Cloning, expression, and nucleotide sequence of a Staphylococcus aureus gene (fbpA) encoding a fibrinogen-binding protein.

Septicemia due to Staphylococcus aureus often begins as a focal infection (e.g., colonized wounds or catheters) from which the organism gains access to the bloodstream. On the basis of recent data from this laboratory, it is likely that S. aureus colonizes catheters and endothelium by using a fibrinogen-binding protein to mediate adhesion to fibrinogen-coated surfaces. To characterize the fibrinogen-reactive protein, we screened a lambda Zap library of S. aureus DB, a clinical isolate, for clones that were reactive with fibrinogen. Of 100,000 plaques screened, 3 were found to react with fibrinogen on immunoblots. Plasmid DNA prepared from clones 14, 30, and 36, upon digestion with EcoR1, which released the insert, revealed fragments of 4.6, 3.6, and 3.2 kb, respectively. To identify the cloned protein expressed in E. coli, cells were fractionated into periplasmic, membrane, and cytoplasmic fractions. Expression studies of clone 14, which comprised approximately two-thirds of the mature molecule, including the C terminus, revealed a 34-kDa fibrinogen-reactive protein in both the periplasmic and membrane fractions. This protein, designated FbpA, could be partially purified on a fibrinogen column. By using both clones 14 and 36 as templates, the complete DNA sequence of the fibrinogen-binding protein was obtained, yielding a molecule with a predicted size of 69,991 Da. Although sequence analysis revealed a high degree of homology with coagulase, there is a unique sequence of 11 amino acids that is not found in three known coagulases as well as two recently cloned fibrinogen-binding proteins. This unique sequence shares homology with a cell wall anchor motif found in other gram-positive surface proteins.     

Isolation of heart- and kidney-binding protein from group A streptococci.

Tritium-labeled, water-soluble components of Streptococcus pyogenes type M6 absorbed to cardiac tissue in vitro. Tissue binding was time dependent, saturable, and reversible. Chromatography of the crude bacterial extract on Bio-Gel P-300 indicated a molecular weight greater than 300,000 for the heart-binding component. Sodium dodecyl sulfate (SDS) dissociated this aggregate into a protein of 18,000 to 20,000 daltons as determined by Sephacryl S-200 chromatography and SDS-polyacrylamide disc gel electrophoresis. The tissue-binding protein was also purified from streptococcal extracts by absorption to immobilized heart components. SDS-polyacrylamide gel electrophoresis of the protein desorbed from tissue revealed a radioactive band of 19,000 daltons. Indirect immunofluorescence tests on cardiac tissue treated with streptococcal extract showed an accumulation of a bacterial antigen on the sarcolemmal sheaths. Streptococcal components also adsorbed to basement membranes of kidney. Antisera prepared to isolated cytoplasmic membranes and water-soluble extracts of S. pyogenes type M6 were the most sensitive reagents for the detection of bacterial components bound to tissue. Antisera prepared to isolated cell walls and to intact bacteria were weakly reactive in these assays.     

Binding of human fibronectin to group A, C, and G streptococci

A total of 387 bacterial strains belonging to 35 species were tested in direct binding experiments for the uptake of purified radiolabeled human fibronectin. Positive binding was found in group A, C, and G streptococci and in Staphylococcus aureus. The group C streptococcal species Streptococcus equisimilis, Streptococcus zooepidemicus, Streptococcus equi, and Streptococcus dysgalactiae were uniformly reactive with fibronectin. Beta-hemolytic bovine group G streptococci showed the same degree of reactivity as human group G strains. In contrast, only 4 out of 15 alpha-hemolytic bovine group G strains were able to bind fibronectin. The uptake of fibronectin measured at room temperature with a human group G streptococcus was a slow, time-dependent process with maximum binding after approximately 1 h. Human polyclonal immunoglobulin G and serum albumin tested in inhibition experiments did not affect the fibronectin binding. Fibronectin seems, therefore, to interact with a surface component that is different from the specific binding sites previously described for human immunoglobulin G and serum albumin.     

Interaction of soluble fibronectin with group B streptococci.

Fibronectin binds to a variety of bacterial species, and we hypothesized that differential fibronectin binding might influence the invasive potential of group B streptococci (GBS). Human plasma fibronectin purified by a standard two-step chromatographic procedure was radiolabeled with 3H. Fifty GBS strains (invasive, colonizing, or bovine) representing serotypes Ia (10 strains), Ib (6 strains), Ia/c (6 strains), II (10 strains), III (11 strains), IV (1 strain), and nontypable (6 strains) were tested. No source or serotype variability was detected among GBS strains, and binding was uniformly less than 1.5% of available fibronectin. Lack of detectable binding occurred at both the log and stationary growth phases and persisted despite treatment with trypsin or neuraminidase or opsonization with immunoglobulin G containing high levels (greater than 40 micrograms/ml) of antibody specific for the Ia, II, or III GBS capsular polysaccharides. Incubation with GBS did not inhibit fibronectin binding to the Cowan 1 strain of Staphylococcus aureus. Strain COH 31-15, an isogenic, type III, capsule-deficient mutant of COH 31r/s, also failed to bind fibronectin. In contrast to other streptococci, GBS do not have readily detectable receptors for soluble fibronectin as part of their surface structures. If present, binding sites for soluble fibronectin are deep to surface structures, obscured from host defense systems, or require the presence of other factors to facilitate their recognition of fibronectin. The uniform ability of GBS to resist binding to soluble fibronectin could be a significant virulence factor in the pathogenesis of invasive infections of infants.     

Adherence of group A streptococci to fibronectin on oral epithelial cells.

The possibility that fibronectin on the surface of oropharyngeal cells may serve as a receptor for the binding of group A streptococci (Streptococcus pyogenes) was investigated. Purified human plasma fibronectin inhibited the adherence of group A streptococci to oral epithelial cells in a dose-dependent manner. The relative amounts of fibronectin available on oral epithelial cells correlated closely with the ability of these cells to bind streptococci. Group A streptococci agglutinated latex beads containing covalently linked fibronectin on their surface, and this agglutination could be inhibited by lipoteichoic acid, the adhesion that mediates attachment of group A streptococci to epithelial cells. Gelatin and the alpha 1 chain of type I collagen partially inhibited both the adherence of streptococci to oral epithelial cells and the binding of radiolabeled fibronectin to streptococci; however, the purified fibronectin-binding peptide of collagen, alpha 1 (I)CB7, inhibited neither. The binding of radiolabeled fibronectin to streptococci was inhibited by lipoteichoic acid. These results suggest that fibronectin on oral epithelial cells serves as a lipoteichoic acid-sensitive receptor for group A streptococci.     

Cloning, sequencing and expression of GroEL-like protein gene of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is an important enteropathogen in regions where much seafood is consumed. Substantial quantity of GroEL-like protein is produced during the heat shock of V. parahaemolyticus and located in periplasmic and extracellular fractions. In this study, the GroEL-like protein gene of this pathogen was cloned and sequenced and its properties were analyzed. The open reading frame consisted of 1647 bp, encoded a 57.6-kDa GroEL-like protein of 548 amino acids. The amino acid sequence, hydrophobicity and antigenic pattern of V. parahaemolyticus GroEL-like protein were similar to the GroEL proteins of other vibrios, Escherichia coli and Salmonella enterica Typhi. The groEL-like gene of V. parahaemolyticus was cloned into the pQE-30 expression vector and the expression of a His-tagged GroEL-like protein was rapidly induced by isopropyl-beta-D-thiogalactopyranoside, largely in an insoluble form. The results of this study facilitate the study of the functions of the GroEL-like protein of V. parahaemolyticus.     

Cloning of the glutamine synthetase gene from group B streptococci.

The glnA gene from the human pathogen Streptococcus agalactiae was cloned from a genomic library prepared with the lambda phage vector lambdaDASHII. A 4.6-kb DNA fragment of one of the recombinant phages was subcloned in pUC18. This Escherichia coli clone expressed a 52-kDa protein encoded by a 1,341-bp open reading frame. The nucleotide sequence of the open reading frame and the deduced amino acid sequence shared a significant degree of homology with the sequences of other glutamine synthetases (GS). The highest homology was between our deduced protein and GS of gram-positive bacteria such as Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus. Plasmids with the cloned streptococcal glnA were able to complement E. coli glnA mutants grown on minimal media. Rabbit antisera to streptococcal GS recombinant protein recognized not only the recombinant protein but also a similar-sized band in mutanolysin extracts of all group B streptococcal strains tested, regardless of polysaccharide type or surface protein profile. The amino acid sequence of the deduced protein had similarities to other streptococcal cell-surface-bound proteins. The possible functional role of the immunological features of streptococcal GS is discussed.     

Biological and immunochemical identity of M protein on group G streptococci with M protein on group A streptococci.

Previous evidence for the presence of an M or M-like protein on group G streptococci has been based on the ability of these strains to survive in human blood. In addition, cross-reactions between group A and group G streptococci have been demonstrated, but they have relied either on whole bacterial cell vaccine-induced polyclonal sera or crude protein extracts of these cells. In this study two monoclonal antibodies prepared against the purified, native group A streptococcal M6 protein demonstrated a high degree of cross-reactivity with group G streptococcal clinical isolates (9 and 19 of 22 strains examined, respectively). Ten of these strains exhibited resistance to phagocytosis when rotated in human blood. In addition, immunoblot analysis of crude mutanolysin extracts of group G streptococci with one of the M6 monoclonal antibodies illustrated a remarkable similarity in the protein pattern of these extracts as compared with those of group A streptococcal M protein. The immunoblots further demonstrated a variation in the relative molecular weights of the extracted proteins from strain to strain over a range of 57,000 to 77,000. In addition, a purified, pepsin-derived fragment (Mr, 43,000) from a group G strain was capable of eliciting rabbit antibodies that were opsonic for group G cells in a bactericidal assay. These functional and immunochemical data, in concert with DNA hybridization between group G streptococcal DNA and a group A M6 gene probe (J. R. Scott, W. M. Pulliam, S. K. Hollingshead, and V. A. Fischetti, Proc. Natl. Acad. Sci. USA 82:1822-1826, 1985), provide strong evidence for the presence of an M protein on these organisms and indicate its probable role as a virulence molecule on the surface of group G streptococci.     

Binding of fibronectin to the surface of group A,C, and G streptococci isolated from human infections

Sixty-nine haemolytic and non-haemolytic streptococcal strains were isolated from various human infections and serogrouped with the coagglutination test. The amount of¹²⁵I-fibronectin bound to bacterial cells in a standard assay was determined. Most of the group A, C, and G strains were able to bind fibronectin. None of the group B or D strains bound significant amounts of fibronectin. Group A, C, and G streptococci appear to preferentially bind the N-terminal region of the fibronectin molecule because the 25K N-terminal fragment of the protein could effectively inhibit the binding of¹²⁵I-fibronectin to cells. Furthermore, the ability of representative strains of group A, C, and G to bind fibronectin was markedly reduced after trypsin treatment of the cells. Fibronectin binding components released from one strain by trypsin treatment inhibited the binding of¹²⁵I-fibronectin to all group A, C, and G streptococci strains. These findings indicate similarities among fibronectin binding proteins of the three groups of streptococci tested. However, the relative susceptibility to trypsin of fibronectin receptors of the three strains differed as did the relative potency of the inhibitory activity of receptors solubilized from different strains. Binding of fibronectin to the cell surface of group A, C, and G streptococci may contribute to virulence, for instance by promoting specific attachment to exposed fibronectin in open wounds and tissue lesions.     

Cloning and expression of the CAMP factor of group B streptococci in Escherichia coli.

The genetic determinant of the CAMP factor from a strain of group B streptococcus (GBS; Streptococcus agalactiae) was cloned in Escherichia coli. Total cell DNA from the GBS strain R268 was used to construct a gene bank with bacteriophage lambda EMBL4 in the E. coli K-12 strain LE392. Recombinant phage plaques were detected by immunoblots by using a specific antiserum raised against purified CAMP factor. Two hybrid phages showing expression of CAMP factor were identified. Subcloning the CAMP gene (cfb) into the high-copy-number vector pUC8 resulted in highly unstable plasmids. Therefore, subcloning was performed with the low-copy-number vector pLG339 resulting in the stable recombinant plasmids pCO61 and pCO62 which lead to expression of CAMP protein first identified by colony immunoblotting. Western blot (immunoblot) analysis revealed a similar CAMP protein pattern in lambda EMBL4 recombinant phage lysates (molecular weight, 22,000 to 24,000) as compared to that obtained from a GBS culture supernatant (molecular weight, 22,000 to 26,000) but a different CAMP protein pattern (molecular weight, 20,000 to 23,000) from lysates of E. coli carrying pCO61 or pCO62. To study the excretion of the CAMP protein we performed a semi-quantitative dot blot analysis of proteins recovered from cell fractions and supernatants of the E. coli recombinant clones. In contrast to GBS R268, where the CAMP factor is readily excreted, the CAMP protein is not excreted in E. coli clones containing pCO61 and pCO62 but is found associated with the cell fractions.     

Isolation and molecular characterization of a novel albumin-binding protein from group G streptococci.

Many streptococcal strains are known to bind the two most abundant plasma proteins, namely, immunoglobulin G and albumin. Protein G isolated from group C and G streptococci has been demonstrated to have separate binding regions for each of these proteins. However, some group G streptococcal strains bind only serum albumin. This report describes the isolation of a 48-kDa albumin-binding protein from such a strain (DG12). The affinity constant of this protein for human serum albumin was determined to be 5 x 10(9) M-1, and the protein interacted strongly also with serum albumin from several other mammalian species. The gene encoding the albumin-binding protein was cloned and expressed in Escherichia coli. DNA sequence analysis of this gene revealed a unique NH2-terminal sequence and three types of repeats in the encoded protein. One of these repeated sequences has significant homology with the albumin-binding domains of protein G, and it was demonstrated that the albumin binding of the DG12 protein was localized within these domains. Another type of repeat is localized in the putative wall-spanning region of the molecule. This repeat sequence, which has the length of only 4 amino acids (LysProGluVal), is repeated 14 times. The relationship of the albumin-binding protein to other cell-wall-associated proteins of pathogenic streptococci is discussed.     

A potent antimitogenic factor from group A streptococci.

A cytoplasmic component from group A streptococci produced complete suppression of human lymphocyte transformation induced by phytohaemagglutinin or the mixed lymphocyte reaction in vitro. It also suppressed antibody-forming cells in mice against sheep erythrocytes. The active substance was eluted as second and third fractions form Sephadex G-200 chromatography of the 100,000 g supernatant of sonically ruptured group A streptococci. The antimitogenic activity was not susceptible to trypsin, pronase, RNase or DNase digestion, but the activity was completely lost when it was sequentially digested, first with RNase and DNase and then with pronase. The active substance was not antigenic nor heat-labile at 56 degrees. It may be a protein component of a nucleoprotein.     

Genetic separation of serum opacity factor from M protein of group A streptococci.

Mutagenized cultures of three strains of group A streptococci which were M positive and produced the extracellular serum opacity factor (OF) were screened for mutants which failed to exhibit OF activity on serum agar plates. All cell fractions from three mutants studied in detail, including culture supernatants, protoplast membranes, and cytoplasmic fractions, were completely devoid of activity. Two OF- isolates also lacked OF antigen, whereas the third continued to produce cross-reacting antigen. All three mutants were resistant to phagocytosis and yielded acid-extractable M antigen, indicating that the M protein of each strain was unaltered by the mutations. The separation of the OF+ and M+ phenotypes by mutation establishes that genes which code for the M protein and OF are distinct; therefore, the activities and antigenic determinants of each must be associated with separate, distinct protein molecules.     

Cloning and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae.

An expression library was constructed from Actinobacillus pleuropneumoniae serotype 7. Escherichia coli transformants expressing recombinant proteins were identified by immunoscreening with porcine convalescent serum. One transformant expressing a 60-kDa protein (60K protein) in aggregated form was identified. Serum raised against the recombinant protein recognized a polypeptide with an indistinguishable electrophoretic mobility in the A. pleuropneumoniae wild type after iron-restricted growth only. The recombinant protein bound transferrin after blotting onto nitrocellulose. Using a competitive enzyme-linked immunosorbent assay (ELISA), the specificity of this binding for the amino-terminal half of iron-saturated porcine transferrin was established. Also, the 60K wild-type protein bound hemin as assessed by hemin-agarose chromatography. Hemin could inhibit transferrin binding of the recombinant protein in the competitive ELISA, whereas hemoglobin and synthetic iron chelators failed to do so. Southern blot analysis of several other A. pleuropneumoniae strains indicated that highly homologous sequence is present in eight of eight isolates of serotype 7 and in some isolates of serotypes 2, 3, and 4.     

Cloning, sequencing, and expression of a gene from Campylobacter jejuni encoding a protein (Omp18) with similarity to peptidoglycan-associated lipoproteins.

A Campylobacter jejuni genomic plasmid library was screened with antiserum generated against whole C. jejuni, revealing two immunoreactive clones. Sequence analysis of the recombinant plasmids revealed a common open reading frame of 498 nucleotides encoding a protein of 165 amino acids with a calculated molecular mass of 18,018 Da. The recombinant product partitioned to the outer membrane fractions of Escherichia coli transformants and has been designated Omp18. The deduced amino acid sequence of the cloned C. jejuni gene exhibits considerable similarity to peptidoglycan-associated lipoproteins from other gram-negative bacteria.     

Role of M protein in adherence of group A streptococci.

The role of the M protein in adherence of group A streptococci to human epithelial cells was directly tested by using an isogenic pair of M+ and M- strains. There was no difference between these strains in the number of streptococcal units that adhered to buccal or tonsillar epithelial cells, indicating the following: (i) that adhesins that are not dependent upon M protein expression are present on the surface of group A streptococci and (ii) that the M protein is not the primary streptococcal adherence ligand. However, the M+ strain adhered to tonsillar epithelial cells as aggregates. This aggregation was dependent on the presence of the M protein, since the isogenic M- strain did not clump. The coaggregation of streptococci suggests that the M protein plays an important role in promoting the formation of microcolonies after initial attachment. Binding to fibronectin, a potential epithelial cell receptor for group A streptococci, was also the same for the isogenic M+ and M- strains as well as for an isogenic strain with a regulatory mutation that decreases the expression of M protein. In summary, the M protein is not the primary streptococcal adhesin, nor is it required to orient the streptococcal adhesin and/or fibronectin receptor.     

Cloning and expression of the gene for an immunoglobulin G Fc receptor protein from a group A streptococcus

DNA containing the 5' end of the M-12 structural gene was used as a probe in colony hybridizations in an attempt to clone the M-76 gene from an M-type 76 group A streptococcal strain. A single positive colony was detected, and Southern hybridization analysis of plasmid DNA isolated from this colony indicated that the insert DNA had homology to the 5' end of the M-12 structural gene. Subclones were constructed to define the limits of the M-76 gene, and sonicates of these subclones were reacted with M-76-specific antiserum in immunodiffusion. A sonicate of one subclone, JM83(pDH56), reacted strongly with the M-76-specific antiserum but also reacted with preimmune rabbit serum. Protein expressed from this subclone bound immunoglobulin from horse and pig, as well as human myeloma immunoglobulin G (IgG) representing all four subclasses and purified human IgG Fc fragments. This indicated that JM83(pDH56) expressed a protein with characteristics previously attributed to the IgG Fc receptor protein from group A streptococci. Western blot analysis indicated that the cloned IgG Fc receptor protein had a molecular weight of approximately 29,000. Binding studies showed that the Fc receptor gene is expressed by the M-type 76 strain from which it was cloned and by an M- variant.     

Association of type II immunoglobulin G-binding protein expression and survival of group A streptococci in human blood.

Expression of immunoglobulin G (IgG)-binding proteins on group A streptococcus strain 64 was monitored on bacteria subjected to sequential passage in human blood. After approximately 10 cycles through human blood, strain 64 demonstrated enhanced levels of IgG-binding protein, including the expression of a type IIa binding molecule with an M(r) of approximately 47,000 present only at very low levels on the parent isolate. Changes in the expression of IgG-binding proteins after passage in human blood were similar to those observed when the same organism was passaged sequentially intraperitoneally in mice. Strain 64, passaged in human blood 23 times, was found to be more virulent than the parent isolate when used to infect mice either intraperitoneally or in a skin air sac. These findings suggest that the expression of IgG-binding proteins may be a common response of group A organisms to pressures exerted by distinct host defense mechanisms.     

Attachment of staphylococci and streptococci on fibronectin, fibronectin fragments, and fibrinogen bound to a solid phase.

The attachment of Staphylococcus aureus (Cowan I) and two strains of group A and G streptococci on glass cover slips coated with fibronectin, fibronectin fragments, or fibrinogen was studied. The attachment was quantitated by counting the attached bacteria on glass surfaces coated with a similar molarity of the proteins. Fibronectin was a more effective attachment factor than fibrinogen for staphylococci, while group G streptococci attached better on fibrinogen- than on fibronectin-coated cover slips. In this system, group A streptococci bound almost exclusively to substrate-bound fibrinogen. Attachment experiments involving the use of staphylococci pretreated with soluble fibronectin or fibrinogen revealed that bacterium-bound fibronectin and fibrinogen were able to enhance the adherence on cover slips coated with fibronectin. The 30-kilodalton NH2-terminal and the 120- to 140-kilodalton COOH-terminal fragments of fibronectin, both of which contain bacterial binding sites, mediated the staphylococcal attachment, suggesting that both parts of the molecule are involved in the attachment mediated by fibronectin.