ultrastructures and interactions of complement factors H and I.

The human complement regulatory protein, factor H, was examined by high resolution transmission electron microscopy. Results of electron microscopy confirm hydrodynamic analysis and indicate that factor H is a monomer of M(r) approximately 155,000. Factor H is an extended flexible molecule with a contour length of 495 A and a cross-sectional diameter of 34 A. Most images of factor H indicate that its polypeptide chain typically folds back on itself with the result that the average length of a factor H molecule is about half its contour length. Only one end of factor H associates with C3b. When bound to C3b, factor H still shows considerable conformational flexibility. Factor I is a bilobal protein of 130 A in length, and its two globular parts have maximal diameters of 54 and 49 A. The results establish that factor I is a two domain protein where the smaller subunit is a protease and the larger one is involved with binding C3b. Factor I binds C3b with a one-to-one stoichiometry in an ionic strength-dependent fashion. In the absence of sodium chloride an affinity constant of 5.7 x 10(5) M-1 was determined for factor I interaction with C3b. Whereas the Scatchard plot of factor I binding to C3b in the absence of factor H is linear, in the presence of factor H a curvilinear graph is obtained. The strong binding sites on C3b for factor I have an affinity at least 15-fold higher in the presence of factor H than in its absence. The results of both electron microscopy and binding studies were combined to compose a scheme envisioning how factors H and I cooperate for the processing of C3b.     

The Staphylococcus aureus Protein Sbi Acts as a Complement Inhibitor and Forms a Tripartite Complex with Host Complement Factor H and C3b

The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1) from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG) as a ligand that interacts with Factor H by a—to our knowledge—new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite Sbi∶C3∶Factor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and β₂-glycoprotein I and interferes with innate immune recognition.     

Chemical Chaperones Curcumin and 4-Phenylbutyric Acid Improve Secretion of Mutant Factor H R127H by Fibroblasts from a Factor H-Deficient Patient

Factor H (FH) is one of the most important regulatory proteins of the alternative pathway of the complement system. Patients with FH deficiency have a higher risk for development of infections and kidney diseases because of the uncontrolled activation and subsequent depletion of the central regulatory component C3 of the complement system. In this study, we investigated the consequences of the Arg(127)His mutation in FH (FH(R127H)) previously described in an FH-deficient patient, on the secretion of this protein by skin fibroblasts in vitro. We observed that, although the patient cells stimulated with IFN-γ were able to synthesize FH(R127H), the mutant protein was largely retained within the endoplasmic reticulum (ER), whereas normal human fibroblasts stimulated with IFN-γ secrete FH without retention in the ER. Moreover, the retention of FH(R127H) provoked enlargement of ER cisterns after treatment with IFN-γ. A similar ER retention was observed in Cos-7 cells expressing the mutant FH(R127H) protein. Despite this deficiency in secretion, we show that the FH(R127H) mutant is capable of functioning as a cofactor in the Factor I-mediated cleavage of C3. We then evaluated whether a treatment could increase the secretion of FH, and observed that the patient's fibroblasts treated with the chemical chaperones 4-phenylbutiric acid or curcumin increased the secretion rate of FH. We propose that these chemical chaperones could be used as alternative therapeutic agents to increase FH plasma levels in FH-deficient patients caused by secretion delay of this regulatory protein.     

Structural and functional analysis of the complement component factor H with the use of different enzymes and monoclonal antibodies to factor H.

The action of six different enzymes on the function and structure of Factor H was investigated by use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, haemagglutination, two enzyme-linked immunosorbent assay systems and an assay for Factor I cofactor activity. Six monoclonal antibodies directed against the 38 kDa tryptic fragment of Factor H [which contains the binding site for C3b (a 180 kDa fragment of the third component of complement) and the cofactor activity] were also used to detect cleavage products derived from the same fragment. Elastase, chymotrypsin A4 or trypsin first cleaved Factor H to 36-38 kDa fragments carrying all six monoclonal anti-(Factor H)-binding sites. In parallel, the interaction of Factor H with surface-bound C3b was lost, whereas the cofactor function was preserved. Further cleavage of the 36-38 kDa fragments into two 13-19 kDa fragments (one carrying the MAH4 and MRC OX 24 epitopes, the other the MAH1, MAH2, MAH3 and MRC OX 23 epitopes) destroyed cofactor activity. Pepsin, bromelain or papain rapidly split off a 13-15 kDa fragment of Factor H carrying the MAH1, MAH2, MAH3 and MRC OX 23 epitopes and destroyed all tested functions of Factor H. Ficin cleaved Factor H into disulphide-linked fragments smaller than 25 kDa, but did not affect the functions of the Factor H molecule. The 38 kDa tryptic fragment of Factor H is the N-terminal end of the Factor H molecule, as determined by N-terminal sequence analysis. A model is presented of the substructure of Factor H.     

Zinc-induced Self-association of Complement C3b and Factor H: IMPLICATIONS FOR INFLAMMATION AND AGE-RELATED MACULAR DEGENERATION*

The sub-retinal pigment epithelial deposits that are a hallmark of age-related macular degeneration contain both C3b and millimolar levels of zinc. C3 is the central protein of complement, whereas C3u is formed by the spontaneous hydrolysis of the thioester bridge in C3. During activation, C3 is cleaved to form active C3b, then C3b is inactivated by Factor I and Factor H to form the C3c and C3d fragments. The interaction of zinc with C3 was quantified using analytical ultracentrifugation and x-ray scattering. C3, C3u, and C3b associated strongly in >100 μm zinc, whereas C3c and C3d showed weak association. With zinc, C3 forms soluble oligomers, whereas C3u and C3b precipitate. We conclude that the C3, C3u, and C3b association with zinc depended on the relative positions of C3d and C3c in each protein. Computational predictions showed that putative weak zinc binding sites with different capacities exist in all five proteins, in agreement with experiments. Factor H forms large oligomers in >10 μm zinc. In contrast to C3b or Factor H alone, the solubility of the central C3b-Factor H complex was much reduced at 60 μm zinc and even more so at >100 μm zinc. The removal of the C3b-Factor H complex by zinc explains the reduced C3u/C3b inactivation rates by zinc. Zinc-induced precipitation may contribute to the initial development of sub-retinal pigment epithelial deposits in the retina as well as reducing the progression to advanced age-related macular degeneration in higher risk patients.     

Gpm1p is a factor H-, FHL-1-, and plasminogen-binding surface protein of Candida albicans

The human pathogenic yeast Candida albicans utilizes host complement regulators for immune evasion. Here we identify the first fungal protein that binds Factor H and FHL-1. By screening a protein array of 4088 proteins of Saccharomyces cerevisiae, phosphoglycerate mutase (ScGpm1p) was identified as a Factor H- and FHL-1-binding protein. The homologous C. albicans Gpm1p (CaGpm1p) was cloned and recombinantly expressed as a 36-kDa His-tagged protein. Purified CaGpm1p binds the host complement regulators Factor H and FHL-1, but not C4BP. The CaGpm1p binding regions in the host proteins were localized; FHL-1 binds via short consensus repeats (SCRs) 6 and 7, and Factor H utilizes two contact regions that are located in SCRs 6 and 7 and in SCRs 19 and 20. In addition, recombinant CaGpm1p binds plasminogen via lysine residues. CaGpm1p is a surface protein as demonstrated by immunostaining and flow cytometry. A C. albicans gpm1(-/-) mutant strain was generated that did not grow on glucose-supplemented but on ethanol- and glycerol-supplemented medium. Reduced binding of Factor H and plasminogen to the null mutant strain is in agreement with the presence of additional binding proteins. Attached to CaGpm1p, each of the three host plasma proteins is functionally active. Factor H and FHL-1 show cofactor activity for cleavage of C3b, and bound plasminogen is converted by urokinase-type plasminogen activator to proteolytically active plasmin. Thus, the surface-expressed CaGpm1p is a virulence factor that utilizes the host Factor H, FHL-1, and plasminogen for immune evasion and degradation of extracellular matrices.     

Plasma kallikrein is activated on dermatan sulfate and cleaves factor H

When human plasma is applied to a dermatan sulfate column, amidase activity is detected in the bound fraction and complement factor H is cleaved [A. Saito, H. Munakata, Factor H is a dermatan sulfate-binding protein: identification of a dermatan sulfate—mediated protease that cleaves factor H, J. Biochem. 137 (2005) 225-233]. Here, the amidase-active fraction was purified by sequential gel filtration and hydroxyapatite chromatography, and the amidase-active protein was identified to be plasma kallikrein by mass spectrometry. The activation of plasma kallikrein was further investigated by Western blotting using plasma deficient in prekallikrein or coagulation factor Xll. The dermatan sulfate column-bound fraction of the prekallikrein- and factor Xll-deficient plasmas did not show any amidase activity and factor H remained intact. Addition of kallikrein, but not activated factor Xll, to factor H purified from plasma resulted in cleavage of factor H. Thus, dermatan sulfate induces contact activation and activates kallikrein-mediated cleavage of FH.     

Differences between the binding sites of the complement regulatory proteins DAF, CR1, and factor H on C3 convertases

Binding studies using purified decay-accelerating factor (DAF), CR1, and Factor H indicate that the primary interaction of DAF with C3 convertases is with the Bb or C2a subunits, whereas CR1 and Factor H interact primarily with the C3b or C4b subunits. The ability of soluble DAF, CR1, or Factor H to decay C3b,Bb bound to zymosan was inhibited by various concentrations of fluid-phase competitors (C3b, Bb, C3b,Bb, C3b,B, C4b, or C4b,C2a) in 0.1% NP-40 at 22 degrees C. The apparent association constants (appKa) for DAF were 0.045, 0.067, 0.91, 0.71, 0.00045, and 0.53 microM-1, respectively. The appKa for CR1 were 0.50, 0.0040, 1, 1, 1, and 1.1 microM-1, respectively. The appKa for Factor H were 4.3, 0.0005, 2.9, 6.3, 0.27, and 0.29 microM-1, respectively. Thus, C3b binds to DAF with a 10-fold lower affinity than to CR1 and a 100-fold lower affinity than to Factor H. The appKa of C3b,Bb for the three proteins were more similar: DAF (0.91 microM-1), CR1 (1 microM-1), and Factor H (2.9 microM-1). DAF binds to Bb with a 50% higher affinity than to C3b, and to C4b,C2a with a 1000-fold higher affinity than to C4b alone. In contrast, CR1 and Factor H bind almost equally well to the C3 convertases and to their noncatalytic subunits. The affinity of DAF for CVF,Bb was similar to its affinity for Bb alone, suggesting that DAF does not recognize conformational determinants unique to Bb in C3 convertases.     

Isolation of rabbit C3, Factor B, and Factor H and comparison of their properties with those of the human analog

The rabbit complement components C3, Factor B, and Factor H were isolated and characterized and were compared to the corresponding proteins of human serum. Chromatographic behavior, chemical properties, and functional interactions show great similarities between the components in both species. By SDS polyacrylamide gel electrophoresis, the m.w. were estimated to be 195,000 for C3, 86,000 for Factor B, and 155,000 for Factor H. The amino acid compositions of the rabbit proteins resembled those of the human analog. The total carbohydrate content of rabbit C3 and Factor H was approximately one-half that of the human proteins. In addition, a qualitative difference in the carbohydrate moieties of the C3 proteins was observed. The serum concentration of the rabbit proteins was markedly lower than that of the human proteins. The rabbit C3b,Bb enzyme resembled the human analog with respect to half-life, control by Factor H, and stabilization by nickel ions.     

Plasminogen is a complement inhibitor

Plasminogen is a 92-kDa single chain glycoprotein that circulates in plasma as a zymogen and when converted to proteolytically active plasmin dissolves preformed fibrin clots and extracellular matrix components. Here, we characterize the role of plasmin(ogen) in the complement cascade. Plasminogen binds the central complement protein C3, the C3 cleavage products C3b and C3d, and C5. Plasminogen binds to C3, C3b, C3d, and C5 via lysine residues, and the interaction is ionic strength-dependent. Plasminogen and Factor H bind C3b; however, the two proteins bind to different sites and do not compete for binding. Plasminogen affects complement action in multiple ways. Plasminogen enhanced Factor I-mediated C3b degradation in the presence of the cofactor Factor H. Plasminogen when activated to plasmin inhibited complement as demonstrated by hemolytic assays using either rabbit or sheep erythrocytes. Similarly, plasmin either in the fluid phase or attached to surfaces inhibited complement that was activated via the alternative and classical pathways and cleaved C3b to fragments of 68, 40, 30, and 17 kDa. The C3b fragments generated by plasmin differ in size from those generated by the complement protease Factor I, suggesting that plasmin-mediated C3b cleavage fragments lack effector function. Plasmin also cleaved C5 to products of 65, 50, 30, and 25 kDa. Thus, plasmin(ogen) regulates both complement and coagulation, the two central cascade systems of a vertebrate organism. This complement-inhibitory activity of plasmin provides a new explanation why pathogenic microbes utilize plasmin(ogen) for immune evasion and tissue penetration.     

Structure of the N-terminal region of complement factor H and conformational implications of disease-linked sequence variations

Factor H is a regulatory glycoprotein of the complement system. We expressed the three N-terminal complement control protein modules of human factor H (FH1-3) and confirmed FH1-3 to be the minimal unit with cofactor activity for C3b proteolysis by factor I. We reconstructed FH1-3 from NMR-derived structures of FH1-2 and FH2-3 revealing an approximately 105-A-long rod-like arrangement of the modules. In structural comparisons with other C3b-engaging proteins, factor H module 3 most closely resembles factor B module 3, consistent with factor H competing with factor B for binding C3b. Factor H modules 1, 2, and 3 each has a similar backbone structure to first, second, and third modules, respectively, of functional sites in decay accelerating factor and complement receptor type 1; the equivalent intermodular tilt and twist angles are also broadly similar. Resemblance between molecular surfaces is closest for first modules but absent in the case of second modules. Substitution of buried Val-62 with Ile (a factor H single nucleotide polymorphism potentially protective for age-related macular degeneration and dense deposit disease) causes rearrangements within the module 1 core and increases thermal stability but does not disturb the interface with module 2. Replacement of partially exposed (in module 1) Arg-53 by His (an atypical hemolytic uremic syndrome-linked mutation) did not impair structural integrity at 37 degrees C, but this FH1-2 mutant was less stable at higher temperatures; furthermore, chemical shift differences indicated potential for small structural changes at the module 1-2 interface.     

Human factor H-related protein 5 (FHR-5). A new complement-associated protein

A novel human plasma protein has been identified as a universal component of complement deposits, when complement is detected immunohistochemically in vivo. The protein is homologous to complement factor H and related proteins and has been designated factor H-related protein 5 (FHR-5). FHR-5 was identified by a monoclonal antibody raised using pathologic human glomerular preparations as the immunogen. FHR-5 was purified by affinity chromatography from complement-lysed erythrocytes, and the peptide sequence was obtained. The cDNA was cloned from a human liver library, and FHR-5 was deduced to be a protein containing 551 amino acids organized into nine short consensus repeat motifs. The short consensus repeats of FHR-5 show homology to Factor H and to other Factor H-related proteins, with some unique features demonstrated. Recombinant FHR-5, expressed in insect cells, was shown to bind C3b in vitro. The strong association of FHR-5 with tissue complement deposits in vivo suggests that this additional member of the Factor H family of proteins has a function in complement regulation.     

Complement factor H-binding protein of Raji cells and tonsil B lymphocytes.

Several reports have indicated that Factor H has specific effects on certain cell populations, suggesting that Factor H receptors may exist. Lambris & Ross [(1982) J. Exp. Med. 155, 1400-1411] purified a protein from Raji B-lymphoblastoid cell culture supernatants, using Factor H-Sepharose affinity chromatography. This species appeared to consist of two disulphide-linked components each of Mr 50,000, with an additional 50,000-Mr chain attached non-covalently. The existence of cell-surface Factor H-binding proteins has now been re-investigated with 125I surface-labelled Raji and tonsil B cells. Non-ionic-detergent extracts of the cells, in 0.1% Nonidet P40/10 mM-sodium phosphate buffer, pH 7.4, were incubated with Factor H-Sepharose in the presence of proteinase inhibitors. After the beads had been washed, bound components were eluted with 50 mM-NaCl. A single radioactive species was eluted from the resin, which migrates identically with Factor H (apparent Mr 170,000) in SDS/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions. Biosynthetic radiolabelling studies confirmed that this species was synthesized by Raji cells. Examination of culture supernatants from biosynthetically radiolabelled Raji cells showed again the presence of a single soluble species that bound to Factor H-Sepharose, but this species was of lower Mr (approx. 105,000) than the membrane-derived protein. The soluble form may be produced by proteolysis of the membrane form, or may be of separate origin. The similarity in size of the cell-surface protein to Factor H was initially confusing, but it is distinct from cell-surface Factor H on the basis of three criteria: (1) it is not recognized by anti-(Factor H) monoclonal antibodies MRC OX23 and MRC OX24, nor by polyclonal F(ab')2 anti-(Factor H); (2) it does not bind to Zn2+-chelate resin, whereas Factor H does; (3) cell-surface Factor H present on U937 cells does not bind to Factor H-Sepharose.     

Localization of the heparin-binding site on complement factor H.

Factor H is a regulator of complement activation and, in this capacity, it prevents activation of the alternative pathway on host cells and tissues when it recognizes markers on these surfaces. This report describes the binding characteristics and location of the site on factor H that is responsible for host recognition. Factor H was found to bind a variety of polyanions, including heparin, heparan sulfate, dextran sulfate, and clusters of sialic acid. In heparin-agarose binding assays it exhibited an affinity for heparin only 2-fold weaker than that of antithrombin III. Factor H exhibited little or no affinity for polyaspartic acid or bacterial colominic acid (polysialic acid). Factor H (Mr 150,000 with approximate dimensions of 30 x 600 A) is composed of 20 highly homologous domains (SCRs) that are arranged as beads on a string. Polyanions were found to block a tryptic cleavage site in domain 15, and a photoaffinity-tagged heparin probe labeled the region between domains 12 and 15. Affinity chromatography of tryptic fragments on heparin-Sepharose confirmed that this region contained the heparin-binding site. CNBr cleavage at Met787 located between SCRs 13 and 14 split the photoaffinity-tagged region. Sequence analysis strongly suggests that domain 13 contains the primary site of polyanion binding. Factor H expresses its complement regulatory function through a site located in domains 4-6 where C3b binds. Thus, the polyanion-binding site that regulates the affinity of factor H for C3b appears to reside more than 200 A away from the C3b-binding site.     

Sequence polymorphism of human complement factor H

Factor H is a major regulatory protein of the complement system. The complete cDNA coding sequence has been derived from overlapping clones, and a polymorphism at base 1277 has been characterized. In four clones there is a T at nucleotide 1277 and in two others there is a C. This T/C change represents a tyrosine/histidine polymorphism at position 384 in the derived amino acid sequence. Protein sequence studies on peptides generated by trypsin digestion of factor H, purified from pooled plasma from 12 donors, confirmed the presence of both tyrosine and histidine at this position. Tyrosine and histidine were observed in a ratio of 2 : 1, respectively, and therefore this polymorphism is likely to represent a sequence difference between the two most abundant charge variants, FH1 and FH2, of factor H.     

Protein S is essential for the activated protein C-catalyzed inactivation of platelet-associated factor Va

The prothrombin-converting activity of Factor Xa was enhanced by thrombin-stimulated Factor V-deficient platelets and supplementary extraneous Factor Va, and also by thrombin-stimulated normal human platelets. Both extraneous Factor Va and intra-platelet Factor Va were equally inactivated by a gamma-carboxyglutamic acid-containing plasma protease, activated protein C. However, a relatively larger amount of activated protein C was required for efficient inactivation of platelet-associated Factor Va as compared with the amount of activated protein C needed for inactivation of phospholipid vesicle-associated Factor Va. Protein S, another gamma-carboxyglutamic acid-containing plasma protein, increased the rate of the inactivation of platelet-associated Factor Va about 25-fold. This stimulating effect was observed only slightly with the thrombin-modified protein S. Thus, it was concluded that protein S is essential for the process of inactivation of platelet-associated Factor Va by activated protein C.     

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8–11 and SCR19–20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19–20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase.