Parvimonas micra: A rare cause of native joint septic arthritis.

Parvimonas micra is a fastidious, anaerobic, gram positive coccus, which is found in normal human oral and gastrointestinal flora. It has also been known as Peptostreptococcus micros and Micromonas micros with its most recent re-classification in 2006. It has been described in association with hematogenous seeding of prosthetic joints [1,2]. Several cases of discitis and osteomyelitis have been described in association with dental procedures and periodontal disease often with a subacute presentation. However, cases of native joint septic arthritis are limited [3-5]. Per our literature review, there is one case of native knee septic arthritis described in 1999, with a prolonged time to diagnosis and treatment due to difficulty culturing P. micra. The previously reported patient experienced significant joint destruction and morbidity [6]. Advances in culture techniques and new methods of organism identification including MALDI-TOF and 16s rRNA sequencing have lead to increased identification of this organism, which may be a more frequent bone and joint pathogen than previously realized.

Incorporating into a Cα Go model the effects of geometrical restriction on Cα atoms caused by side chain orientations.

Coarse-grained Go models have been widely used for studying protein-folding mechanisms. Despite the simplicity of the model, these can reproduce the essential features of the folding process of a protein. However, it is also known that side chains significantly contribute to the folding mechanism. Hence, it is desirable to incorporate the side chain effects into a coarse-grained Go model. In this study, to distinguish the effects of side chain orientation and to understand how these effects contribute to folding mechanisms, we incorporate into a Cα Go model not only heterogeneous contact energies but also geometrical restraints around two Cα atoms in contact with each other. We confirm that the heterogeneity of contact energies governs the folding pathway of a protein and that the geometric constraints attributed to side chains reproduce cooperative transitions in folding.

Regioselective covalent immobilization of recombinant antibody-binding proteins A, G, and L for construction of antibody arrays.

Immobilized antibodies are useful for the detection of antigens in highly sensitive microarray diagnostic applications. Arrays with the antibodies attached regioselectively in a uniform orientation are typically more sensitive than those with random orientations. Direct regioselective immobilization of antibodies on a solid support typically requires a modified form of the protein. We now report a general approach for the regioselective attachment of antibodies to a surface using truncated forms of antibody-binding proteins A, G, and L that retain the structural motifs required for antibody binding. The recombinant proteins have a C-terminal CVIX protein farnesyltransferase recognition motif that allows us to append a bioorthogonal azide or alkyne moiety and use the Cu(I)-catalyzed Huisgen cycloaddition to attach the binding proteins to a suitably modified glass surface. This approach offers several advantages. The recombinant antibody-binding proteins are produced in Escherichia coli, chemoselectively modified posttranslationally in the cell-free homogenate, and directly attached to the glass surface without the need for purification at any stage of the process. Complexes between immobilized recombinant proteins A, G, and L and their respective strongly bound antibodies were stable to repeated washing with PBST buffer at pH 7.2. However, the antibodies could be stripped from the slides by treatment with 0.1 M glycine·HCl buffer, pH 2.6, for 30 min and regenerated by shaking with PBS buffer, pH 7.2, at 4 °C overnight. The recombinant forms of proteins A, G, and L can be used separately or in combination to give glass surfaces capable of binding a wide variety of antibodies.

Crystal structure of a bacterial albumin-binding domain at 1.4 A resolution.

The albumin-binding domain, or GA module, of the peptostreptococcal albumin-binding protein expressed in pathogenic strains of Finegoldia magna is believed to be responsible for the virulence and increased growth rate of these strains. Here we present the 1.4A crystal structure of this domain, and compare it with the crystal structure of the GA-albumin complex. An analysis of protein-protein interactions in the two crystals, and the presence of multimeric GA species in solution, indicate the GA module is "sticky", and is capable of forming contacts with a range of protein surfaces. This might lead to interactions with different host proteins.

Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins.

BACKGROUND: Peptostreptococcus magnus protein L (PpL) is a multidomain, bacterial surface protein whose presence correlates with virulence. It consists of up to five homologous immunoglobulin binding domains that interact with the variable (VL) regions of kappa light chains found on two thirds of mammalian antibodies. RESULTS: We refined the crystal structure of the complex between a human antibody Fab fragment (2A2) and a single PpL domain (61 residues) to 2.7 A. The asymmetric unit contains two Fab molecules sandwiching a single PpL domain, which contacts similar VL framework regions of two light chains via independent interfaces. The residues contacted on VL are remote from the hypervariable loops. One PpL-Vkappa interface agrees with previous biochemical data, while the second is novel. Site-directed mutagenesis and analytical-centrifugation studies suggest that the two PpL binding sites have markedly different affinities for VL. The PpL residues in both interactions are well conserved among different Peptostreptococcus magnus strains. The Fab contact positions identified in the complex explain the high specificity of PpL for antibodies with kappa rather than lambda chains. CONCLUSIONS: The PpL-Fab complex shows the first interaction of a bacterial virulence factor with a Fab light chain outside the conventional combining site. Structural comparison with two other bacterial proteins interacting with the Fab heavy chain shows that PpL, structurally homologous to streptococcal SpG domains, shares with the latter a similar binding mode. These two bacterial surface proteins interact with their respective immunoglobulin regions through a similar beta zipper interaction.

Statistical theory for protein combinatorial libraries. Packing interactions, backbone flexibility, and the sequence variability of a main-chain structure.

Combinatorial experiments provide new ways to probe the determinants of protein folding and to identify novel folding amino acid sequences. These types of experiments, however, are complicated both by enormous conformational complexity and by large numbers of possible sequences. Therefore, a quantitative computational theory would be helpful in designing and interpreting these types of experiment. Here, we present and apply a statistically based, computational approach for identifying the properties of sequences compatible with a given main-chain structure. Protein side-chain conformations are included in an atom-based fashion. Calculations are performed for a variety of similar backbone structures to identify sequence properties that are robust with respect to minor changes in main-chain structure. Rather than specific sequences, the method yields the likelihood of each of the amino acids at preselected positions in a given protein structure. The theory may be used to quantify the characteristics of sequence space for a chosen structure without explicitly tabulating sequences. To account for hydrophobic effects, we introduce an environmental energy that it is consistent with other simple hydrophobicity scales and show that it is effective for side-chain modeling. We apply the method to calculate the identity probabilities of selected positions of the immunoglobulin light chain-binding domain of protein L, for which many variant folding sequences are available. The calculations compare favorably with the experimentally observed identity probabilities.

Mapping of the immunoglobulin light chain-binding site of protein L.

Protein L is a cell surface protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus. The molecule binds specifically and with high affinity to immunoglobulins (Ig) of a wide range of animal species. The Ig-binding activity is mediated through five highly homologous domains, each 72 to 76 amino acid residues long, which interact with framework regions in the variable domain of Ig light chains. The interaction does not interfere with the antigen binding capacity of the antibody. The fold of the Ig light chain-binding domains of Protein L is comprised of an alpha-helix packed against a four stranded beta-sheet and is similar to the fold of the IgG heavy chain-binding domains of streptococcal protein G, despite the fact that the two proteins show no significant sequence homology. In the present work, heteronuclear NMR spectroscopy has been utilized to define the interaction between the N-terminal Ig-binding domain of Protein L and the variable domain of a human Ig kappa light chain. The Ig-binding region of the Protein L domain involves most of the residues in the second beta-strand, the C-terminal residues of the alpha-helix and the loop connecting the alpha-helix with the third beta-strand. The Ig light chain-binding surface of Protein L thus resembles the surface of Protein G which binds to the C gamma 1 domain of IgG, but is different from the portion of Protein G involved in the contact with the C gamma 2-C gamma 3 interface region. The data suggest that the global fold shared by the Ig-binding domains of Proteins L and G provide bacteria with a flexible template for the evolution of surface structures capable of interacting with different conserved parts of Ig molecules of the infected host.

A breakdown of symmetry in the folding transition state of protein L.

The 62 residue IgG binding domain of protein L consists of a central alpha-helix packed on a four-stranded beta-sheet formed by N and C-terminal beta-hairpins. The overall topology of the protein is quite symmetric: the beta-hairpins have similar lengths and make very similar interactions with the central helix. Characterization of the effects of 70 point mutations distributed throughout the protein on the kinetics of folding and unfolding reveals that this symmetry is completely broken during folding; the first beta-hairpin is largely structured while the second beta-hairpin and helix are largely disrupted in the folding transition state ensemble. The results are not consistent with a "hydrophobic core first" picture of protein folding; the first beta-hairpin appears to be at least as ordered at the rate limiting step in folding as the hydrophobic core.

Differences in backbone dynamics of two homologous bacterial albumin-binding modules: implications for binding specificity and bacterial adaptation.

Proteins G and PAB are bacterial albumin-binding proteins expressed at the surface of group C and G streptococci and Peptostreptococcus magnus, respectively. Repeated albumin-binding domains, known as GA modules, are found in both proteins. The third GA module of protein G from the group G streptococcal strain G148 (G148-GA3) and the second GA module of protein PAB from P.magnus strain ALB8 (ALB8-GA) exhibit 59% sequence identity and both fold to form three-helix bundle structures that are very stable against thermal denaturation. ALB8-GA binds human serum albumin with higher affinity than G148-GA3, but G148-GA3 shows substantially broader albumin-binding specificity than ALB8-GA. The (15)N nuclear magnetic resonance spin relaxation measurements reported here, show that the two GA modules exhibit mobility on the picosecond-nanosecond time scale in directly corresponding regions (loops and termini). Most residues in G148-GA3 were seen to be involved in conformational exchange processes on the microsecond-millisecond time scale, whereas for ALB8-GA such motions were only identified for the beginning of helix 2 and its preceding loop. Furthermore, and more importantly, hydrogen-deuterium exchange and saturation transfer experiments reveal large differences between the two GA modules with respect to motions on the second-hour time scale. The high degree of similarity between the two GA modules with respect to sequence, structure and stability, and the observed differences in dynamics, binding affinity and binding specificity to different albumins, suggest a distinct correlation between dynamics, binding affinity and binding specificity. Finally, it is noteworthy in this context that the module G148-GA3, which has broad albumin-binding specificity, is expressed by group C and G streptococci known to infect all mammalian species, whereas P.magnus with the ALB8-GA module has been isolated only from humans.

Robustness of protein folding kinetics to surface hydrophobic substitutions.

We use both combinatorial and site-directed mutagenesis to explore the consequences of surface hydrophobic substitutions for the folding of two small single domain proteins, the src SH3 domain, and the IgG binding domain of Peptostreptococcal protein L. We find that in almost every case, destabilizing surface hydrophobic substitutions have much larger effects on the rate of unfolding than on the rate of folding, suggesting that nonnative hydrophobic interactions do not significantly interfere with the rate of core assembly.

The GA module, a mobile albumin-binding bacterial domain, adopts a three-helix-bundle structure.

We present the first study of the secondary structure and global fold of an albumin-binding domain. Our data show that the GA module from protein PAB, an albumin-binding protein from the anaerobic bacterial species Peptostreptococcus magnus, is composed of a left-handed three-helix bundle. The helical regions were identified by sequential and medium range NOEs, values of NH-C alpha H coupling constants, chemical shift indices, and the presence of slowly exchanging amide protons, as determined by NMR spectroscopy. In addition, circular dichroism studies show that the module is remarkably stable with respect to both pH and temperature.

Purification of antibodies using protein L-binding framework structures in the light chain variable domain.

Protein L from the bacterial species Peptostreptococcus magnus binds specifically to the variable domain of Ig light chains, without interfering with the antigen-binding site. In this work a genetically engineered fragment of protein L, including four of the repeated Ig-binding repeat units, was employed for the purification of Ig from various sources. Thus, IgG, IgM, and IgA were purified from human and mouse serum in a single step using protein L-Sepharose affinity chromatography. Moreover, human and mouse monoclonal IgG, IgM, and IgA, and human IgG Fab fragments, as well as a mouse/human chimeric recombinant antibody, could be purified from cultures of hybridoma cells or antibody-producing bacterial cells, with protein L-Sepharose. This was also the case with a humanized mouse antibody, in which mouse hypervariable antigen-binding regions had been introduced into a protein L-binding kappa subtype III human IgG. These experiments demonstrate that it is possible to engineer antibodies and antibody fragments (Fab, Fv) with protein L-binding framework regions, which can then be utilized in a protein L-based purification protocol.

Total chemical synthesis of the B1 domain of protein L from Peptostreptococcus magnus.

Reported here is a native chemical ligation strategy for the total chemical synthesis of the B1 domain of protein L. A synthetic construct of this 76 amino acid protein domain was prepared by the chemoselective ligation of two unprotected polypeptide fragments, one containing an N-terminal cysteine residue and one containing a C-terminal thioester moiety. The polypeptide fragments utilized in the ligation reaction were readily prepared by stepwise solid phase peptide synthesis (SPPS) methods for Boc-chemistry. The milligram quantities of protein required for conventional biophysical studies were readily accessible using the synthetic protocol described here. The folding properties of the synthetic protein L construct were also determined and found to be very similar to those of a similar wild-type protein L constructs prepared by recombinant-DNA methods. This work facilitates future unnatural amino acid mutagenesis experiments on this model protein system to further dissect the molecular basis of its folding and stability.

On the origin of the thermostabilization of proteins induced by sodium phosphate.

The B1 domain of protein L shows a linear rise in thermostability with increasing concentrations of sodium phosphate. Equal behavior is observed for a set of mutant proteins where surface lysines are mutated to noncharged residues, but the mutant's thermostabilities show different sensitivities to phosphate, encoded in the varying slopes observed (mi). The melting temperature in the absence of the cosolute also correlates linearly with mi. The stabilizing effect of the phosphate ion reaches a saturation point, which has been experimentally determined for protein L (1610 mM phosphate). These results indicate that the phosphate-induced stabilization is an inherent property of the protein, encoded in the amino acid sequence. Changes in stability upon mutation are attributed to a redistribution of the overall network of solvated surface charges. Stabilization by phosphate is understood in terms of interactions with the protein surface, reducing the unfavorable contacts between like charges, maximizing the number of accessible conformations of the surface-charged side chains, and optimizing solvation.

Synthesis and screening of a rationally designed combinatorial library of affinity ligands mimicking protein L from Peptostreptococcus magnus.

Rational design and combinatorial chemistry were utilized to search for lead protein L (PpL) mimetics for application as affinity ligands for the purification of antibodies and small fragments, such as Fab and scFv, and as potential diagnostic or therapeutic agents. Inspection of the key structural features of the complex between PpL and human Fab prompted the de novo design and combinatorial synthesis of a 169-membered solid-phase ligand library, which was assessed for binding to human IgG and subsequent selectivity for the Fab fragment. Eight ligands were selected, chemically characterized and compared with a commercial PpL-adsorbent for binding pure immunoglobulin fractions. The most promising lead, ligand 8/7, when immobilized on an agarose support, behaved in a similar fashion to PpL in isolating Fab fragments from papain digests of human IgG to a final purity of 97%.

Identification of oral peptostreptococcus isolates by PCR-restriction fragment length polymorphism analysis of 16S rRNA genes.

Oral Peptostreptococcus isolates tentatively identified by conventional microbiological culture methods were identified to the species level by a combination of PCR amplification of 16S rRNA genes and restriction enzyme analysis of the amplified products. This method is a reliable and rapid alternative to conventional methods for identification of these bacterial species.

Estimates of methyl 13C and 1H CSA values (Deltasigma) in proteins from cross-correlated spin relaxation.

Simple pulse schemes are presented for the measurement of methyl (13)C and (1)H CSA values from (1)H-(13)C dipole/(13)C CSA and (1)H-(13)C dipole/(1)H CSA cross-correlated relaxation. The methodology is applied to protein L and malate synthase G. Average (13)C CSA values are considerably smaller for Ile than Leu/Val (17 vs 25 ppm) and are in good agreement with previous solid state NMR studies of powders of amino acids and dipeptides and in reasonable agreement with quantum-chemical DFT calculations of methyl carbon CSA values in peptide fragments. Small averaged (1)H CSA values on the order of 1 ppm are measured, consistent with a solid state NMR determination of the methyl group (1)H CSA in dimethylmalonic acid.

Three new species of the genus Peptostreptococcus isolated from humans: Peptostreptococcus vaginalis sp. nov., Peptostreptococcus lacrimalis sp. nov., and Peptostreptococcus lactolyticus sp. nov.

We describe three new species of the genus Peptostreptococcus which were isolated from human specimens and were tentatively identified as Peptostreptococcus prevotii. These three organisms were not homologous with previously described type strains of the genus Peptostreptococcus. A total of 12 strains that were identified biochemically as P. prevotii were divided into five independent DNA similarity groups; 10 of these strains were divided into three similarity groups which exhibited significant phenotypic differences from previously described species. Therefore, we propose the following new species: Peptostreptococcus vaginalis for group 1 strains, Peptostreptococcus lacrimalis for group 2 strains, and Peptostreptococcus lactolyticus for group 3 strains. The type strain of P. vaginalis is strain GIFU 12669 (= JCM 8138), the type strain of P. lacrimalis is strain GIFU 7667 (= JCM 8139), and the type strain of P. lactolyticus is strain GIFU 8586 (= JCM 8140).

Phospholipid analogue profiles of Peptostreptococcus, Micromonas, and Finegoldia species analysed by fast atom bombardment mass spectrometry.

Species of Peptostreptococcus cause a variety of infections, primarily abscesses of soft tissues, joints, and mucous membranes. The aim of this study was to compare the phospholipid analogue profiles of Peptostreptococcus species, represented by P. anaerobius, P. asaccharolyticus, P. indolicus, P. lacrimalis, and P. prevotii; Micromonas micros (P. micros) and Finegoldia magna (P. magnus). After anaerobic growth on blood-FAA, lipids extracted by chloroform-methanol (2:1 v/v) were purified, then analysed by fast atom bombardment mass spectrometry (FAB-MS) in negative ion mode. The major peaks with mass to charge (m/z) 719, 721, and 749, corresponded to phosphatidylglycerol analogues, namely PG (32:1), PG (32:0), and PG (34:0), which have been found previously in Lactobacillus spp., Clostridium difficile, and Staphylococcus spp. Other major peaks observed, with m/z 619, 647, 665, 675, 677, 687, 691, 693, 701, 703, 707, 733, and 746 have also been reported in one or more of these three species. However, other major peaks found here in Peptostreptococcus, Micromonas, and Finegoldia have not been described elsewhere; these are 501, 514, 515, 618, 659, 673, 676, 688, 690, 692, 694, 700, 706, 715, 718, 722, and 750. We conclude that Peptostreptococcus, Micromonas, and Finegoldia isolates are chemically unique.

Chi1 torsion angle dynamics in proteins from dipolar couplings.

Experiments are presented for the measurement of one-bond carbon-proton dipolar coupling values at CH and CH2 ositions in 13C-labeled, approximately 50% fractionally deuterated proteins. 13Cbeta-1Hbeta dipolar couplings have been measured for 38 of 49 possible residues in the 63-amino-acid B1 domain of peptostreptococcal protein L in two aligning media and interpreted in the context of side-chain chi1 torsion angle dynamics. The beta protons for 18 of the 25 beta-methylene-containing amino acids for which dipolar data are available can be unambiguously stereoassigned, and for those residues which are best fit to a single rotamer model the chi(1) angles obtained deviate from crystal structure values by only 5.2 degrees (rmsd). The results for 11 other residues are significantly better fit by a model that assumes jumps between the three canonical (chi1 approximately -60 degrees, 60 degrees, 180 degrees ) rotamers. Relative populations of the rotamers are determined to within +/-6% uncertainty on average and correlate with dihedral angles observed for the three molecules in the crystal asymmetric unit. Entropic penalties for quenching chi1 jumps are considered for six mobile residues thought to be involved in binding to human immunoglobulins. This study demonstrates that dipolar couplings may be used to characterize both the conformation of static residues and side-chain motion with high precision.