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1.
Infect Immun ; 73(1): 126-34, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15618147

ABSTRACT

Matrix metalloproteinases (MMPs) are induced from host tissues in response to Borrelia burgdorferi. Upregulation of MMPs may play a role in the dissemination of the organism through extracellular matrix tissues, but it can also result in destructive pathology. Although mice are a well-accepted model for Lyme arthritis, there are significant differences compared to human disease. We sought to determine whether MMP expression could account for some of these differences. MMP expression patterns following B. burgdorferi infection were analyzed in primary human chondrocytes, synovial fluid samples from patients with Lyme arthritis, and cartilage tissue from Lyme arthritis-susceptible and -resistant mice by using a gene array, real-time PCR, an enzyme-linked immunosorbent assay, and immunohistochemistry. B. burgdorferi infection significantly induced transcription of MMP-1, -3, -13, and -19 from primary human chondrocyte cells. Transcription of MMP-10 and tissue inhibitor of metalloprotease 1 was increased with B. burgdorferi infection, but protein expression was only minimally increased. The synovial fluid levels of MMPs from patients with high and low spirochete burdens were consistent with results seen in the in vitro studies. B. burgdorferi-susceptible C3H/HeN mice infected with B. burgdorferi showed induction of MMP-3 and MMP-19 but no other MMP or tissue inhibitor of metalloprotease. As determined by immunohistochemistry, MMP-3 expression was increased only in chondrocytes near the articular surface. The levels of MMPs were significantly lower in the more Lyme arthritis-resistant BALB/c and C57BL/6 mice. Differences between human and murine Lyme arthritis may be related to the lack of induction of collagenases, such MMP-1 and MMP-13, in mouse joints.


Subject(s)
Lyme Disease/enzymology , Matrix Metalloproteinases/biosynthesis , Animals , Cartilage/enzymology , Cells, Cultured , Chondrocytes/enzymology , Enzyme Induction , Gene Expression Profiling , Humans , Joints/enzymology , Matrix Metalloproteinases/genetics , Mice , Mice, Inbred Strains , Species Specificity , Synovial Fluid/enzymology
2.
Microbiology (Reading) ; 150(Pt 2): 391-397, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14766917

ABSTRACT

Phospholipids are an important component of bacterial membranes. Borrelia burgdorferi differs from many other bacteria in that it contains only two major membrane phospholipids: phosphatidylglycerol (PG) and phosphatidylcholine (PC). B. burgdorferi appears to lack enzymes required for synthesis of PC through the well-described methylation pathway. However, B. burgdorferi does contain a gene (BB0249) with significant identity to a recently described phosphatidylcholine synthase gene (pcs) of Sinorhizobium meliloti. B. burgdorferi also contains a gene (BB0721) with significant identity to the gene (pgs) encoding phosphatidylglycerolphosphate synthase, an enzyme in the synthetic pathway of PG. Activity of BB0249 was confirmed by cloning the gene into Escherichia coli, which does not produce PC. Transformation with a plasmid carrying BB0249 resulted in production of PC by E. coli, but only in the presence of exogenously supplied choline, as would be predicted for a Pcs. Because loss of Pgs activity is lethal to E. coli, activity of BB0721 was confirmed by the ability of BB0721 to complement an E. coli Pgs(-) mutant. A plasmid containing BB0721 was transformed into a Pgs(-) mutant of E. coli containing a copy of the native gene on a temperature-regulated plasmid. The temperature-regulated plasmid was exchanged for a plasmid containing BB0721 and it was shown that BB0721 was able to replace the lost Pgs function and restore bacterial growth. This study has established the existence and function of two critical enzymes in the synthesis of PC and PG in B. burgdorferi. Understanding of the biosynthetic pathways of PC and PG in B. burgdorferi is the first step in delineating the role of these phospholipids in the pathogenesis of Lyme disease.


Subject(s)
Borrelia burgdorferi/enzymology , Borrelia burgdorferi/genetics , Phospholipids/biosynthesis , Transferases (Other Substituted Phosphate Groups)/genetics , Base Sequence , Gene Expression Regulation, Bacterial/genetics , Kinetics , Plasmids/genetics , Transferases (Other Substituted Phosphate Groups)/metabolism
3.
J Bacteriol ; 186(1): 51-60, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14679224

ABSTRACT

The Borrelia burgdorferi genome encodes five orthologues of the substrate binding protein oligopeptide permease A (OppA). It was previously shown that these genes are under the control of separate promoters and are differentially expressed under various environmental conditions. We were interested in determining whether there are also differences in substrate specificities among the proteins. The substrate specificities of recombinant proteins were determined by screening for high-affinity peptides by use of a combinatorial phage display heptapeptide library. Different heptapeptides with high affinities for OppA-1, OppA-2, and OppA-3 were identified. No heptapeptide binding OppA-4 or OppA-5 could be identified. Competitive binding assays were performed under various conditions to determine the substrate preferences of the OppA proteins. OppA-1 retained maximal activity over a broad range of pHs (5.5 to 7.5), whereas OppA-2 and OppA-3 showed peak activities at pHs below 5.5. OppA-1 and OppA-2 showed preferences for tripeptides over dipeptides and longer-chain peptides. Although a wide variety of amino acyl side chains were tolerated by all three OppA proteins, OppA-1 showed the broadest substrate specificity and was able to accommodate peptides composed of bulky hydrophobic residues; OppA-2 and OppA-3 showed preferences for peptides composed of small nonpolar amino acids. All three OppA proteins showed preferences for peptides composed of L- rather than D-amino acids. OppA-3 showed the greatest tolerance for changes in stereochemistry. Substantial differences in the substrate specificities of the OppA proteins of B. burgdorferi suggest that they may have distinct functions in the organism.


Subject(s)
Borrelia burgdorferi/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Lipoproteins/chemistry , Lipoproteins/metabolism , Oligopeptides/metabolism , Operon , Bacterial Proteins , Binding, Competitive , Borrelia burgdorferi/genetics , Carrier Proteins/genetics , Carrier Proteins/isolation & purification , Combinatorial Chemistry Techniques , Gene Expression Regulation, Bacterial , Hydrogen-Ion Concentration , Lipoproteins/genetics , Lipoproteins/isolation & purification , Peptide Library , Stereoisomerism , Substrate Specificity
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