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• W2046869263 abstract "The anaerobic bacterium Peptostreptococcus magnus is a human commensal and pathogen. Previous work has shown that strains of P. magnus isolated from patients with gynecological disease (vaginosis) frequently express an immunoglobulin (Ig) light chain-binding protein called protein L. Here we report that strains isolated from localized suppurative infections bind human serum albumin (HSA), whereas commensal isolates bind neither Ig nor HSA. The HSA-binding protein PAB was extracted from the bacterial surface or isolated from the culture supernatant of the P. magnus strain ALB8. Protein PAB was shown to have two homologous HSA-binding domains, GA and uGA. GA is absent in the sequence of a related protein from another P. magnus strain and shows a high degree of homology to the HSA-binding domains of streptococcal protein G. Therefore GA is believed to have recently been shuffled as a module from genes of other bacterial species into the protein PAB gene. This GA module was shown to exhibit a much higher affinity for HSA than uGA and was also found to be present in all of the isolates tested from localized suppurative infections, indicating a role in virulence. Moreover, when peptostreptococci or streptococci expressing the GA module were grown in the presence of HSA, the growth rate was substantially increased. Thus, the HSA binding activity of the GA module adds selective advantages to the bacteria, which increases their virulence in the case of P. magnus strains. The anaerobic bacterium Peptostreptococcus magnus is a human commensal and pathogen. Previous work has shown that strains of P. magnus isolated from patients with gynecological disease (vaginosis) frequently express an immunoglobulin (Ig) light chain-binding protein called protein L. Here we report that strains isolated from localized suppurative infections bind human serum albumin (HSA), whereas commensal isolates bind neither Ig nor HSA. The HSA-binding protein PAB was extracted from the bacterial surface or isolated from the culture supernatant of the P. magnus strain ALB8. Protein PAB was shown to have two homologous HSA-binding domains, GA and uGA. GA is absent in the sequence of a related protein from another P. magnus strain and shows a high degree of homology to the HSA-binding domains of streptococcal protein G. Therefore GA is believed to have recently been shuffled as a module from genes of other bacterial species into the protein PAB gene. This GA module was shown to exhibit a much higher affinity for HSA than uGA and was also found to be present in all of the isolates tested from localized suppurative infections, indicating a role in virulence. Moreover, when peptostreptococci or streptococci expressing the GA module were grown in the presence of HSA, the growth rate was substantially increased. Thus, the HSA binding activity of the GA module adds selective advantages to the bacteria, which increases their virulence in the case of P. magnus strains. As a rule, anaerobic infections are caused by bacteria that are part of the indigenous flora of mucosal surfaces and the skin. Peptostreptococcus magnus is such a commensal, and it belongs to the major group of anaerobic bacterial species causing clinically significant infections (1Bourgault A.-M. Rosenblatt J.E. Fitzgerald R.H. Ann. Intern. Med. 1980; 93: 244-248Crossref PubMed Scopus (66) Google Scholar). Still little is known about the virulence factors of this bacterium. Increased oxygen tolerance, as seen among clinical P. magnus isolates, could contribute to the pathogenic potential (2Ezaki, T., Oyaizu, H., Yabuuchi, E., (1992) The Prokaryotes, (Balows, E., Trüper, H. G., Dworkin, M., Harder, W., Schleifer, K.-H., eds), 2nd Ed., Vol 2, p. 1879, Springer-Verlag New York Inc., New York.Google Scholar). Other possible virulence factors, apart from the herein discussed bacterial surface proteins, include encapsulation (3Brook I. Walker R.I. Can. J. Microbiol. 1985; 31: 176-180Crossref PubMed Scopus (25) Google Scholar) and collagenase production (4Krepel C.J. Gohr C.M. Walker A.P. Farmer S.G. Edmiston C.E. J. Clin. Microbiol. 1992; 30: 2330-2334Crossref PubMed Google Scholar). Numerous Gram-positive bacterial species and human pathogens express structurally related surface proteins that interact mainly with soluble host proteins (5Kehoe M.A. New Compr. Biochem. 1994; 27: 217-261Crossref Scopus (131) Google Scholar). Protein A of Staphylococcus aureus and protein G of human group C and G streptococci both interact with the Fc region of IgG (6Reis K.J. Ayoub E.M. Boyle M.D.P. J. Immunol. 1984; 132: 3091-3097PubMed Google Scholar, 7Forsgren A. Sjöquist J. J. Immunol. 1966; 97: 822-827PubMed Google Scholar, 8Björck L. Kronvall G. J. Immunol. 1984; 133: 969-974PubMed Google Scholar). Protein G also has affinity for human serum albumin (HSA) 1The abbreviations used are: HSAhuman serum albuminPCRpolymerase chain reactionPBSphosphate-buffered salinePVDFpolyvinylidene difluoridePAGEpolyacrylamide gel electrophoresisFFAfree fatty acidsTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. (9Björck L. Kastern W. Lindahl G. Widebäck K. Mol. Immunol. 1987; 24: 1113-1122Crossref PubMed Scopus (91) Google Scholar), as do members of the M protein family expressed by Streptococcus pyogenes (10Åkesson P. Schmidt K.-H. Cooney J. Björck L. Biochem. J. 1994; 300: 877-886Crossref PubMed Scopus (136) Google Scholar, 11Retnoningrum D.S. Cleary P.P. Infect. Immun. 1994; 62: 2387-2394Crossref PubMed Google Scholar, 12Schmidt K.-H. Wadström T. Zentralbl. Bakteriol. 1990; 273: 216-228Crossref PubMed Scopus (43) Google Scholar). Albumin binding has also been described for protein PAB (peptostreptococcal albumin:binding) of the anaerobic commensal and pathogen P. magnus (13de Château M. Björck L. J. Biol. Chem. 1994; 269: 12147-12151Abstract Full Text PDF PubMed Google Scholar). Protein L is another surface protein of certain strains of P. magnus that binds to immunoglobulin light chains (14Björck L. J. Immunol. 1988; 140: 1194-1197PubMed Google Scholar). Protein L has been shown to be a virulence determinant in bacterial vaginosis (15Kastern W. Holst E. Nielsen E. Sjöbring U. Björck L. Infect. Immun. 1990; 58: 1217-1222Crossref Google Scholar), perhaps due to its histamine-releasing activity (16Patella V. Casolaro V. Björck L. Marone G. J. Immunol. 1990; 145: 3054-3061PubMed Google Scholar). M proteins contribute to the virulence of S. pyogenes by their antiphagocytic property (17Fischetti V.A. Clin. Microbiol. Rev. 1989; 2: 285-314Crossref PubMed Scopus (638) Google Scholar), and also IgGFc-binding proteins of these bacteria have been reported to be virulence factors (18Reader R. Boyle M.D.P. Infect. Immun. 1993; 61: 3696-3702Crossref PubMed Google Scholar). Experiments with deletion mutants have likewise shown that protein A of S. aureus plays a role in virulence, possibly by inhibiting opsonophagocytosis (19Foster T.J. FEMS Microbiol. Lett. 1994; 118: 199-206Crossref PubMed Scopus (163) Google Scholar). human serum albumin polymerase chain reaction phosphate-buffered saline polyvinylidene difluoride polyacrylamide gel electrophoresis free fatty acids N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Host protein binding cell wall proteins of Gram-positive bacteria share common primary structure motifs, including (from the distal NH2 terminus) a signal sequence, a variable NH2-terminal region, a varying number of repeated domains that independently bind different plasma proteins, and a proline-rich region supposedly intercalating the protein in the Gram-positive cell wall, followed by a COOH-terminal cell wall sorting signal required to anchor the protein to the cell wall (20Schneewind O. Model P. Fischetti V.A. Cell. 1992; 70: 267-281Abstract Full Text PDF PubMed Scopus (441) Google Scholar). The gene structure of the albumin-binding protein PAB has been shown to contain a centrally located functional domain of 45 amino acid residues responsible for the binding of HSA (Fig. 1). This domain has been subject to module shuffling between bacterial species, and was subsequently named the GA module, protein G-related albumin binding module (13de Château M. Björck L. J. Biol. Chem. 1994; 269: 12147-12151Abstract Full Text PDF PubMed Google Scholar). Such shuffling of modules seems to be a persistent activity among this group of genes, and when a consensus sequence of 15 nucleotides (called recer sequence) flanking the different modules in the P. magnus family of surface proteins was identified, a model for the shuffling of modules was proposed (21de Château M. Björck L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8490-8495Crossref PubMed Scopus (34) Google Scholar). The albumin-binding protein G of group G streptococcal strain G148 carries three GA modules in the NH2-terminal part of the protein showing up to 60% identity to the shuffled module of protein PAB, indicating that protein G might be the source of the GA module in protein PAB. The secondary structure and global fold of the GA module in protein PAB have been determined by NMR and were shown to adopt a 3-helix bundle (22Johansson M.U. de Château M. Björck L. Forsén S. Drakenberg T. Wikström M. FEBS Lett. 1995; 374: 257-261Crossref PubMed Scopus (25) Google Scholar), and the third GA module of protein G was recently shown to exhibit the same structure (23Kraulis P.J. Jonasson P. Nygren P.-Å. Uhlén M. Jendeberg L. Nilsson B. Kördel J. FEBS Lett. 1996; 378: 190-194Crossref PubMed Scopus (63) Google Scholar). The predecessor of protein PAB has also been identified. This protein, called urPAB, is expressed by a strain that binds less albumin to its surface. Protein urPAB lacks the shuffled GA module (21de Château M. Björck L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8490-8495Crossref PubMed Scopus (34) Google Scholar), but in the NH2-terminal region, a domain (uGA) was identified showing 38% identity to the GA module. Likewise, sequence comparison between proteins urPAB and PAB revealed an analogous uGA domain in protein PAB, thus indicating the presence of a second HSA-binding region in protein PAB (Fig. 1). HSA binding has been described for a number of additional surface proteins of group C and G streptococci as well as for a P. magnus protein that, in addition to several GA modules, also contains Ig light chain-binding domains closely related to those of protein L (24Sjöbring U. Infect. Immun. 1992; 60: 3601-3608Crossref PubMed Google Scholar, 25Murphy J.P. Duggleby C.J. Atkinson M.A. Trowern A.R. Atkinson T. Goward C.R. Mol. Microbiol. 1994; 12: 911-920Crossref PubMed Scopus (25) Google Scholar, 26Jonsson H. Frykberg L. Rantamäki L. Guss B. Gene (Amst.). 1994; 143: 85-89Crossref PubMed Scopus (27) Google Scholar, 27Jonsson H. Lindmark H. Guss B. Infect. Immun. 1995; 63: 2968-2975Crossref PubMed Google Scholar). Apart from the HSA-binding proteins of S. pyogenes that belong to the M protein family, the other HSA-binding proteins all contain GA-related sequences. In the case of M proteins, the binding of HSA is located to the so-called C repeats (10Åkesson P. Schmidt K.-H. Cooney J. Björck L. Biochem. J. 1994; 300: 877-886Crossref PubMed Scopus (136) Google Scholar, 11Retnoningrum D.S. Cleary P.P. Infect. Immun. 1994; 62: 2387-2394Crossref PubMed Google Scholar), which show no homology to the GA module, suggesting that these structures have evolved separately. The aims of the present study were to further characterize the HSA binding properties of protein PAB, to localize its HSA-binding regions, and to compare the affinities of the shuffled GA module with that of the uGA domains, i.e. the NH2-terminal HSA-binding domains of proteins PAB and urPAB. It was believed that this information could help to explain why the GA module has been shuffled into protein PAB. The biological consequences of HSA binding have also been investigated, and the results demonstrate that essential properties such as bacterial growth and virulence are affected by the interaction with albumin. P. magnus strains were clinical isolates from the Department of Clinical Microbiology, Lund University Hospital. The peptostreptococci were grown under strict anaerobic conditions at 37°C in Todd-Hewitt broth (Difco). HSA and human IgG were purified from human plasma (28de Château M. Nilson B.H.K. Erntell M. Myhre E. Magnusson C.G.M. Åkerström B. Björck L. Scand. J. Immunol. 1993; 37: 399-405Crossref PubMed Scopus (59) Google Scholar). Other serum albumins and sera were from Sigma. Protein L was prepared from peptostreptococcal growth medium (15Kastern W. Holst E. Nielsen E. Sjöbring U. Björck L. Infect. Immun. 1990; 58: 1217-1222Crossref Google Scholar), and recombinant protein G was from Escherichia coli lysates (9Björck L. Kastern W. Lindahl G. Widebäck K. Mol. Immunol. 1987; 24: 1113-1122Crossref PubMed Scopus (91) Google Scholar). Proteins were radiolabeled with 125I using the Bolton-Hunter reagent (Amersham Corp., Buckinghamshire, Great Britain), chloramine T, or lactoperoxidase. Bacteria were suspended, heat killed (80°C, 5 min), and washed in phosphate-buffered saline (PBS) containing 0.02% NaN3 and 0.5% Tween 20. Bacterial suspensions of different concentrations in a volume of 100 μl were mixed with 900 μl of Staphylococcus epidermidis strain L603 (109 bacteria/ml). 200 μl of these mixed bacterial suspensions were then incubated with 104 cpm of 125I-labeled HSA or IgG for 30 min. Cells were spun down, and the radioactivity of the pellet was measured in a γ counter and expressed as percentage of added radioactivity. Peptostreptococci were grown as described until the stationary phase was reached and then harvested by centrifugation. Subsequently, 10% solutions of the bacteria were boiled in HCl, pH 2.0, or NaOH, pH 11.0, vortexed in PBS, and treated with mutanolysin or trypsin to extract surface proteins. Acid and alkali extractions were performed by boiling the bacteria for 3 min in 0.1 M HCl or NaOH, whereafter the sample was neutralized by the addition of 0.1 volume of 0.1 M Tris-HCl, pH 8.0. Extraction was done by vortexing the bacterial suspension in PBS. Mutanolysin digestions were performed in 0.01 M phosphate buffer, pH 6.8, and blocked with NaHCO3 to pH 7.5 and by cooling on ice. Trypsin digestions were done at pH 6.1 in 0.05 M phosphate buffer containing 5 mM EDTA. Reactions were inhibited by adding benzamidine to a final concentration of 5 mM. Enzyme incubations were at 37°C for 1-2 h. Culture medium from peptostreptococci (strain ALB8) expressing protein PAB or periplasmic E. coli lysates of clones expressing the 5′-end (nucleotides 1-857) of the protein PAB gene (pab) were used as starting materials and subjected to affinity chromatography on HSA-Sepharose CL-4B (Pharmacia Biotech Inc., Uppsala, Sweden). Columns of Sepharose, coupled with 3-5 mg of HSA/ml of packed gel, were equilibrated in PBS. The sample, in 0.01 M phosphate buffer, pH 7.5, was applied, and the column was rinsed with PBS and then eluted with 0.1 M glycine buffer, pH 2.0. Eluates were immediately neutralized by the addition of 0.1 volume of unbuffered 1 M Tris. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) (29Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and transferred to PVDF membranes (30Towbin H. Staehlin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). Agarose gel electrophoresis was performed as described (31Johansson B.G. Scand. J. Clin. Lab. Invest. Suppl. 1972; 29: 7-19Crossref Scopus (511) Google Scholar). Transfer of proteins from agarose gels was done by applying PVDF membranes on top of the gels under pressure (1-2 kg). P. magnus bacteria of strain 312 (109 cells) were incubated in 1 ml of human plasma for 2 h at 37°C. The cells were spun down and washed five times in PBS. The bacterial pellet was then resuspended in 50 μl of PBS and boiled in an equal volume of SDS sample buffer. Cell debris was spun down, and the supernatant was subjected to SDS-PAGE and Western blot analysis (9Björck L. Kastern W. Lindahl G. Widebäck K. Mol. Immunol. 1987; 24: 1113-1122Crossref PubMed Scopus (91) Google Scholar). Equilibrium constants were determined by incubating constant amounts of Immunobeads (Bio-Rad), coupled with HSA, together with 125I-labeled protein PAB or GA and varying amounts of non-labeled proteins for 16 h at room temperature. After washing, the amount of bound radioactivity was measured. Using the formula of Scatchard, the calculations were done as previously reported (32Åkerström B. Björck L. J. Biol. Chem. 1989; 264: 19740-19746Abstract Full Text PDF PubMed Google Scholar). 10% (v/v) solutions of strain ALB8 peptostreptococci were incubated for 2 h at 37°C with mutanolysin in 0.01 M phosphate buffer, pH 6.8. The reaction was blocked with NaHCO3 to pH 7.5 followed by cooling on ice. Cells were spun down, and material from the supernatant was applied to PVDF membranes using a dot blot apparatus from Schleicher & Schuell (Dassel, Germany). Incubation with radiolabeled HSA was followed by autoradiography to visualize binding. Five oligonucleotides were synthesized, all of which include restriction site linkers (NarI and SalI) for cloning in the expression vector pHD 389 (33Dalböge H. Bech Jensen E. Töttrup H. Grubb A. Abrahamson M. Olafsson I. Carlsen S. Gene (Amst.). 1989; 79: 325-332Crossref PubMed Scopus (74) Google Scholar): RXN1, dGCT CAG GCG CGC CGG ACG AAC CCG GGG CAC CCA A; RXC3, dCAG CAG GTC GAC TTA TTA AGC GTG TGC TTT TAA AAT TTC GTT; 1118, dCAG GTC GAC TTA TTA TTC AGC TTC TAC TGG TGA TAA TAC; RKC1, dGGT GTT CTA GAT TAT TAT (T/G)TT T(T/C)A GCT (T/G)TT TCT TCT TCT TTT; RXN0”, dGCG AAT TCG GCG CAT GAA AAT TAA TAA GAA ATT ATT. These oligonucleotides were used as primers with genomic DNA from P. magnus strains ALB8 and ALB1B as templates to generate inserts for cloning. PCR products were purified by chloroform/phenol extraction and ethanol precipitation and were subjected to restriction enzyme cleavage before a second purification and ligation to the vector. Subsequently, the ligation mixes were transformed into competent E. coli strain JM109 cells. Clones were selectively grown on ampicillin-containing plates and were screened by PCR and for expression of HSA binding peptides. HSA binding peptides were then purified by affinity chromatography on HSA-Sepharose and on a gel chromatography column, Superose 12 for fast protein liquid chromatography (Pharmacia). The purification of the GA module has also been described elsewhere in more detail (22Johansson M.U. de Château M. Björck L. Forsén S. Drakenberg T. Wikström M. FEBS Lett. 1995; 374: 257-261Crossref PubMed Scopus (25) Google Scholar). Chromosomal DNA was isolated from 15 HSA-binding strains of suppurative infections and from 10 non-binding, non-pathogenic isolates. These chromosomal preparations were used as templates in PCRs using the following oligonucleotides as primers: RX2N, dTTA AAG AAC GCT AAA GAA GAT GCA AT; 769, dGCT ACC AGC TTT TGG TAA. Forty-eight P. magnus strains were isolated from patients with localized suppurative infections (n = 30) or vaginosis (n = 8). Ten commensal isolates were from healthy carriers. The ability of these strains to bind HSA and human IgG was tested (Fig. 2). Among the 30 suppurative infection isolates, 16 (53%) were HSA binding, whereas only one isolate showed significant IgG binding. In the vaginosis group, on the other hand, five of the eight isolates bound IgG and none bound HSA. Finally, neither HSA nor IgG binding activity could be detected among the commensal isolates. Thus, when the group of strains causing suppurative infections was compared with the commensal strains, there was a significant difference in the prevalence of HSA binding as tested with the χ2 test (p < 0.0001), implying a role for protein PAB in virulence. The binding capacity of the HSA-binding strains was shown to be saturable and varied among the strains between 15 and 71% as tested by direct binding of radiolabeled HSA to bacterial cells (Fig. 3). Among the HSA-binding strains, strain ALB8 showing maximal binding (70%) and strain ALB1B showing intermediate binding (27%) were chosen for further isolation and characterization of the HSA-binding proteins PAB and urPAB, respectively.Fig. 3Binding of radiolabeled HSA to different strains of P. magnus. The binding of 125I-labeled HSA to six different strains of P. magnus was measured at bacterial concentrations varying from 105 to 109/ml. Strain 312 (♦) expresses protein L, ALB8 (▴) protein PAB, and ALB1B protein urPAB (∘). Albumin-binding strains ALB15 (•) and ALB18 (⋄), as well as the non-binding strain 505 (□), are also included.View Large Image Figure ViewerDownload (PPT) After anaerobic growth of P. magnus strain ALB8 bacteria for 3 days in Todd-Hewitt broth, cells were collected by centrifugation and washed in PBS. Different procedures to solubilize HSA binding materials were tested. Boiling of the bacteria at low or high pH in HCl and NaOH, respectively, released substantial amounts of fragmented HSA-reactive material in the range of 14-45 kDa (Fig. 4). Similarly, degraded material could be obtained by treatment of intact bacteria with the muranolytic enzyme mutanolysin, whereas trypsin digestion resulted in extensive degradation. A higher proportion of larger fragments could be obtained by simply vortexing the bacteria in PBS. Still, a full-length molecule of 47 kDa was only seen when the HSA-binding protein was isolated from the culture supernatant by affinity chromatography on HSA-Sepharose. The protein yield from the culture supernatant was approximately 1 mg/liter. The NH2-terminal sequence of this 47-kDa material has been shown to correspond to that of the NH2-terminal sequence of protein PAB as deduced from the gene sequence (13de Château M. Björck L. J. Biol. Chem. 1994; 269: 12147-12151Abstract Full Text PDF PubMed Google Scholar). Mild treatment of the material coming off the HSA-Sepharose column with trypsin resulted in a single HSA binding fragment of 23 kDa. Protein PAB-expressing bacteria were incubated with human plasma. After incubation and washing, the bacteria were boiled in SDS-PAGE sample buffer. Cells were spun down, and the supernatant was run on a gel that was also blotted onto a PVDF membrane and probed with radiolabeled protein PAB. As seen in Fig. 5, only a single band at 66 kDa representing HSA could be adsorbed to the bacterial surface, and this band also reacted with protein PAB in the Western blot experiment. The conclusion is that neither the bacteria nor protein PAB will interact with any other plasma protein than HSA. Subsequent tests of the binding of 125I-labeled protein PAB to a number of purified proteins (i.e. IgG, IgA, fibrinogen, and fibronectin) in slot binding experiments were also negative (not shown). In order to further analyze the specificity of the interaction between protein PAB and HSA, human plasma was run on agarose gels. After transfer to PVDF membranes, the plasma proteins were probed with either protein L or protein PAB. As seen in Fig. 6, protein L bound to the cathodal immunoglobulin region of the plasma sample, whereas protein PAB bound to the anodal albumin region. Proteins PAB and L were also applied to the gel and were found to migrate in opposite directions to their respective ligands. These results reflect that protein PAB is a basic protein (net charge of +7, pI of 9.78), and protein L is an acidic protein (net charge of −54).Fig. 6Analysis of the interactions between peptostreptococcal proteins L and PAB and human plasmaproteins. Samples of diluted plasma and purified IgG, HSA, and proteins PAB and L were separated by agarose gel electrophoresis at pH 8.6. Two duplicates of the first three samples were also transferred to PVDF membranes and probed with radiolabeled proteins PAB and L, respectively. Lane a, human plasma diluted 1:15; lane b, polyclonal IgG; lane c, HSA; lane d, protein L (recombinant B1-B4); lane e, protein PAB (recombinant RX1N/4C).View Large Image Figure ViewerDownload (PPT) Sera from 10 different mammalian species were subjected to SDS-PAGE. The separated proteins were blotted onto a PVDF membrane and probed with radiolabeled protein PAB (Fig. 7). Binding to the albumin band in serum from man, baboon, rhesus monkey, rat, and cat was seen. Fig. 8 demonstrates that, also among purified albumins from 12 mammalian species, protein PAB has affinity for albumin from man, baboon, rhesus monkey, and rat (purified cat serum albumin was not available).Fig. 8Binding of protein PAB to albumin from different species. Dilution series (5, 1, 0.2, and 0.04 μg) of albumin from different species were applied to a PVDF membrane in slots. The membrane was probed with radiolabeled protein PAB.View Large Image Figure ViewerDownload (PPT) If P. magnus strain ALB8 bacteria are grown in Todd-Hewitt broth until stationary phase, HSA binding activity can be detected not only on the surface of the bacteria but also in the culture supernatant. This material can be adsorbed to HSA-Sepharose and then eluted at pH 2.0. The eluted proteins were separated by SDS-PAGE and blotted onto two PVDF membranes. One was probed with radiolabeled HSA, and the results demonstrated that the majority of the bands still bound HSA (not shown). The other membrane was stained with Coomassie Blue, and as seen in Fig. 9, the HSA binding material coming off of the column is often size heterogeneous. This could be due to partial hydrolysis at low pH or due to proteolytic activity at the bacterial surface or in the culture medium. The albumin binding bands at 47 (I), 24 (II), and 16 kDa (III) were cut out and subjected to NH2-terminal sequencing. All three bands had the same amino acid sequence, identical to the absolute NH2 terminus of protein PAB. This shows that the fragments are derived from protein PAB and that there must be an HSA-binding site in the most NH2-terminal 130-140 amino acids, giving an approximate molecular mass of 16 kDa. The same material was then subjected to mild trypsin treatment, yielding a single major albumin-binding protein species at 23 kDa (IV). The NH2-terminal sequence of this fragment was MTIDQ which corresponds to the five amino acid residues at positions 213-217 of protein PAB. This sequence is found immediately in the NH2-terminal direction of the GA module. From this, a second albumin-binding region in protein PAB is implied as fragments III and IV do not overlap. In the region of protein PAB covered by fragment I, at positions 57-101, a region exhibiting 42% identity to the GA module has been found. This GA-related domain was designated uGA since its counterpart was found in the same position in the protein PAB predecessor, protein urPAB (Fig. 1) (21de Château M. Björck L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8490-8495Crossref PubMed Scopus (34) Google Scholar). To show that the recently introduced GA module contributes to the pathogenicity of the HSA-binding strains causing localized suppurative infections, 25 different P. magnus strains were tested for the presence of the GA module. Chromosomal DNA from the different strains was used as templates in polymerase chain reactions. An oligonucleotide (RX2N) representing the 5′-end of the recently introduced GA module of protein PAB and a second oligonucleotide spanning the very well conserved cell wall anchor motif (769) were used as primers in these reactions. DNA from the protein PAB strain, which was used as a positive control, yielded a product of approximately 450 base pairs. All 15 HSA-binding strains yielded products of equal or near equal (±100 base pairs) sizes, whereas none of the 10 non-binding strains yielded any PCR products with these primers. To find an explanation to why a second GA module has been introduced into protein PAB, the affinity of this GA module was determined and compared with the relative affinities of the uGA regions of proteins PAB and urPAB. Recombinant fragments of proteins PAB and urPAB covering the three resident GA modules were expressed in E. coli and purified by affinity chromatography on HSA-Sepharose, followed by gel filtration. Fragment AC corresponds to amino acid residues 27-195 of protein PAB, containing the uGA and C domains. A second fragment (GA) covers residues 213-265 of protein PAB containing the GA module, whereas uGA-K is a fragment corresponding to the uGA region (positions 1-102) of protein urPAB (Fig. 1). These fragments together with proteins urPAB and G were run on a Tricine-SDS-PAGE gel to show sizes and purity (Fig. 10A). They were also radiolabeled and allowed to bind to HSA in slot binding experiments (Fig. 10B). In these experiments, the AC fragment bound weakly, the GA module bound almost as strong as intact protein PAB (compare with Fig. 8), and the uGA-K bound to an intermediate degree. A competitive binding assay was utilized to determine the equilibrium constant of the interactions between HSA and fragment GA of protein PAB, as well as the relative affinities of the three peptostreptococcal GA modules. The affinity of fragment GA (the shuffled module) for HSA was determined to be 6.7 × 109M−1. Furthermore, HSA was coupled to polyacrylamide beads and incubated with the radiolabeled GA fragment in the presence of different amounts of non-labeled competing proteins (Fig. 10D). The AC fragment could only interfere with the binding of radiolabeled GA when added at a molar excess of >1000:1. Protein urPAB was a much more efficient inhibitor but still not as efficient as GA itself. Based on the amount of inhibitor needed to elicit th" @default.
• W2046869263 created "2016-06-24" @default.
• W2046869263 creator A5005892505 @default.
• W2046869263 creator A5027419998 @default.
• W2046869263 creator A5032209580 @default.
• W2046869263 date "1996-10-01" @default.
• W2046869263 modified "2023-09-30" @default.
• W2046869263 title "Protein PAB, an Albumin-binding Bacterial Surface Protein Promoting Growth and Virulence" @default.
• W2046869263 cites W1235877488 @default.
• W2046869263 cites W1486311063 @default.
• W2046869263 cites W1498894824 @default.
• W2046869263 cites W1578846813 @default.
• W2046869263 cites W1604588480 @default.
• W2046869263 cites W1607549514 @default.
• W2046869263 cites W1935933547 @default.
• W2046869263 cites W1968347832 @default.
• W2046869263 cites W1975085160 @default.
• W2046869263 cites W1996233811 @default.
• W2046869263 cites W1998045538 @default.
• W2046869263 cites W2008918642 @default.
• W2046869263 cites W2009387111 @default.
• W2046869263 cites W2009624023 @default.
• W2046869263 cites W2018491773 @default.
• W2046869263 cites W2038193703 @default.
• W2046869263 cites W2042344055 @default.
• W2046869263 cites W2070149310 @default.
• W2046869263 cites W2079021059 @default.
• W2046869263 cites W2085545970 @default.
• W2046869263 cites W2100837269 @default.
• W2046869263 cites W2101108802 @default.
• W2046869263 cites W2103880491 @default.
• W2046869263 cites W2125441279 @default.
• W2046869263 cites W2134523720 @default.
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