Karina Persson

The changing faces of Streptococcus antigen I/II polypeptide family adhesins.

Streptococcus mutans antigen I/II (AgI/II) protein was one of the first cell wall‐anchored adhesins identified in Gram‐positive bacteria. It mediates attachment of S. mutans to tooth surfaces and has been a focus for immunization studies against dental caries. The AgI/II family polypeptides recognize salivary glycoproteins, and are also involved in biofilm formation, platelet aggregation, tissue invasion and immune modulation. The genes encoding AgI/II family polypeptides are found among Streptococcus species indigenous to the human mouth, as well as in Streptococcus pyogenes, S. agalactiae and S. suis. Evidence of functionalities for different regions of the AgI/II proteins has emerged. A sequence motif within the C‐terminal portion of Streptococcus gordonii SspB (AgI/II) is bound by Porphyromonas gingivalis, thus promoting oral colonization by this anaerobic pathogen. The significance of other epitopes is now clearer following resolution of regional crystal structures. A new picture emerges of the central V (variable) region, predicted to contain a carbohydrate‐binding trench, being projected from the cell surface by a stalk formed by an unusual association between an N‐terminal α‐helix and a C‐terminal polyproline helix. This presentation mode might be important in determining functional conformations of other Gram‐positive surface proteins that have adhesin domains flanked by α‐helical and proline‐rich regions.

Two intramolecular isopeptide bonds are identified in the crystal structure of the Streptococcus gordonii SspB c-terminal domain

Streptococcus gordonii is a primary colonizer and is involved in the formation of dental plaque. This bacterium expresses several surface proteins. One of them is the adhesin SspB, which is a member of the Antigen I/II family of proteins. SspB is a large multi-domain protein that has interactions with surface molecules on other bacteria and on host cells, and is thus a key factor in the formation of biofilms. Here, we report the crystal structure of a truncated form of the SspB C-terminal domain, solved by single-wavelength anomalous dispersion to 1.5 A resolution. The structure represents the first of a C-terminal domain from a streptococcal Antigen I/II protein and is comprised of two structurally related beta-sandwich domains, C2 and C3, both with a Ca(2+) bound in equivalent positions. In each of the domains, a covalent isopeptide bond is observed between a lysine and an asparagine, a feature that is believed to be a common stabilization mechanism in Gram-positive surface proteins. S. gordonii biofilms contain attachment sites for the periodontal pathogen Porphyromonas gingivalis and the SspB C-terminal domain has been shown to have one such recognition motif, the SspB adherence region. The motif protrudes from the protein, and serves as a handle for attachment. The structure suggests several additional putative binding surfaces, and other binding clefts may be created when the full-length protein is folded.

Sequence analyses of fimbriae subunit FimA proteins on Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus with variant carbohydrate binding specificities.

BackgroundActinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers) with variant Galβ binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking.ResultsHere we have sequenced fimA genes from strains of A.naeslundii genospecies 1 (n = 4) and genospecies 2 (n = 4), both of which harboured variant Galβ-dependent hemagglutination (HA) types, and from A.odontolyticus PK984 with a sialic acid-dependent HA pattern. Three unique subtypes of FimA proteins with 63.8–66.4% sequence identity were present in strains of A. naeslundii genospecies 1 and 2 and A. odontolyticus. The generally high FimA sequence identity (>97.2%) within a genospecies revealed species specific sequences or segments that coincided with binding specificity. All three FimA protein variants contained a signal peptide, pilin motif, E box, proline-rich segment and an LPXTG sorting motif among other conserved segments for secretion, assembly and sorting of fimbrial proteins. The highly conserved pilin, E box and LPXTG motifs are present in fimbriae proteins from other Gram-positive bacteria. Moreover, only strains of genospecies 1 were agglutinated with type-2 fimbriae antisera derived from A. naeslundii genospecies 1 strain 12104, emphasizing that the overall folding of FimA may generate different functionalities. Western blot analyses with FimA antisera revealed monomers and oligomers of FimA in whole cell protein extracts and a purified recombinant FimA preparation, indicating a sortase-independent oligomerization of FimA.ConclusionThe genus Actinomyces involves a diversity of unique FimA proteins with conserved pilin, E box and LPXTG motifs, depending on subspecies and associated binding specificity. In addition, a sortase independent oligomerization of FimA subunit proteins in solution was indicated.

Crystal structure of the variable domain of the Streptococcus gordonii surface protein SspB.

The Antigen I/II (AgI/II) family of proteins are cell wall anchored adhesins expressed on the surface of oral streptococci. The AgI/II proteins interact with molecules on other bacteria, on the surface of host cells, and with salivary proteins. Streptococcus gordonii is a commensal bacterium, and one of the primary colonizers that initiate the formation of the oral biofilm. S. gordonii expresses two AgI/II proteins, SspA and SspB that are closely related. One of the domains of SspB, called the variable (V‐) domain, is significantly different from corresponding domains in SspA and all other AgI/II proteins. As a first step to elucidate the differences among these proteins, we have determined the crystal structure of the V‐domain from S. gordonii SspB at 2.3 Å resolution. The domain comprises a β‐supersandwich with a putative binding cleft stabilized by a metal ion. The overall structure of the SspB V‐domain is similar to the previously reported V‐domain of the Streptococcus mutans protein SpaP, despite their low sequence similarity. In spite of the conserved architecture of the binding cleft, the cavity is significantly smaller in SspB, which may provide clues about the difference in ligand specificity. We also verified that the metal in the binding cleft is a calcium ion, in concurrence with previous biological data. It was previously suggested that AgI/II V‐domains are carbohydrate binding. However, we tested that hypothesis by screening the SspB V‐domain for binding to over 400 glycoconjucates and found that the domain does not interact with any of the carbohydrates.

Structure of the sortase AcSrtC-1 from Actinomyces oris

The crystal structure of the sortase AcSrtC-1 from the oral microorganism Actinomyces oris has been determined to 2.4 Å resolution. AcSrtC-1 is a cysteine transpeptidase that is responsible for the formation of fimbriae by the polymerization of a shaft protein. Similar to other pili-associated sortases, the AcSrtC-1 active site is protected by a flexible lid. The asymmetric unit contains five AcSrtC-1 molecules and their catalytic Cys-His-Arg triads are trapped in two different conformations. It is also shown that the thermostability of the enzyme is increased by the presence of calcium.

The Pilin Protein FimP from Actinomyces oris: Crystal Structure and Sequence Analyses

The Actinomyces oris type-1 pili are important for the initial formation of dental plaque by binding to salivary proteins that adhere to the tooth surface. Here we present the X-ray structure of FimP, the protein that is polymerized into the type-1 pilus stalk, assisted by a pili-specific sortase. FimP consists of three tandem IgG-like domains. The middle and C-terminal domains contain one autocatalyzed intramolecular isopeptide bond each, a feature used by Gram-positive bacteria for stabilization of surface proteins. While the N-terminal domain harbours all the residues necessary for forming an isopeptide bond, no such bond is observed in the crystal structure of this unpolymerized form of FimP. The monomer is further stabilized by one disulfide bond each in the N- and C-terminal domains as well as by a metal-coordinated loop protruding from the C-terminal domain. A lysine, predicted to be crucial for FimP polymerization by covalent attachment to a threonine from another subunit, is located at the rim of a groove lined with conserved residues. The groove may function as a docking site for the sortase-FimP complex. We also present sequence analyses performed on the genes encoding FimP as well as the related FimA, obtained from clinical isolates.

Structure of the C-terminal domain of the surface antigen SpaP from the caries pathogen Streptococcus mutans

The structure of the C-terminal domain of the S. mutans surface adhesin SpaP has been determined to 2.2 Å resolution.

Role of Mfa5 in Expression of Mfa1 Fimbriae in Porphyromonas gingivalis

Fimbriae are protein-based filamentous appendages that protrude from the bacterial cell surface and facilitate host adhesion. Two types of fimbriae, FimA and Mfa1, of the periodontal pathogen Porphyromonas gingivalis are responsible for adherence to other bacteria and to host cells in the oral cavity. Both fimbrial forms are composed of 5 proteins, but there is limited information about their polymerization mechanisms. Here, the authors evaluated the function of Mfa5, one of the Mfa1 fimbrial accessory proteins. Using mfa5 gene disruption and complementation studies, the authors revealed that Mfa5 affects the incorporation of other accessory proteins, Mfa3 and Mfa4, into fibers and the expression of fimbriae on the cell surface. Mfa5 is predicted to have a C-terminal domain (CTD) that uses the type IX secretion system (T9SS), which is limited to this organism and related Bacteroidetes species, for translocation across the outer membrane. To determine the relationship between the putative Mfa5 CTD and the T9SS, mutants were constructed with in-frame deletion of the CTD and deletion of porU, a C-terminal signal peptidase linked to T9SS-mediated secretion. The ∆CTD-expressing strain presented a similar phenotype to the mfa5 disruption mutant with reduced expression of fimbriae lacking all accessory proteins. The ∆porU mutants and the ∆CTD-expressing strain showed intracellular accumulation of Mfa5. These results indicate that Mfa5 function requires T9SS-mediated translocation across the outer membrane, which is dependent on the CTD, and subsequent incorporation into fibers. These findings suggest the presence of a novel polymerization mechanism of the P. gingivalis fimbriae.

Small molecules containing hetero-bicyclic ring systems compete with UDP-Glc for binding to WaaG glycosyltransferase

The α-1,3-glucosyltransferase WaaG is involved in the synthesis of the core region of lipopolysaccharides in E. coli. A fragment-based screening for inhibitors of the WaaG glycosyltrasferase donor site has been performed using NMR spectroscopy. Docking simulations were performed for three of the compounds of the fragment library that had shown binding activity towards WaaG and yielded 3D models for the respective complexes. The three ligands share a hetero-bicyclic ring system as a common structural motif and they compete with UDP-Glc for binding. Interestingly, one of the compounds promoted binding of uridine to WaaG, as seen from STD NMR titrations, suggesting a different binding mode for this ligand. We propose these compounds as scaffolds for the design of selective high-affinity inhibitors of WaaG. Binding of natural substrates, enzymatic activity and donor substrate selectivity were also investigated by NMR spectroscopy. Molecular dynamics simulations of WaaG were carried out with and without bound UDP and revealed structural changes compared to the crystal structure and also variations in flexibility for some amino acid residues between the two WaaG systems studied.

High-yield expression and purification of a monotopic membrane glycosyltransferase.

Membrane proteins are essential to many cellular processes. However, the systematic study of membrane protein structure has been hindered by the difficulty in obtaining large quantities of these proteins. Protein overexpression using Escherichia coli is commonly used to produce large quantities of protein, but usually yields very little membrane protein. Furthermore, optimization of the expressing conditions, as well as the choice of detergent and other buffer components, is thought to be crucial for increasing the yield of stable and homogeneous protein. Herein we report high-yield expression and purification of a membrane-associated monotopic protein, the glycosyltransferase monoglucosyldiacylglycerol synthase (alMGS), in E. coli. Systematic optimization of protein expression was achieved through controlling a few basic expression parameters, including temperature and growth media, and the purifications were monitored using a fast and efficient size-exclusion chromatography (SEC) screening method. The latter method was shown to be a powerful tool for fast screening and for finding the optimal protein-stabilizing conditions. For alMGS it was found that the concentration of detergent was just as important as the type of detergent, and a low concentration of n-dodecyl-beta-D-maltoside (DDM) (approximately 1x critical micelle concentration) was the best for keeping the protein stable and homogeneous. By using these simply methods to optimize the conditions for alMGS expression and purification, the final expression level increase by two orders of magnitude, reaching 170 mg of pure protein per litre culture.