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Biomedical subjects

P E Lindgren

Publications and source records attributed to P E Lindgren.

13 recordsLinked to original sources
PubMed2001

Interaction of Staphylococcus epidermidis from infected hip prostheses with neutrophil granulocytes.

This study focuses on the interaction of Staphylococcus epidermidis isolated from granulation tissue covering infected hip prostheses and neutrophil granulocytes. Bacterial strains isolated from normal flora were used as controls. The bacteria were well characterized with routine methods and further characterized with random amplified polymorphic DNA analyses and slime tests. Phagocytosis and chemiluminescence (CL) assays were used in the neutrophil interaction studies. The prostheses strains were ingested to a lesser extent than strains from normal flora (p < or = 0.001). There was no significant difference between the prostheses strains and the normal flora strains in terms of total CL response. However, the extracellular CL response from the neutrophils was lower in comparison with the normal flora when interacting with the prostheses strains. The results of this study support the notion that S. epidermidis strains isolated from infected hip prostheses have an enhanced capacity to resist phagocytosis and that most of these strains elicit a reduced inflammatory response, measured as the production of extracellular oxidative metabolites from the neutrophils, compared to normal flora.

DNA, Bacterial↗
PubMed2001

Quantification of ammonia-oxidizing bacteria in arable soil by real-time PCR.

Real-time PCR was used to quantify populations of ammonia-oxidizing bacteria representing the beta subdivision of the class Proteobacteria in samples of arable soil, both nitrogen fertilized and unfertilized, from Mellby, Sweden. Primers and probes targeting a 16S ribosomal DNA region of the ammonia-oxidizing bacteria were designed and used. In the fertilized soil there were approximately 6.2 x 10(7) ammonia-oxidizing bacteria per g of soil, three times more than the number of bacteria in the unfertilized soil. The lytic efficiency of bead beating in these soils was investigated by using populations of free or loosely attached bacteria, bacteria tightly bound to particles, and bacteria in nonfractionated samples. The shapes of the curves generated in these tests showed that the concentration of template DNA released at various times remained constant after 10 to 100 s of bead beating.

Ammonia↗
PubMed2000

Streptococcal opacity factor: a family of bifunctional proteins with lipoproteinase and fibronectin-binding activities.

The serum opacity factor (SOF) of Streptococcus pyogenes is a type-specific lipoproteinase of unknown biological significance. We have sequenced the sof gene and characterized the corresponding SOF protein from a strain of type M63. It was found that sof63 is related to sof22 and that, similar to SOF22 [25], SOF63 binds fibronectin. Moreover, we demonstrate opacity factor activity in a Streptococcus dysgalactiae fibronectin-binding protein FnBA that is structurally related to the SOF proteins of S. pyogenes. Sequence analysis of these three SOF proteins showed a unique periodical pattern of conserved and variable regions. The enzymatically active part of SOF63 was localized to the fragment corresponding to the entire set of conserved and variable sequences, while for fibronectin-binding a single repeat in the C terminal part of the protein was sufficient. The results show that streptococcal SOF proteins form a novel family of bifunctional proteins with lipoproteinase and fibronectin-binding activities.

Adhesins, Bacterial↗
PubMed1999

Characterization of the molybdate transport system ModABC of Staphylococcus carnosus.

Transposon mutagenesis of Staphylococcus carnosus led to the identification of three genes, modABC, which encode an ABC transporter that is involved in molybdate transport. It was shown by [14C]palmitate labeling that ModA represents a lipoprotein that in gram-positive bacteria is the counterpart of the periplasmic binding proteins of gram-negative organisms. The sequence characteristics identify ModB as the integral-membrane, channel-forming protein and ModC as the ATP-binding energizer for the transport system. Mutants defective in modABC had only 0.4% of the wild-type nitrate reductase activity. Molybdate at a non-physiologically high concentration (100 microM) fully restored nitrate reductase activity, suggesting that at least one other system is able to transport molybdate, but with lower affinity. The expression of modA (and most likely of modBC) was independent of oxygen and nitrate. To date, there are no indications for molybdate-specific regulation of modABC expression since in a modB mutant, modA expression was unchanged in comparison to the wild-type.

ATP-Binding Cassette Transporters↗
PubMed1999

PCR detection of genes encoding nitrite reductase in denitrifying bacteria.

Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplify cd1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships of nir genes were also established. The cd1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.

Alcaligenes↗
PubMed1998

Identification and characterization of the Staphylococcus carnosus nitrate reductase operon.

Physiological and genetic characterization of Staphylococcus carnosus nitrate reductase-negative mutants led to the identification of the nitrate reductase operon, narGHJI. Transcription from the nar promoter was stimulated by anaerobiosis, nitrate, and nitrite. This is in accordance with the nitrate reductase activities determined with benzyl viologen as electron donor. However, in the presence of oxygen and nitrate, high transcriptional initiation but low nitrate reductase activity was observed. Since the alphabeta complex of the nitrate reductase formed during anaerobic growth was insensitive to oxygen, other oxygen-sensitive steps (e.g., post-transcriptional mechanisms, molybdenum cofactor biosynthesis) must be involved. The nitrate-reducing system in S. carnosus displays similarities to the dissimilatory nitrate reductases of Escherichia coli. However, in the S. carnosus nar promoter, no obvious Fnr and integration host factor recognition sites are present; only one site that is related to the E. coli NarL consensus sequence was found. Studies to determine whether the E. coli proteins NarL and Fnr are functional at the S. carnosus narGHJI promoter indicated that the promoter is not functional in E. coli.

Amino Acid Sequence↗
PubMed1994

A fibronectin-binding protein from Streptococcus equisimilis: characterization of the gene and identification of the binding domain.

We have cloned and characterized a gene, fnb, from S. equisimilis, encoding a fibronectin-binding protein, FnB. A genomic library containing chromosomal DNA from S. equisimilis strain Se165 in the pUC18 vector in E. coli TG1 was screened using a DNA fragment of the gene fnbB from S. dysgalactiae strain S2 as probe. The complete gene was sequenced. The molecular mass of the protein, calculated from the deduced amino acid sequence is 120 kDa, which coincides with that determined by gel electrophoresis and Western blotting of the gene product. The fibronectin-binding activity was localized to a region of three repeated units, each 36 amino acids long. The COOH-terminal part of FnB from S. equisimilis, including the repeated Fn-binding domain, is very similar to the corresponding part of FnBB from S. dysgalactiae, indicating a common origin of the regions encoding Fn-binding activity of the respective genes.

Adhesins, Bacterial↗
PubMed1994

Biochemical properties of a novel metalloprotease from Staphylococcus hyicus subsp. hyicus involved in extracellular lipase processing.

Two extracellular proteases from Staphylococcus hyicus subsp. hyicus, ShpI and ShpII, have been characterized. ShpI is a neutral metalloprotease with broad substrate specificity; the gene has been cloned and sequenced. ShpII, characterized here, is mainly produced in the late logarithmic growth phase in contrast to ShpI, which is mainly produced in the late stationary growth phase. ShpII was purified from culture medium of S. hyicus by ammonium sulfate precipitation and DEAE-Sepharose chromatography. The molecular mass, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 34 kDa. The temperature optimum of ShpII was 55 degrees C, and the pH optimum was 7.4. ShpII, a neutral metalloprotease, was strongly inhibited by zinc and calcium chelators. The amino-terminal sequence of the active enzyme was similar to the corresponding region of a Staphylococcus epidermidis metalloprotease. The substrate specificity of ShpII was similar to that of thermolysin-like proteases, with the exception that ShpII also recognized aromatic amino acids. We demonstrated in vitro that ShpII, but not ShpI, cleaved the 86-kDa S. hyicus subsp. hyicus prolipase between Thr-245 and Val-246 to generate the mature 46-kDa lipase. Results of additional in vivo experiments supported the model that ShpII is necessary for the extracellular processing and maturation of S. hyicus subsp. hyicus lipase.

Amino Acid Sequence↗
PubMed1993

Fibronectin receptors from Streptococcus dysgalactiae and Staphylococcus aureus. Involvement of conserved residues in ligand binding.

The nucleotide sequence of two genes encoding fibronectin (Fn) receptors FnBA and FnBB of Streptococcus dysgalactiae S2 revealed the presence of repeated motifs (called RA1-A3 and RB1-B3, respectively) which encode Fn binding activity (Lindgren, P.-E., McGavin, M. J., Signäs, C., Guss, B., Gurusiddappa, S., Höök, M., and Lindberg, M. (1993) Eur. J. Biochem. 214, 819-827). Synthetic peptides of 32-37 amino acids, corresponding to individual repeated motifs, were assayed for the ability to inhibit Fn binding to cells of S. dysgalactiae. Within the RA motifs, peptide A2 was 10-fold more active than either A1 or A3, while in the RB motifs, only B3 was active. The same level of activity is observed when these synthetic peptides were assayed for inhibition of Fn binding to cells of Staphylococcus aureus. Likewise, synthetic peptides corresponding to the RD1-D3 motifs, which comprise a ligand binding domain in a Fn receptor from S. aureus, inhibit binding of Fn to both S. aureus and S. dysgalactiae. Assays of chemically modified peptides and peptide fragments derived from chemical or proteolytic cleavage suggest that a conserved core sequence, defined as ED(T/S) (X9,10)GG(X3,4)(I/V)DF, within a 30-amino acid-long segment is present in the active RA and RD motifs. Analyses of the importance of individual residues of this core sequence indicate that the ED(T/S) motif is nonessential, whereas the GG and the (I/V)DF together with additional acidic residues in the C-terminal half of the peptide are required for activity.

Amino Acid Sequence↗
PubMed1993

Two different genes coding for fibronectin-binding proteins from Streptococcus dysgalactiae. The complete nucleotide sequences and characterization of the binding domains.

The binding of Streptococcus dysgalactiae to fibronectin involves fibronectin-binding protein(s) present on the bacterial surface. Previously, we reported the cloning of two different genes coding for cell-wall-associated fibronectin-binding proteins from S. dysgalactiae strain S2 [Lindgren, P.-E., Speziale, P., McGavin, M. J., Monstein, H.-J., Höök, M., Visai, L., Kostiainen, T., Bozzini, S. & Lindberg, M. (1992) J. Biol. Chem. 267, 1924-1931]. The two genes, fnbA and fnbB, have now been sequenced and the primary amino acid sequences of the two fibronectin-binding proteins, FnBA and FnBB, have been deduced. The two proteins have predicted molecular masses of 117 kDa and 122 kDa, respectively, and are organized in a similar way. The fibronectin-binding activities are localized in repeated motifs, 32-37 amino acids long, in the COOH-terminal regions of the proteins. The two fibronectin-binding proteins have heterologous amino acid sequences, except for the COOH-terminal ends which include the fibronectin-binding repeats. The fibronectin-binding regions of the genes have been fused to IgG-binding domains of protein A, utilizing the IgG-binding capacity of the resulting fusion proteins, to facilitate isolation of the fibronectin-binding domains.

Adhesins, Bacterial↗
PubMed1992

Cloning and expression of two different genes from Streptococcus dysgalactiae encoding fibronectin receptors.

Binding of bacteria to fibronectin has been implicated as a mechanism of bacterial adhesion to the host tissue. In this report we have analyzed the binding of a strain of Streptococcus dysgalactiae to fibronectin. The cells bind to a site in the NH2-terminal domain of the protein via trypsin-sensitive cell surface components. Furthermore, a lysate prepared by sonication of streptococcal cells contained fibronectin-binding proteins that inhibit the binding of the ligand to intact bacteria. When the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to an Immobilon-P filter, and probed with 125I-labeled fibronectin, a 140-kDa fibronectin-binding protein was identified along with a number of smaller binding proteins. A genomic DNA library was constructed and screened for the expression of fibronectin-binding proteins. Two clones were isolated and shown to contain unrelated inserts by restriction mapping and cross-hybridization experiments. The two encoded proteins were also immunologically distinct although both bound to the same region of the fibronectin molecule, and both effectively inhibited the binding of 125I-fibronectin to bacterial cells. Immunological analyses showed that only one of the two proteins tentatively identified as fibronectin receptors was expressed in detectable quantities in the Streptococcus dysgalactiae strain under the culture conditions employed.

Bacterial Adhesion↗
PubMed1989

Nucleotide sequence of the gene for a fibronectin-binding protein from Staphylococcus aureus: use of this peptide sequence in the synthesis of biologically active peptides.

Binding of cells of Staphylococcus aureus to fibronectin, which may represent a mechanism of host tissue adherence, involves a fibronectin-receptor protein present on the bacterial surface. Cloning of a gene coding for a staphylococcal fibronectin-binding protein and construction of a fusion protein with fibronectin-binding properties was previously reported from our laboratory. We have now sequenced the gene and deduced a primary sequence of the fibronectin-binding protein. The protein resembles other cell-wall-associated proteins on Gram-positive bacteria in that it (i) appears to be anchored in the cell membrane via its C-terminal end, (ii) contains a proline-rich repeating unit outside the membrane anchor, and (iii) contains a long (36-amino acid) signal sequence at the N terminus. The fibronectin-binding activity has been localized to a domain composed of a 38-amino acid unit repeated completely three times and partially a fourth time; the identity between the three 38-amino acid sequences varies from 42 to 87%. Three synthetic peptides mimicking the structure of each 38-amino acid unit were constructed. All three peptides interacted with fibronectin, as indicated by their ability to inhibit binding of fibronectin to staphylococcal cells, whereas an unrelated 37-amino acid peptide showed no inhibitory activity.

Amino Acid Sequence↗