Borrelia burgdorferi Infection-Associated Surface Proteins ErpP,ErpA, and ErpC Bind Human Plasminogen

Host-derived plasmin plays a critical role in mammalian infection by Borrelia burgdorferi. The Lyme disease spirochete expresses several plasminogen-binding proteins. Bound plasminogen is converted to the serine protease plasmin and thereby may facilitate the bacterium''s dissemination throughout the host by degrading extracellular matrix. In this work, we demonstrate plasminogen binding by three highly similar borrelial outer surface proteins, ErpP, ErpA, and ErpC, all of which are expressed during mammalian infection. Extensive characterization of ErpP demonstrated that this protein bound in a dose-dependent manner to lysine binding site I of plasminogen. Removal of three lysine residues from the carboxy terminus of ErpP significantly reduced binding of plasminogen, and the presence of a lysine analog, ɛ-aminocaproic acid, inhibited the ErpP-plasminogen interaction, thus strongly pointing to a primary role for lysine residues in plasminogen binding. Ionic interactions are not required in ErpP binding of plasminogen, as addition of excess NaCl or the polyanion heparin did not have any significant effect on binding. Plasminogen bound to ErpP could be converted to the active enzyme, plasmin. The three plasminogen-binding Erp proteins can also bind the host complement regulator factor H. Plasminogen and factor H bound simultaneously and did not compete for binding to ErpP, indicating separate binding sites for both host ligands and the ability of the borrelial surface proteins to bind both host proteins.Lyme disease is the most commonly reported arthropod-borne disease in the United States (8). Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to its hosts through the bites of infected Ixodes ticks. In the earliest stage of Lyme disease, a bull''s-eye-shaped rash, erythema migrans, occurs as the spirochete spreads outward from the site of the tick bite. If left untreated, serious clinical outcomes can occur, including arthritis, neuropathies, and carditis (48).The bacterium disseminates from the bite site to other host tissues. B. burgdorferi can traverse the epithelium and invade vascular walls but is rarely abundant in blood (1). In addition, B. burgdorferi can pass through the blood-brain barrier to enter the central nervous system (58). The spirochete, unlike many invasive pathogens, lacks surface protease activities (12, 26). Therefore, binding of host proteases to the surface of the bacterium may aid in the spirochete''s dissemination. Indeed, B. burgdorferi binds plasminogen, a component of the host''s fibrinolytic system (12, 19). Plasminogen circulates in the plasma as an inactive proenzyme and is activated by tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA) to plasmin (55). Plasminogen binding is an important virulence factor for invasive pathogens such as group A streptococci and Staphylococcus, as well as Borrelia species (10, 43, 55). The binding of plasminogen to bacteria and its subsequent activation allow bacteria to degrade the host''s extracellular matrix and basement membranes either through the direct protease activity of plasmin or by plasmin''s activation of host matrix metalloproteases (MMPs). B. burgdorferi has previously been shown to bind plasminogen, which is rapidly converted to active plasmin in the presence of host plasminogen activator (11). In vitro, plasmin-coated B. burgdorferi is able to penetrate endothelial cell monolayers (12). Surface-associated plasmin on B. burgdorferi can directly degrade fibronectin, a major component of the extracellular matrix, as well as laminin and vitronectin (11, 19). B. burgdorferi induces the release of MMP-9 (gelatinase) and MMP-1 (collagenase) from human cells, and plasmin-coated B. burgdorferi activates pro-MMP-9 (20), initiating a cascade that leads to degradation of basement membranes. Plasminogen has previously been shown to be important in B. burgdorferi pathogenesis. Although not strictly required for infection, plasminogen was required for efficient dissemination in ticks, and its absence decreased spirochetemia in plasminogen-deficient mice (10).Plasminogen-binding proteins of B. burgdorferi have previously been identified, including the outer-surface lipoprotein OspA (19). A role for OspC in plasminogen binding has also been suggested (31). However, OspA is generally not expressed during human infection, and OspC production ceases within the first few days of mammalian infection (13, 24, 25, 34, 42). Other, unidentified plasminogen-binding proteins have been observed in B. burgdorferi, including a protein(s) with an approximate molecular mass of 20 kDa, which is close to the size of several Erp proteins (12, 19). The members of the Erp family of outer-surface lipoproteins are expressed at high levels during mammalian infection (15, 23, 38-41).Lyme disease spirochetes contain numerous DNA elements, including the main chromosome as well as linear and circular plasmids (6). Infectious isolates carry several distinct yet homologous elements called cp32s, circular prophages of approximately 32 kb (54). All cp32 elements encode one or two Erp proteins, which can vary widely in amino acid sequence (50). However, all erp loci are preceded by nearly identical promoter regions (36, 53). Hence, most of the erp genes analyzed follow the same pattern of expression, being repressed in the tick vector but synthesized during mammalian infection (15, 21, 23, 35, 37-41). Roles for most of the Erp proteins have yet to be defined. ErpX has been demonstrated to bind host laminin (our unpublished results and reference 3). Three Erp proteins bind the host complement regulator factor H and factor H-related protein 1: ErpP, ErpC, and ErpA (22, 28, 29). Some factor H binding proteins of other human pathogens have been demonstrated to bind multiple ligands, including plasminogen (30, 47). These data, and the presence of unidentified plasminogen-binding proteins in B. burgdorferi, prompted us to examine if Erp proteins are able to bind plasminogen.     

Transient Human Monoclonal Immunoglobulins

Fourteen cases of transient serum monoclonal immunoglobulins are reported. Seven of the patients were young children. In 8 cases (including 5 children) a primary or secondary immunodeficiency could be ascertained. The level of the peak was under 1 g % in all cases except one, where the monoclonal Ig level was 2.5 g %. In 9 cases the monoclonal Ig disappeared in less than 2 months. The significance of these transient homogeneous components is discussed at both the clinical and aetiological level.     

Inhibition of Rosette Formation by Spleen Cells of Immunized Mice by Antibodies to Aggregated Mouse Immunoglobulins

The effect of rabbit antiserum to heat-aggregated mouse immunoglobulins (AAS) upon rosette formation by mouse spleen cells was examined before and after immunization with sheep erythrocytes. Pretreatment with AAS of spleen cells obtained during the first five days after immunization resulted in up to 707. reduction in detectable rosette-forming cells (RFC). RFC sensitive to AAS treatment did not contain 4tH antigen and carried Ig receptors. Spontaneous RFC from normal mice, just like those observed later than five days after immunization were not inhibited by AAS. The data obtained are considered as indication that aggregated Ig appearing on B cells during the initial period following primary immunization are, probably, immune complexes which serve as antigen-binding receptors.     

Immunoglobulins on Cultured Human Lymphocytes

Established, long-term cultured human T-lymphocytes were examined for the presence of cell surface immunoglobulin M and immunoglobulin G. Cells of the T-cell line, MOLT-4F, and of the B-cell line, RPMI 8392, were labeled with 125-iodine by the lactoperoxidase-catalyzed method. The labeled cells were lysed with 0.5% Triton X-100 and the lysates were further treated with 5% Triton X-100. For the IgM determination, a known monoclonal IgM protein labeled with 131-iodine was added to the supernatant as an internal reference standard and the mixture then treated with goat anti-human v chain antiserum and subsequently by sheep anti-goat γG antiserum. The immunoprecipitates were reduced and alkylated and examined by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. No μ or L chains were detected in the T-cell lysates. Only lysates from the B cells showed significant peaks of 125-iodine radioactivity corresponding to the 131-iodine labeled μ and L chains. Similar experiments were carried out for the detection of immunoglobulin G on these cells with negative results for both classes of cells.     

Ability of Staphylococcus aureus Cells to Bind Normal Human Immunoglobulin G

Staphylococcus aureus cells suspended in a human immunoglobulin G (IgG) solution adsorbed nearly 90% of the IgG; electron micrographs showed the cells enclosed in a thick, dense envelope of IgG.     

Studies on Fd Fragments of Human Immunoglobulins

Earlier studies on antisera against Fab of pooled human IgG and IgA myeloma proteins disclosed the presence of class-specific Fd antibodies, the demonstration of which required interaction of heavy and light chains. To extend our knowledge about the antigenic structure of the Fd fragment of human immunoglobulins, antisera were prepared in rabbits against γ chains of pooled IgG and of four IgG1 myeloma proteins, an Fdγ fragment and a cyanogen bromide-produced CH1 preparation of an IgG1 myeloma protein, and an Fd_α fragment of an IgA1 myeloma protein. No antigenic determinants exclusively confined to the CH1 region of intact human IgG could be demonstrated with these antisera. The antigenic structure of CH1 in intact immunoglobulins may thus only be defined by light-chain-dependent determinants.     

Bence Jones Proteins and Light Chains of Immunoglobulins XIV.

The region on the light chain molecule responsible for expression of the kappa and lambda antigenic determinants was determined by comparative immunochemical analyses of intact Bence Jones proteins and naturally occurring or enzymatically derived fragments of Bence Jones proteins that lacked extensive portions of the V region or part of the C region. The reactivity of these fragments with numerous antisera having specificity for light-chain antigenic determinants indicated the essentiality of the intact light polypeptide chain for expression of the kappa and lambda antigenic determinants. The conformational dependency of the kappa and lambda antigenic determinants was also evidenced by denaturation-renaturation studies on kappa and lambda chains. The V domain, C domain, and interdomain 'switch' region contribute to the expression of kappa and lambda antigenicity and to certain isotypic and allotypic specificities.     

Immunoglobulins and Other Serum Proteins in Feces from Infants and Children

IgA in variable amounts was found in extracts of feces from all of 14 infants and children with no apparent diseases involving the gastrointestinal tract or the immune system, whereas IgG and IgM were only found in some. No age variation was evident's even one-month-old infants seemed fully able to secrete intestinal immunoglobulins. Freeze-drying of the feces facilitated the extraction, and the amounts of immunoglobulins were related to the dry weight of fecas. Most of the immunoglobulins were easily dissolved in saline. Of other serum proteins α₁ antitrypsin was constantly present; α₁ antichymotrypsin and α₂ macroglobulin were found in many. Only traces of albumin were sometimes demonstrated.     

Identification and Characterization of Porphyromonas gingivalis Client Proteins That Bind to Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase

Coaggregation of Porphyromonas gingivalis and oral streptococci is thought to play an important role in P. gingivalis colonization. Previously, we reported that P. gingivalis major fimbriae interacted with Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and that amino acid residues 166 to 183 of GAPDH exhibited strong binding activity toward P. gingivalis fimbriae (H. Nagata, M. Iwasaki, K. Maeda, M. Kuboniwa, E. Hashino, M. Toe, N. Minamino, H. Kuwahara, and S. Shizukuishi, Infect. Immun. 77:5130–5138, 2009). The present study aimed to identify and characterize P. gingivalis components other than fimbriae that interact with S. oralis GAPDH. A pulldown assay was performed to detect potential interactions between P. gingivalis client proteins and S. oralis recombinant GAPDH with amino acid residues 166 to 183 deleted by site-directed mutagenesis. Seven proteins, namely, tonB-dependent receptor protein (RagA4), arginine-specific proteinase B, 4-hydroxybutyryl-coenzyme A dehydratase (AbfD), lysine-specific proteinase, GAPDH, NAD-dependent glutamate dehydrogenase (GDH), and malate dehydrogenase (MDH), were identified by two-dimensional gel electrophoresis followed by proteomic analysis using tandem mass spectrometry. Interactions between these client proteins and S. oralis GAPDH were analyzed with a biomolecular interaction analysis system. S. oralis GAPDH showed high affinity for five of the seven client proteins (RagA4, AbfD, GAPDH, GDH, and MDH). Interactions between P. gingivalis and S. oralis were measured by a turbidimetric method and fluorescence microscopy. RagA4, AbfD, and GDH enhanced coaggregation, whereas GAPDH and MDH inhibited coaggregation. Furthermore, the expression of luxS in P. gingivalis was upregulated by RagA4, AbfD, and GDH but was downregulated by MDH. These results indicate that the five P. gingivalis client proteins function as regulators in P. gingivalis biofilm formation with oral streptococci.     

Evolution of Maternofetal Transport of Immunoglobulins During Human Pregnancy

PROBLEM: We determined the evolution of the maternal-fetal transport of immunoglobulins during human pregnancy. METHOD: Paired blood samples were collected between 17–41 weeks of gestation (WG) by puncture of a peripheral maternal vein and by cordocentesis (17–36 WG, n=91) or directly at delivery (37–41 WG, n=16) from the umbilical vein. Additional maternal samples were collected from the same individual (n=16) at 10,20,30 WG, and at term. The concentration of IgG and its four subclasses and of IgA were determined in the sera using ELISA method. RESULTS: The mean level of IgG and IgA in maternal sera at 9–16 WG was 13.72±2.53 g/L and 3.95±1.23 g/L, respectively. Both, IgG and IgA throughout pregnancy decreased to a level of 60–70% (37–41 WG) of the initial concentration in early pregnancy. The ratio of IgG₁:IgG₂ in the maternal circulation was 2–3 and remained constant throughout pregnancy (17–41 WG). IgG₃ and IgG₄ levels remained constant and together were less than 10% of total IgG. In the fetal circulation a continuous rise in the level of both IgG and IgA was observed between 17 and 41 WG. Fetal level of IgG at 17–22 WG was only 5–10% of the maternal level and at term exceeded the maternal level reaching a value of 11.98±2.18 g/L. IgG₁ at 17–22 WG was 0.93±0.42 g/L, which is approximately three times higher than IgG₂. IgG, showed an exponential rise and at 37–41 WG its concentration was seven times higher than IgG₂. IgG₃ and IgG₄ also showed an exponential rise and at term reached a similar level as in the maternal circulation. Striking was the difference in results for IgG₂ with a slow linear rise throughout gestation. The fetal IgG₂ level at term remained significantly below the maternal concentration. The IgG subclasses when characterized according to the differences in transport capacity gave the following sequence: IgG₁>IgG₄>IgG₃>IgG₂. Fetal IgA showed a slow linear rise with fetal levels at term remaining approximately 1,000 times lower than the concentration in the maternal circulation. CONCLUSIONS: Comparison of fetal and maternal levels of Immunglobulines indicate that the human placenta during pregnancy develops a specific transport mechanism for IgG. There are differences for the four subclasses with preferential transfer of IgG₁ while the slowest transfer is seen for IgG₂.     

Human Anti-Ro Autoantibodies Bind Peptides Accessible to the Surface of the Native Ro Autoantigen

The relationship between fine specificity of linear epitopes and conformational determinants has been explored in a naturally arising human autoimmune response. In particular, the hypothesis tested is that the linear epitopes of the human Ro autoantigen are components of its conformational epitopes. Twenty groups among the 531 overlapping octapeptides 60kDa Ro are variably bound by anti-Ro precipitin positive lupus sera whose reactivity was easily distinguished from sera of normal controls and of anti-Ro precipitin negative lupus patients. The specific activities of anti-peptide antibodies and of anti-native Ro autoantibodies are similarly increased after affinity enrichment using native human Ro as ligand. Moreover, affinity-enriched anti-native Ro autoantibodies bind virtually the same 20 groups of epitopes recognized by whole anti-Ro positive sera. Two peptides (residues 274–290 and 480–494) from the defined 60 kDa Ro octapeptide epitopes have been prepared and used as ligands for affinity purification of peptide specific autoantibodies. The binding of both whole IgG and affinity-enriched peptide specific autoantibodies is inhibited by native Ro autoantigen. Thus, none of the available data can be construed to support the existence of cryptic linear epitopes in this system. Indeed, the data are only consistent with the conclusion that all of the anti-Ro octapeptide autoantibodies are part of the population of anti-native Ro autoantibodies in this naturally arising autoimmune response.     

Immunoglobulins to Group A Streptococcal Surface Molecules Decrease Adherence to and Invasion of Human Pharyngeal Cells

The M protein is one of the most important virulence factors of group A streptococci (Streptococcus pyogenes) and may play an important role in the first steps of streptococcal infection. Since acute pharyngitis is a frequently occurring infectious disease caused by these bacteria, we wished to know whether antibodies to the M protein or other surface components inhibit adherence and internalization of streptococci to pharyngeal cells. We investigated the role of whole human secretory immunoglobulin A (sIgA), M6 protein-specific sIgA, and M6 protein-specific serum IgG in the inhibition of streptococcal adherence and internalization to cultured human pharyngeal cells. S. pyogenes D471, which produces a type 6 M protein (M+), and its isogenic M-negative (M−) derivative JRS75 were tested. Purified whole sIgA, M protein-specific sIgA, and sIgA preabsorbed with M protein were able to decrease significantly the adherence of streptococci to pharyngeal cells. Purified IgG against the M6 protein did not diminish the attachment of streptococci to the pharyngeal cells but did reduce internalization. Thus, our data suggest that secretory IgA may play a key role in preventing streptococcal infection at mucosal surfaces by blocking adherence while affinity-purified anti-M protein-specific IgG blocks epitopes responsible for invasion.     

Modulation of Pokeweed Mitogen-Induced Human B-Cell Differentiation by Aggregated IgG

The modulatory effect of human heat-aggregated IgG on human B-cell differentiation induced by pokeweed mitogen was investigated with three experimental protocols. Pulse exposure to aggregated IgG, followed by extensive washings before culture, of peripheral blood mononuclear cell suspensions rigorously depleted of platelets and containing less than 4% monocytes resulted in a selective decrease of the numbers of IgG-containing cells and IgG-secreting cells, whereas a simultaneous decrease of the numbers of cells producing IgG and, to a lesser extent, of those producing IgM or IgA was observed when the pulsed suspensions contained platelets and more than 4% monocytes. This non-isotype-specific suppression was shown to be more pronounced when aggregated IgG and platelets were present in the cell suspensions throughout the cultures. The results suggest that two distinct suppressor pathways can be triggered by aggregated IgG. The first one is restricted to cells producing the matching isotype, in the absence of platelets, with few monocytes in the cell suspensions. The second one leads to a nonspecific suppression of the three major Ig classes. It requires the presence of platelets and/or a high percentage of monocytes and, although it remains to be demonstrated, is probably mediated by prostaglandin E2.     

Non-Immune Binding of Human Protein Fv to Immunoglobulins from Various Mammalian and Non-Mammalian Species

Reactivity of the secretory protein Fv with immunoglobulins (Ig) from various species of vertebrates was investigated. Binding was observed throughout all taxonomic classes: mammalian, avian, reptilian, amphibian and fish. Contrasting with this wide spectrum, no significant binding was found with most mammalian ungulates, such as horse (Perissodactyl), cow, sheep and goat (Artiodactyls). Nevertheless, disruption of the hydrogen bonds of Ig allowed these non-reactive molecules to bind. Such a conserved reactivity during evolution, and our previous data on the effect of the cleavage of the intra-chain disulphide bonds, suggest that protein Fv recognizes a discontinuous framework structure involving both the FR1 and FR3 regions in the variable domain of the heavy chain of Ig.     

Immunoglobulins in Feces from Infants Fed Human or Bovine Milk

Concentrations of human immunoglobulins in extracts of feces from infants receiving human milk were higher than in feces from infants fed bovine milk. This was most valid for IgA. The antibody activity, tested with agglutinins to rabbit erythrocytes as markers, was also higher in feces from 1-month-old infants fed human milk than in feces from infants given bovine milk. No difference in agglutinin activity was found later in infancy, Isoagglutinins in human milk to blood group antigens A and B were recovered in feces from infants receiving this milk, regardless of the infants' own blood group. Gel filtration studies, as well as inhibition of agglutination and antiglobulin test, gave evidence that IgA was largely responsible for the fecal agglutinin activity. Unlike human milk in which also IgM reacted with rabbit erythrocytes, shown by immunization of rabbits, only IgA in the fecal extracts seemed to have such activity. IgA antibodies in human milk will thus retain some of their activity with passage through the infant gut     

Purification of J Chain After Mild Reduction of Human Immunoglobulins

Two methods are described for the purification of J chain from polymeric IgA after mild reduction without the use of alkylating or dissociating reagents. The released peptide was separated from other protein components by immunoadsorption combined with gel filtration or anionic-exchange chromatography, or both. J chain was thus obtained in a yield of about 30% of the total release. Most of it consisted of dimers (molecular weight, approximately 25,000 to 30,000) or larger polymers, but re-reduction and alkylation produced a quite homogeneous fraction that sedimented slightly more slowly than egg-white lysozyme. The purity was high enough for successful immunization. When J chain coupled to bovine serum albumin was used as an antigen, all of five rabbits showed a good immune response. Although the same principle could be used for the purification of J chain from IgM and colostral IgA, high purity was more difficult to achieve and the yield was much lower. These preparations contained an unidentified slow-moving component, and the J chain was more prone to become rapidly degraded to smaller fragments.