Identification of a human lactoferrin-binding protein in Gardnerella vaginalis

Previous studies have shown that Gardnerella vaginalis can utilize iron-loaded human lactoferrin as a sole source of iron. In this study, G. vaginalis cells were shown to bind digoxigenin (DIG)-labeled human lactoferrin in a dot blot assay. Using the DIG-labeled human lactoferrin, a 120-kDa human lactoferrin-binding protein was detected by Western blot analysis of G. vaginalis proteins. The lactoferrin-binding activity of this protein was found to be heat stable. Competition studies indicated that this binding activity was specific for human lactoferrin. Treatment of G. vaginalis cells with proteases suggested that this protein was surface exposed. An increase in lactoferrin binding by the 120-kDa protein was observed in G. vaginalis cells grown under iron-restrictive conditions, suggesting that this activity may be iron regulated.     

Identification and characterization of the human lactoferrin-binding protein from Neisseria meningitidis.

Lactoferrin-binding activity in Neisseria meningitidis was detected by a solid-phase binding assay with horseradish peroxidase-conjugated human lactoferrin (HRP-lactoferrin). Expression of lactoferrin-binding activity was regulated by the level of iron in the medium, so that growth in the presence of the iron chelator EDDA (ethylenediamine di-ortho-hydroxyphenylacetic acid) resulted in a greater than 350-fold increase in binding activity, which was reversed by addition of excess iron. A maximal level of expression could be obtained at reasonable culture densities by using either intermediate levels of EDDA or high levels of EDDA and moderate levels of complexed iron sources such as hemoglobin and transferrin. Competition binding assays demonstrated that the binding of lactoferrin was specific for human lactoferrin in that neither bovine lactoferrin, human transferrin, nor human hemoglobin was able to block binding of HRP-lactoferrin. The binding specificity for human lactoferrin correlated with growth studies in which human but not bovine lactoferrin could support the growth of iron-starved cells. Binding of lactoferrin was not dependent on its level of iron saturation, since iron-saturated lactoferrin and apolactoferrin were equally effective at blocking binding of HRP-lactoferrin in competitive binding assays. The lactoferrin-binding protein was identified as a 105,000-molecular-weight iron-regulated outer membrane protein in three different meningococcal strains by a batch affinity method with biotinylated human lactoferrin and streptavidin-agarose.     

Identification of a Staphylococcus aureus extracellular matrix-binding protein with broad specificity.

A staphylococal surface protein capable of binding several extracellular matrix glycoproteins was purified as a result of our attempts to identify a receptor(s) for bone sialoprotein (BSP) on Staphylococcus aureus cells. Proteins from different staphylococcal strains were solubilized in sodium lauryl sulfate, separated by polyacrylamide gel electrophoresis, blotted onto Immobilon P membranes, and probed with 125I-BSP. Several bacterial proteins bound the radiolabeled ligand, and various strains expressed different repertoirs of BSP-binding proteins. Major BSP-binding proteins with apparent M(r)s of 72,000 or 60,000 were present on most strains, and these proteins were further studied. The 72- and 60-kDa proteins were preferentially expressed when bacteria were cultured in Luria broth compared with when they were cultured on tryptic soy broth, and the abundance of the proteins could be correlated to an increased 125I-BSP binding. Both the 72-kDa and the 60-kDa proteins were solubilized by extraction of cells with 1 M LiCl and were purified by cation-exchange chromatography. Amino acid composition analysis of the purified 72-kDa protein indicated a high content of lysine (11.9%) and hydrophobic amino acids (28.0% combined). In Western ligand blotting (immunoblotting) experiments, the 72-kDa protein bound not only BSP but also radiolabeled fibronectin, fibrinogen, vitronectin, thrombospondin, and, to some extent, collagen. Addition of the purified 60-kDa protein to S. aureus cells did not inhibit binding of the different ligands but in some cases resulted in an augmentation of the binding of 125I-ligand. Purified 60-kDa protein could hemagglutinate sheep erythrocytes at a concentration of 61 micrograms/ml. The agglutination reaction was inhibited by high concentrations of fucose, mannose, or melibiose. These data suggest that the purified proteins may serve as bacterial receptors with broad specificity for matrix glycoproteins and that the proteins may act as carbohydrate-binding proteins.     

Identification of pneumococcal surface protein A as a lactoferrin-binding protein of Streptococcus pneumoniae

Lactoferrin (Lf), an iron-sequestering glycoprotein, predominates in mucosal secretions, where the level of free extracellular iron (10(-18) M) is not sufficient for bacterial growth. This represents a mechanism of resistance to bacterial infections by prevention of colonization of the host by pathogens. In this study we were able to show that Streptococcus pneumoniae specifically recognizes and binds the iron carrier protein human Lf (hLf). Pretreatment of pneumococci with proteases reduced hLf binding significantly, indicating that the hLf receptor is proteinaceous. Binding assays performed with 63 clinical isolates belonging to different serotypes showed that 88% of the tested isolates interacted with hLf. Scatchard analysis showed the existence of two hLf-binding proteins with dissociation constants of 5.7 x 10(-8) and 2.74 x 10(-7) M. The receptors were purified by affinity chromatography, and internal sequence analysis revealed that one of the S. pneumoniae proteins was homologous to pneumococcal surface protein A (PspA). The function of PspA as an hLf-binding protein was confirmed by the ability of purified PspA to bind hLf and to competitively inhibit hLf binding to pneumococci. S. pneumoniae may use the hLf-PspA interaction to overcome the iron limitation at mucosal surfaces, and this might represent a potential virulence mechanism.     

Isolation of a fibronectin-binding protein from Staphylococcus aureus.

Fibronectin ("cold-insoluble globulin") has been suggested to play a role in cell-to-cell and cell-to-substratum adhesions. The 70-kilodalton terminal part of human fibronectin has recently been shown to bind to Staphylococcus aureus. In the present study, a fibronectin-binding protein was purified from sonicated S. aureus strain E2371 by affinity chromatography on fibronectin-Sepharose. The fibronectin-binding protein was isolated from an extract of sonicated S. aureus containing at least 57 different proteins as determined by crossed immunoelectrophoresis in antibodies to sonicated S. aureus. The fibronectin-binding protein was released from fibronectin-Sepharose by carbamide (8 M). No impurities in the final preparation could be detected when tested in crossed immunoelectrophoresis. By polyacrylamide gel electrophoresis in both reduced and unreduced gels, the protein showed two bands with relative molecular masses of 197,000 and 60,000, respectively. A complex between the purified S. aureus protein and fibronectin could be demonstrated by crossed immunoelectrophoresis both in monospecific antibodies against fibronectin and in S. aureus polyspecific antibody.     

Immunological response to a Staphylococcus aureus fibronectin-binding protein.

A protein (gal-FnBP), constructed by fusion of the genes encoding beta-galactosidase of Escherichia coli and the binding domains of fibronectin-binding protein (FnBP) of Staphylococcus aureus was used. FnBP is a surface protein responsible for attachment of bacteria to extracellular matrix of various host tissues. Gal-FnBP is more stable and can be produced in larger quantities than native FnBP. The binding specificity of this fusion protein was established in a Western blot analysis. Treatment of gal-FnBP with formalin inactivated the binding capacity of the protein but immunogenicity was retained. Immunisation of mice with formalin-treated gal-FnBP resulted in high antibody titres against the fibronectin-binding part of this fusion protein. These antibodies were measured by their ability to block the specific binding of fibronectin to gal-FnBP in a blocking assay. Sera raised against formalin-treated gal-FnBP and non-treated gal-FnBP blocked this binding to 40 and 25% respectively, thereby indicating the usefulness of gal-FnBP as a vaccine component.     

Identification of a chromosomal determinant of alpha-toxin production in Staphylococcus aureus.

Production of alpha-toxin (the Hla+ phenotype, controlled by the Hla gene and scored as alpha-hemolytic activity) is a property of some isolates of Staphylococcus aureus NCTC 8325 and not of others. Genetic transformation between strains differing in the Hla phenotype revealed that the hla+ gene resides in the following sequence: purB110-bla+-hla+-ilv-129-pig-131; previously, the enterotoxin A (entA) gene of strain S-6 was shown to map very close to hla+. The hla+ mutations occurring naturally in strain Ps6 and after various mutagenic treatments in strains 8325 and 233 also mapped between bla+ and ilv-129. Among the isolates of strain 8325, the Hla+ phenotype was always associated with fibrinolytic activity, whereas Hla- isolates were non-fibrinolytic. This relationship was also observed among transformants selected for their Hla+ or Hla- phenotypes. The failure of Hla- strains and mutants to revert to hla+ at detectable frequencies, the instability of the Hla+ phenotype, and the previously observed pattern of recombination of the hla+ and entA+ determinants lend support to the view that hla+ may reside on a transposon; according to this view, Hla- mutants have lost the hla+-bearing transposon. It remains unclear whether hla+ is the structural gene for alpha-toxin.     

Binding of human immunoglobulin G to protein A in encapsulated Staphylococcus aureus.

Recent studies of the mechanism of resistance to phagocytosis in encapsulated Staphylococcus aureus have suggested that the capsule is readily penetrated by high-molecular-weight proteins such as antibodies and complement components. S. aureus strains contain a cell wall protein, protein A, that reacts with the Fc portion of immunoglobulins. The binding of immunoglobulin G (IgG) to encapsulated and unencapsulated S. aureus strains has been studied to assess the penetrability of the S. aureus capsule by IgG. Encapsulated S. aureus strains M and Smith diffuse bound large amounts of human IgG which were comparable to amounts bound by the unencapsulated strains Cowan I, M variant, and Smith compact. Trypsin treatment of bacteria reduced their ability to bind IgG. Bound IgG was not removed by extensive washing of bacteria with buffer. A non-protein A-containing, coagulase-negative, encapsulated staphylococcal strain did not bind IgG. These observations suggest that IgG is binding to cell wall protein A in encapsulated S. aureus. No differences in the rates of IgG binding by encapsulated and unencapsulated S. aureus strains were observed. It is concluded that the S. aureus capsule is freely permeable to IgG. This is of importance in considerations of the mechanisms of resistance to phagocytosis and antigen masking in encapsulated microorganisms.     

Identification of a new repetitive element in Staphylococcus aureus

The Staphylococcus aureus repeat (STAR) element is a sequence identified in two intergenic regions in S. aureus. The element is found in 13 to 21 copies in individual S. aureus strains, and elements in the homologous intergenic location are variable in length. The element sequence consists of several small and unusually GC-rich direct repeats with recurring intervening sequences. In addition, STAR-like elements may be present in related staphylococcal species.     

Occurrence of protein A in Staphylococcus aureus and closely related Staphylococcus species.

All but 1 of 143 strains of Staphylococcus aureus were positive for protein A, whereas all 34 strains of Staphylococcus hyicus and 123 of 127 strains of Staphylococcus intermedius were devoid of this cell wall component.     

Activation of human B and T lymphocytes by protein A of Staphylococcus aureus.

Protein A from Staphylococcus aureus, in soluble form or coupled to Sepharose beads, acts as a polyclonal B cell activator (PBA) for human lymphocytes in blood and spleen. PBA activity was demonstrated in spleen cells by the ability of protein A to induce the formation of intracellular immunoglobulin synthesis and to activate polyclonal antibody secretion demonstrated against fluorescein isothiocyanate-coupled sheep erythrocytes in a modified hemolysis in gel assay. More plaqueforming cells (PFC) were seen in unseparated cells than in purified B cells. In blood lymphocytes, only few PFC were activated by soluble protein A. Protein A increased DNA synthesis in blood and spleen cells. At a concentration of 100 microgram/ml the peak response was on day 4 or 5, but at 1 microgram/ml the peak response occurred later. On day 4 of culture, high mitogenic activity was seen in unseparated lymphocytes or mixtures of separated B and T cells, whereas in enriched B and T cell suspensions activity was low. On day 7, however, DNA synthesis in both the enriched B and T cells was higher than in mixtures of B and T cells. Protein A stimulated DNA synthesis in thymus cells with a peak response on day 6. It is concluded that protein A alone or as an IgG complex can activate both B and T cells, though the mechanism of activation is not known and may be different for B and T cells.     

Lymphocyte stimulation by protein A of Staphylococcus aureus.

Protein A from Staphylococcus aureus (SpA) is known to bind to the Fc region of most mammalian IgG classes. In the present article data are presented showing that SpA is a highly efficient mitogen for human peripheral B lymphocytes, with no detectable activity for T lymphocytes. In order to achieve optimal stimulating conditions SpA should be presented to the lymphocytes on an insoluble matrix, such as the SpA-positive bacteria themselves or SpA covalently attached to Sephadex or Sepharose beads. Using such conditions SpA is equivalent with regard to stimulatory capacity for B lymphocytes as phytohemagglutinin is for the human T lymphocytes. Specificity controls proved beyond doubt that SpA and not any other contaminating product is the B cell mitogen. It is concluded that SpA as an inducer of human B lymphocyte division might serve as a highly useful assay in the clinical assessment of B lymphocyte function. It should also be a suitable tool in the fine analysis of B lymphocyte activation via the specific interactions with surface IgG molecules.     

Stimulation of human lymphocyte subpopulations by protein A from Staphylococcus aureus

Protein A from Staphylococcus aureus is known to stimulate human lymphocytes. Using 3H-thymidine incorporation, virus plaque assay and induction of cytotoxic T lymphocytes (CTL), this study showed that soluble or insoluble protein A stimulated different lymphocyte subpopulations. Soluble protein A is highly mitogenic to T lymphocytes. In both 3H-thymidine incorporation and virus plaque assay, its maximum stimulation was as high as the stimulation by the nonspecific mitogens phytohemagglutinin and concanavalin A, and a higher CTL response than that induced by phytohemagglutinin or concanavalin A was induced. Mitogenic activity to B lymphocytes was negligible. S. aureus (Cowan I strain) is itself considered to be an insoluble form of protein A and is 3--4 times more mitogenic to B lymphocytes than pokeweed mitogen without any increase in virus plaque-forming cells. No mitogenicity was noted to T lymphocytes. Sepharose CL-4B-protein A, also known as insoluble protein A, stimulated both T and B lymphocytes effectively, but its mitogenicity to T lymphocytes was considered to be due to the soluble protein A released from the Sepharose CL-4B beads.     

Staphylococcus aureus binding to human nasal mucin.

Colonization of human nasal mucosa with Staphylococcus aureus sets the stage for subsequent systemic infection. This study characterizes S. aureus adhesion to nasal mucosa in vitro and investigates the interaction of S. aureus with human nasal mucin. S. aureus binding to cell-associated and cell-free mucus was greater than to nonmucin-coated epithelial cells. Scanning electron microscopy of S. aureus incubated with human nasal mucosal tissue showed minimal binding to ciliated respiratory epithelium. In a solid-phase assay, S. aureus bound to purified human nasal mucin-coated wells significantly more than to bovine serum albumin-coated microtiter wells. Binding to mucin was saturable in a dose- and time-dependent fashion. Staphylococcal adherence to human nasal mucin was inhibited by bovine submaxillary mucin but not by fibrinogen. Pretreatment of mucin with periodate but not with pronase reduced adherence. Trypsin treatment of the bacteria significantly reduced adherence to mucin. 125I-labelled nasal mucin bound to two surface proteins (138 and 127 kDa) of lysostaphin-solubilized S. aureus. Binding to human nasal mucin occurs in part via specific adhesin-receptor interactions involving bacterial proteins and the carbohydrate moiety in mucin. These experiments suggest that S. aureus binding to mucin may be critical for colonization of the nasopharyngeal mucosa.     

A radioimmunoassay of human leukocyte interferon using protein A-containing Staphylococcus aureus

We report the establishment of a radioimmune assay for human alpha-interferon using highly purified alpha-interferon labeled with iodine-125, a purified rabbit immunoglobulin directed against human interferon and the Cowan strain of Staphylococcus aureus. Radiolabeling conditions used do not change the antigenicity of interferon molecules. The regression of counts bound upon log. dose was found to be linear down to 10 units/ml of alpha-interferon (6-7 X10-12 M). This assay was specific for alpha-interferons derived from human peripheral blood leukocytes and from a continuous line of lymphoblastoid cells. No cross-reaction was found with either human beta-interferon or murine interferon. Neither human serum nor plasma interfered with the assay. Correlation between biological assay and radioimmune assay was found to be significant.     

Expression of a cloned Staphylococcus aureus alpha-hemolysin determinant in Bacillus subtilis and Staphylococcus aureus.

A DNA sequence encoding Staphylococcus aureus alpha-hemolysin, which had been previously cloned and mapped in Escherichia coli K-12, was introduced into Bacillus subtilis BD170 and several strains of S. aureus by using plasmid vectors, some of which could replicate in all three organisms. The determinant was cloned on a 3.3-kilobase pair DNA fragment into B. subtilis by using the vector plasmid pXZ105 to form the hybrid plasmid pXZ111. B. subtilis cells harboring pXZ111 produced large zones of alpha-hemolysis after 18 h of growth at 37 degrees C on rabbit blood agar plates, and alpha-hemolysin activity was detected in supernatants prepared from growing cultures of this strain. The alpha-hemolysin was apparently secreted across the B. subtilis cell envelope. Polypeptides of molecular weights 34,000 and 33,000 were precipitated with anti-alpha-hemolysin serum from lysates prepared from BD170 cells harboring pXZ111. A hybrid replicon which could replicate in both E. coli and S. aureus was constructed in E. coli by ligating a HindIII fragment encoding the replication functions and chloramphenicol resistance genes of S. aureus plasmid pCW59 to the pBR322 alpha-hemolysin hybrid plasmid pDU1150. The DNA of this plasmid, pDU1212, was prepared in E. coli and used to transform protoplasts prepared from a non-alpha-hemolytic, nonrestricting strain of S. aureus RN4220. Some of the transformants contained plasmids which had suffered extensive deletions. Some plasmids, however, were transformed intact into RN4220. Such plasmids were subsequently maintained in a stable manner. pDU1212 DNA was prepared from RN4220 and transformed into alpha-hemolytic S. aureus 8325-4 and two mutant derivatives defective in alpha-hemolysin synthesis. All three strains expressed alpha-hemolysin when harboring pDU1212.     

Fibrinogen-binding protein/clumping factor from Staphylococcus aureus.

The binding of staphylococcal components to fibrinogen was studied. Fibrinogen-binding material from lysed staphylococcal cells or culture supernatants was affinity purified on fibrinogen-Sepharose and analyzed on Western (immuno-) blots by the use of fibrinogen and antifibrinogen antibodies. Two main bands of 87 and 19 kilodaltons (kDa) and a weaker band of 35 kDa bound specifically to fibrinogen. A monoclonal antibody bound to all three bands, indicating that these were of the same origin. The yield of these components was much higher in the culture supernatant than on washed cells, suggesting that these molecules are essentially extracellular products. In a plasma coagulase test, the 87-kDa band, but not the 19-kDa band, clotted rabbit plasma, demonstrating that the 87-kDa molecule is coagulase. This was further confirmed by the fact that the 87-kDa band binds specifically to prothrombin. It was shown that the 87- and the 19-kDa molecules were present on the cell surface by surface labeling the cells with 125I. In addition, the fact that killed and washed cells could induce plasma clotting demonstrates that staphylococci have coagulase exposed on the surface. It was concluded that cell-bound coagulase has affinity for fibrinogen also in the absence of prothrombin and thus is responsible for the clumping of staphylococci in fibrinogen.     

Aggregation of human granulocytes by Staphylococcus aureus lipase.

The effects of purified extracellular lipase from Staphylococcus aureus on human granulocytes were studied in vitro with a turbidimetric technique. Within the concentration range 0.6-4.4 micrograms/ml, lipase caused monophasic aggregation accompanied by the release of lactoferrin; the corresponding concentrations of the solvent in which it was suspended, Triton X100, had no effect. Lipase-induced aggregation did not occur in the presence of autologous plasma.     

Determination of protein A in Staphylococcus aureus strains from a collection

The author used the haemagglutination method to estimate A protein in 153 strains of Staphylococcus aureus from collections. Protein A was detected in 66% of coagulase positive strains deposited in the course of time in the collection in 1983-1988. All strains were kept in skimmed milk at -20 degrees C. For evaluation of the reaction a semiquantitative scale was proposed. The author observed a diminished capacity to form protein A with the time of storage of the strains. In strains deposited for a shorter time in the collection (only three years) the percentage of positive results rose to 92.5%. In coagulase negative strains protein A was not detected.     

Radioimmunoassays for protein A of Staphylococcus aureus

Radioimmunoassays have been developed that can detect nanogram amounts of protein A (SpA), a product generated by Staphylococcus aureus that binds selectively to the Fc region of IgG from most mammalian species. Competition assays for fluid phase SpA utilize antibodies produced in chickens, 125I-labeled SpA as the tracer molecule, and either F(ab')2 fragments of rabbit IgG anti-chicken IgG or 40% ammonium sulfate as the precipitating agent to separate antigen-antibody complexes from free antigen. The double antibody assay could be carried out in serum from species that form only soluble complexes with SpA (e.g., rabbit), that react poorly with SpA (e.g., rat), or under appropriate conditions in serum from species (e.g., dog) that show high reactivity with SpA and form precipitating complexes. Chicken antibodies prepared by affinity chromatography on SpA-Sepharose and labeled with 125I were used in a direct binding assay for SpA present either on the cell wall of Cowan strain I or Wood 46 bacteria, in insoluble complexes prepared from SpA and whole serum or purified IgG, or in Clq binding complexes that were formed by passage of serum from normal or tumor bearing humans or dogs over SpA-collodion charcoal. Since both types of assays could detect SpA even in the presence of serum or IgG, they offer advantages over other techniques in which the SpA-Fc interaction may interfere.