Biomedical subjects

Sauli Haataja

Publications and source records attributed to Sauli Haataja.

11 recordsLinked to original sources

PubMed2006

Iron incorporation in Streptococcus suis Dps-like peroxide resistance protein Dpr requires mobility in the ferroxidase center and leads to the formation of a ferrihydrite-like core.

The Dps-like peroxide resistance protein (Dpr) is a dodecameric protein that protects the human and swine pathogen Streptococcus suis from hydrogen peroxide by removing free Fe2+ from the cytosol. Subsequent oxidation of iron by Dpr results in the deposition of Fe3+ inside the protein's central cavity. Structural changes that occur in the ferroxidase center were studied by X-ray crystallography after soaking Dpr crystals with Fe2+ in the presence of sodium dithionite. Twelve iron-binding sites were identified with each site formed by residues Asp74 and Glu78 from one subunit, and Asp63, His47 and His59 from a 2-fold symmetry-related subunit. Compared to the iron-free Dpr, Asp74 and Glu78 were found to be the most flexible amino acid residues and able to adopt a variety of conformations in different subunits. The crystal structure of an Asp74Ala Dpr mutant soaked with a Fe2+ -solution revealed variations in the Asp63 position and no iron bound to the ferroxidase center. These results indicate an intrinsic flexibility in the active site that may be important for the catalytic reaction and subsequent nucleation events. Two iron cores with remarkably different features were identified in Dpr using X-ray absorption spectroscopy. Purified Dpr was found to have a small-size iron core with only approximately 16 iron atoms/dodecamer forming a ferritin-like ferrihydrite structure. Because of its size, this core represents the smallest iron core identified so far in ferritins and other Dps-like proteins. A large-size core (approximately 180 iron atoms/dodecamer) formed after incubating the protein with a ferrous solution shows differences in iron coordination compared to the small size core. Characterization of the two iron cores in Dpr could provide insights into nucleation events and the mechanism of iron core growth in the Dps family of proteins.

Animals↗

PubMed2006

Use of flow cytometry for the adhesion analysis of Streptococcus pyogenes mutant strains to epithelial cells: investigation of the possible role of surface pullulanase and cysteine protease, and the transcriptional regulator Rgg.

BACKGROUND: Flow cytometry based adherence assay is a potentially powerful but little used method in the study of bacterial binding to host structures. We have previously characterized a glycoprotein-binding activity in Streptococcus pyogenes called 'strepadhesin' binding to thyroglobulin, submaxillar mucin, fetuin and asialofetuin. We have identified surface-associated pullulanase (PulA) and cysteine protease (SpeB) as carriers of strepadhesin activity. In the present paper, we investigated the use of flow cytometry as a method to study the binding of Rgg, SpeB and PulA knock-out strains to cultured human epithelial cells. RESULTS: Streptococcal mutants were readily labelled with CFDA-SE and their binding to epithelial cells could be effectively studied by flow cytometry. A strain deficient in Rgg expression showed increased binding to the analyzed epithelial cell lines of various origin. Inactivation of SpeB had no effect on the adhesion, while PulA knock-out strains displayed decreased binding to the cell lines. CONCLUSION: These results suggest that the flow cytometric assay is a valuable tool in the analysis of S. pyogenes adherence to host cells. It appears to be an efficient and sensitive tool for the characterization of interactions between the bacteria and the host at the molecular level. The results also suggest a role for Rgg regulated surface molecules, like PulA, in the adhesion of S. pyogenes to host cells.

Bacterial Adhesion↗

PubMed2005

Structure-activity relationships of galabioside derivatives as inhibitors of E. coli and S. suis adhesins: nanomolar inhibitors of S. suis adhesins.

Four collections of Gal alpha1-4Gal derivatives were synthesised and evaluated as inhibitors of the PapG class II adhesin of uropathogenic Escherichia coli and of the P(N) and P(O) adhesins of Streptococcus suis strains. Galabiosides carrying aromatic structures at C1, methoxyphenyl O-galabiosides in particular, were identified as potent inhibitors of the PapG adhesin. Phenylurea derivatisation at C3' and methoxymethylation at O2' of galabiose provided inhibitors of the S. suis strains type P(N) adhesin with remarkably high affinities (30 and 50 nM, respectively). In addition, quantitative structure-activity relationship models for E. coli PapG adhesin and S. suis adhesin type P(O) were developed using multivariate data analysis. The inhibitory lead structures constitute an advancement towards high-affinity inhibitors as potential anti-adhesion therapeutic agents targeting bacterial infections.

Adhesins, Bacterial↗

PubMed2005

Dps/Dpr ferritin-like protein: insights into the mechanism of iron incorporation and evidence for a central role in cellular iron homeostasis in Streptococcus suis.

The Dps family members constitute a distinct group of multimeric and ferritin-like iron binding proteins (up to 500 iron atoms/12-mer) that are widespread in eubacteria and archaea and implicated in oxidative stress resistance and virulence. Despite the wealth of structural knowledge, the mechanism of iron incorporation has remained elusive. Here, we provide evidence on Dpr of the swine and human pathogen Streptococcus suis that: (i) iron incorporation proceeds by Fe(II) binding, Fe(II) oxidation and subsequent storage as Fe(III); (ii) Fe(II) atoms enter the 12-mer cavity through four hydrophilic pores; and (iii) Fe(II) atoms are oxidized inside the 12-mer cavity at 12 identical inter-subunit sites, which are structurally different but functionally equivalent to the ferroxidase centres of classical ferritins. We also provide evidence, by deleting and ectopically overexpressing Dpr, that Dpr affects cellular iron homeostasis. The key residues responsible for iron incorporation in S. suis Dpr are well conserved throughout the Dps family. A model for the iron incorporation mechanism of the Dps/Dpr ferritin-like protein is proposed.

Amino Acid Sequence↗

PubMed2004

Inhibition of Streptococcus suis adhesion by dendritic galabiose compounds at low nanomolar concentration.

A series of mono-, di-, and tetravalent galabiose (Galalpha1-4Gal) compounds were synthesized in good yields by coupling of a general carboxylic acid-bearing sugar building block to dendritic scaffolds based on the 3,5-di-(2-aminoethoxy)benzoic acid branching unit. Furthermore, a poly(amidoamine)- (PAMAM-) based dendritic galabioside was synthesized containing eight galabiose units. All galabiosides were tested in a hemagglutination assay and a surface plasmon resonance (SPR) competition assay in order to establish their potency in the binding to the bacterial Gram-positive pathogen Streptococcus suis. A monovalent galabioside containing a short spacer was used as a reference compound in all the assays. Variations in the scaffold as well as in the spacer arms were introduced to determine their influence on the inhibition. The best inhibitor of hemagglutination was an octavalent galabioside with a minimal inhibitory concentration (MIC) of 0.3 nM, to the best of our knowledge the first example of inhibition of bacterial binding by a soluble carbohydrate at a subnanomolar concentration.

Bacterial Adhesion↗

PubMed2004

Construction of antibody mimics from a noncatalytic enzyme-detection of polysialic acid.

We have used a conceptually novel way to construct antibody mimics based on the binding of a noncatalytic enzyme to its substrate. Bacteriophage-derived endosialidase cleaves polysialic acid (polySia), an important oncofetal and bacterial antigen, which is poorly immunogenic. We fused to green fluorescent protein (GFP) a catalytically inactive endosialidase known to bind but not degrade polysialic acid. The fusion protein is a convenient single-step reagent in fluorescence microscopy, binding assays and immunoblots. It efficiently and specifically detected polysialic acid in developing brain, neuroblastoma cells and bacteria causing meningitis. Enzyme-substrate interactions represent an unexploited source of molecular recognition events. Some of these could be used in designing well-defined substitute antibodies for the study of target molecules which are difficult to purify, available in low quantities, are unstable or have poor immunogenity.

Animals↗

PubMed2004

Crystal structure of Streptococcus suis Dps-like peroxide resistance protein Dpr: implications for iron incorporation.

The Dps-like peroxide resistance protein (Dpr) is an aerotolerance and hydrogen peroxide resistance agent found in the meningitis-associated pathogen Streptococcus suis. Dpr is believed to act by binding free intracellular iron to prevent Fenton chemistry-catalysed formation of toxic hydroxyl radicals. The crystal structure of Dpr has been determined to 1.95 A resolution. The final model has an Rcyst value of 18.5% (Rfree = 22.4%) and consists of 12 identical monomers (each of them comprising a four alpha-helix bundle) that form a hollow sphere obeying 23 symmetry. Structural features show that Dpr belongs to the Dps family of bacterial proteins. Twelve putative ferroxidase centers, each formed at the interface of neighboring monomer pairs, were identified in the Dpr structure with structural similarities to those found in other Dps family members. Dpr was crystallized in the absence of iron, hence no bound iron was found in the structure in contrast to other Dps family members. A novel metal-binding site approximately 6A from the ferroxidase centre was identified and assigned to a bound calcium ion. Two residues from the ferroxidase centre (Asp63 and Asp74) were found to be involved in calcium binding. Structural comparison with other family members revealed that Asp63 and Asp74 adopt different conformation in the Dpr structure. The structure of Dpr presented here shows potential local conformational changes that may occur during iron incorporation. A role for the metal-binding site in iron uptake is proposed.

Amino Acid Sequence↗

PubMed2003

Streptococcus pyogenes glycoprotein-binding strepadhesin activity is mediated by a surface-associated carbohydrate-degrading enzyme, pullulanase.

The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel antiadhesive drugs and vaccines. We have previously identified strepadhesin, a novel glycoprotein-binding activity in Streptococcus pyogenes binding to thyroglobulin, submaxillar mucin, fetuin, and asialofetuin. The activity is known to be regulated by Mga, a regulator of streptococcal virulence factors, and is carried by the surface-associated streptococcal cysteine protease, SpeB. In the present study, we focused on the high strepadhesin activity in an S. pyogenes strain (NZ131rgg) lacking SpeB expression. By extracting surface proteins from the bacteria, a new strepadhesin protein was identified, and mass spectrometric analysis and database search identified it as a putative pullulanase. The gene was cloned, and the recombinant pullulanase (PulA) exhibited pullulanase and starch hydrolyzing activity, as well as strepadhesin activity. Sequencing of the pulA gene revealed an open reading frame with 3,498 bp encoding a protein of 1,165 amino acids with a predicted molecular mass of 129 kDa. PulA exhibited properties typical for a gram-positive surface protein with a putative signal sequence and LPKTGE cell wall anchoring motif and contained the four highly conserved regions common to pullulanases. Mutant bacteria deficient in PulA expression showed diminished strepadhesin activity on bacterial dot blot assay and reduced adherence to thyroglobulin immobilized on microtiter plates. Thus, S. pyogenes strepadhesin activity is carried by a surface-bound pullulanase, which combines glycoprotein-binding and carbohydrate-degrading activities in the same molecule.

Adhesins, Bacterial↗

PubMed2002

Molecular basis of H2O2 resistance mediated by Streptococcal Dpr. Demonstration of the functional involvement of the putative ferroxidase center by site-directed mutagenesis in Streptococcus suis.

H(2)O(2) is an unavoidable cytotoxic by-product of aerobic life. Dpr, a recently discovered member of the Dps protein family, provides a means for catalase-negative bacteria to tolerate H(2)O(2). Potentially, Dpr could bind free intracellular iron and thus inhibit the Fenton chemistry-catalyzed formation of toxic hydroxyl radicals (H(2)O(2) + Fe(2+) --> (.)OH + (-)OH + Fe(3+)). We explored the in vivo function of Dpr in the catalase- and NADH peroxidase-negative pig and human pathogen Streptococcus suis. We show that: (i) a Dpr allelic exchange knockout mutant was hypersensitive ( approximately 10(6)-fold) to H(2)O(2), (ii) Dpr incorporated iron in vivo, (iii) a putative ferroxidase center was present in Dpr, (iv) single amino acid substitutions D74A or E78A to the putative ferroxidase center abolished the in vivo iron incorporation, and (v) the H(2)O(2) hypersensitive phenotype was complemented by wild-type Dpr or by a membrane-permeating iron chelator, but not by the site-mutated forms of Dpr. These results demonstrate that the putative ferroxidase center of Dpr is functionally active in iron incorporation and that the H(2)O(2) resistance is mediated by Dpr in vivo by its iron binding activity.

Amino Acid Sequence↗

PubMed2002

Expression, purification and crystallization of Dpr, a ferritin-like protein from the Gram-positive meningitis-associated bacterium Streptococcus suis.

Ferritin-like proteins form a novel family of bacterial proteins with diverse functions, such as DNA binding, iron storage and cell activation. A common structural feature of these proteins is their ability to form spherical dodecamers. Dpr is a ferritin-like protein from the Gram-positive bacterium Streptococcus suis. Full-length and truncated Dpr were expressed and purified as 6xHis-tag fusion proteins. Crystals of truncated Dpr suitable for X-ray diffraction analysis were obtained after the removal of the N-terminal affinity tag by thrombin cleavage. A complete data set to 2.3 A resolution was collected using synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 104.3, b = 137.6, c = 142.1 A and 12 molecules in the asymmetric unit.

Bacterial Proteins↗

PubMed2000

Identification of a novel glycoprotein-binding activity in Streptococcus pyogenes regulated by the mga gene.

The interaction between Streptococcus pyogenes and the host cell surface is not completely understood. Characterization of the adhesion mechanisms of the bacterium to the host cell surface is needed in order to develop new vaccines and anti-adhesion drugs. The presence of glycoprotein-binding activities among streptococcal strains was investigated. An activity binding to thyroglobulin, fetuin, asialofetuin and mucin but not non-glycosylated proteins was found to be present in the majority of the S. pyogenes strains studied. Cross-inhibition experiments suggested that the glycoproteins share a common structure recognized by the bacteria. The glycoprotein-binding activity was found to be proteinaceous, tightly attached to the bacterial surface and it also mediated the adherence of bacteria to solid surfaces coated with glycoproteins. The activity was found by transposon mutagenesis and complementation to be regulated by the multiple-gene regulator Mga, which has been implicated as a regulator of S. pyogenes virulence factors.

Bacterial Adhesion↗