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1.
J Clin Microbiol ; 37(4): 987-92, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10074514

ABSTRACT

The quantity of Borrelia burgdorferi organisms in tissue samples is an important determinant for infection studies in the mouse model of Lyme disease. This report presents the development of a rapid and sensitive external-standard-based PCR assay for the absolute quantification of B. burgdorferi in mouse tissue samples. The assay uses a double-stranded DNA dye to continuously monitor product formation and in less than an hour was able to quantify samples ranging up to 6 log units in concentration. The PCR efficiencies of the sample and the standard were matched by using a standard composed of purified B. burgdorferi chromosome mixed with tissue-matched mouse genome lacking bacterial DNA. Normalization of B. burgdorferi quantities to the mouse nidogen gene allowed comparison of B. burgdorferi numbers in samples isolated from different tissues and strains. PCR analysis of the chromosomal gene recA in cultured B. burgdorferi was consistent with a single recA per bacterium. The parameters defined in this assay should be applicable to quantification of other organisms, even infectious agents for which no ready source of DNA standard is available. In summary, this report presents a rapid external-standard-based PCR method for the quantification of B. burgdorferi in mouse DNA samples.


Subject(s)
Borrelia burgdorferi Group/genetics , Borrelia burgdorferi Group/isolation & purification , Lyme Disease/microbiology , Organic Chemicals , Polymerase Chain Reaction/methods , Animals , Base Sequence , Benzothiazoles , Chromosomes, Bacterial/genetics , Colony Count, Microbial , DNA Primers/genetics , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Diamines , Disease Models, Animal , Fluorescent Dyes , Genes, Bacterial , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Polymerase Chain Reaction/standards , Polymerase Chain Reaction/statistics & numerical data , Quinolines , Rec A Recombinases/genetics , Reference Standards , Sensitivity and Specificity , Tissue Distribution
2.
J Immunol ; 162(2): 948-56, 1999 Jan 15.
Article in English | MEDLINE | ID: mdl-9916719

ABSTRACT

A spectrum of disease severity has been observed in patients with Lyme disease, with approximately 60% of untreated individuals developing arthritis. The murine model of Lyme disease has provided strong evidence that the genetic composition of the host influences the severity of arthritis following infection with Borrelia burgdorferi: infected C3H mice develop severe arthritis while infected C57BL/6N mice develop mild arthritis. Regions of the mouse genome controlling arthritis severity and humoral responses during B. burgdorferi infection were identified in the F2 intercross generation of C3H/HeNCr and C57BL/6NCr mice. Rear ankle swelling measurements identified quantitative trait loci (QTL) on chromosomes 4 and 5, while histopathological scoring identified QTL on a unique region of chromosome 5 and on chromosome 11. The identification of QTL unique for ankle swelling or histopathological severity suggests that processes under distinct genetic control are responsible for these two manifestations of Lyme arthritis. Additional QTL that control the levels of circulating Igs induced by B. burgdorferi infection were identified on chromosomes 6, 9, 11, 12, and 17. Interestingly, the magnitude of the humoral response was not correlated with the severity of arthritis in infected F2 mice. This work defines several genetic loci that regulate either the severity of arthritis or the magnitude of humoral responses to B. burgdorferi infection in mice, with implications toward understanding the host-pathogen interactions involved in disease development.


Subject(s)
Antibodies, Bacterial/biosynthesis , Arthritis/genetics , Arthritis/immunology , Lyme Disease/genetics , Lyme Disease/immunology , Quantitative Trait, Heritable , Animals , Arthritis/microbiology , Arthritis/pathology , Borrelia burgdorferi Group/immunology , Chromosome Mapping , Crosses, Genetic , DNA, Bacterial/metabolism , Female , Genetic Linkage , Genetic Markers , Heart/microbiology , Lyme Disease/microbiology , Lyme Disease/pathology , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Severity of Illness Index
3.
Biotechniques ; 24(6): 954-8, 960, 962, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9631186

ABSTRACT

Continuous fluorescence observation of amplifying DNA allows rapid and accurate quantification of initial transcript copy number. A simple and generic method for monitoring product synthesis with the double-stranded DNA dye, SYBR Green I provides initial template copy number estimation limited only by stochastic effects. To reach this degree of sensitivity, two methods were used. First, specific products generally have a higher melting temperature than nonspecific products, and therefore, specific product formation was monitored by fluorescence acquisition at temperatures at which only specific products are double-stranded. Second, anti-Taq antibodies were used to reduce nonspecific product generation. The log-linear portion of the fluorescence vs. cycle plot was extended to determine a fractional cycle number at which a threshold fluorescence was obtained. These fractional cycle numbers were plotted against the log of starting template copies to give linear standard curves from purified PCR products, allowing easy estimation of cDNA unknowns over a 10(6)-fold range. A single template molecule per reaction could be distinguished from the absence of template, although stochastic effects increased the variance of concentration estimates below 10 copies. Above 10 copies per reaction, typical replicate coefficients of variation were 6%-37%, with better precision at higher copy numbers.


Subject(s)
Fluorescent Dyes/metabolism , Gene Amplification , Organic Chemicals , Transcription, Genetic , Animals , Benzothiazoles , DNA Primers/chemical synthesis , Diamines , Fibroblasts/chemistry , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Polymerase Chain Reaction/methods , Quinolines , Rats , Sensitivity and Specificity , Synovial Membrane/cytology , Synovial Membrane/metabolism , Templates, Genetic , Transcription, Genetic/genetics
4.
J Immunol ; 160(11): 5485-92, 1998 Jun 01.
Article in English | MEDLINE | ID: mdl-9605151

ABSTRACT

Borrelia burgdorferi possesses membrane lipoproteins that exhibit stimulatory properties and, consequently, have been implicated in the pathology related to Lyme disease. As CD14 has been shown to mediate signaling by a number of lipid-modified bacterial products, the involvement of CD14 in signaling mediated by two B. burgdorferi lipoproteins, outer surface protein A (OspA) and OspC, was determined. Lipoprotein-mediated induction of nuclear factor-kappaB nuclear translocation and production of IL-8 and IL-6 in HUVEC was enhanced in the presence of serum or soluble rCD14. CD14-specific Abs that block LPS-mediated signaling also inhibited lipoprotein-dependent signaling in HUVEC and neutrophils. The formation of stable complexes between OspA and CD14 was demonstrated by native gel electrophoresis. LPS was found to compete with OspA for binding with CD14, suggesting that LPS and OspA bind similar regions on CD14. The similarity in binding was further supported by the finding that a mutant soluble CD14, lacking the LPS binding site, did not facilitate lipoprotein signaling, nor did it form a complex with OspA. Binding of OspA to CD14 was dependent on the lipid modification, as unlipidated OspA did not form a complex with CD14 or stimulate cells. In contrast, the lipopeptide remaining after proteinase K digestion both formed a complex with CD14 and retained stimulatory properties. These findings indicate that CD14 facilitates bacterial lipoprotein signaling in mammalian cells.


Subject(s)
Antigens, Surface/physiology , Bacterial Outer Membrane Proteins/physiology , Borrelia burgdorferi Group/immunology , Lipopolysaccharide Receptors/physiology , Signal Transduction/immunology , Adjuvants, Immunologic/physiology , Antigens, Surface/chemistry , Antigens, Surface/metabolism , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Bacterial Vaccines , Binding, Competitive/immunology , Cells, Cultured , Culture Media, Serum-Free/pharmacology , Drug Stability , Endothelium, Vascular/cytology , Humans , Immune Sera/pharmacology , Lipopolysaccharide Receptors/immunology , Lipopolysaccharide Receptors/metabolism , Lipopolysaccharides/pharmacology , Lipoproteins/antagonists & inhibitors , Lipoproteins/physiology , Macromolecular Substances , Protein Binding/immunology , Umbilical Veins
5.
J Bacteriol ; 179(17): 5543-50, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9287011

ABSTRACT

CheA, a cytoplasmic histidine autokinase, in conjunction with the CheW coupling protein, forms stable ternary complexes with the cytoplasmic signaling domains of transmembrane chemoreceptors. These signaling complexes induce chemotactic movements by stimulating or inhibiting CheA autophosphorylation activity in response to chemoeffector stimuli. To explore the mechanisms of CheA control by chemoreceptor signaling complexes, we examined the ability of various CheA fragments to interfere with receptor coupling control of CheA. CheA[250-654], a fragment carrying the catalytic domain and an adjacent C-terminal segment previously implicated in stimulatory control of CheA activity, interfered with the production of clockwise flagellar rotation and with chemotactic ability in wild-type cells. Epistasis tests indicated that CheA[250-654] blocked clockwise rotation by disrupting stimulatory coupling of CheA to receptors. In vitro coupling assays confirmed that a stoichiometric excess of CheA[250-654] fragments could exclude CheA from stimulatory receptor complexes, most likely by competing for CheW binding. However, CheA[250-654] fragments, even in vast excess, did not block receptor-mediated inhibition of CheA, suggesting that CheA[250-654] lacks an inhibitory contact site present in native CheA. This inhibitory target is most likely in the N-terminal P1 domain, which contains His-48, the site of autophosphorylation. These findings suggest a simple allosteric model of CheA control by ternary signaling complexes in which the receptor signaling domain conformationally regulates the interaction between the substrate and catalytic domains of CheA.


Subject(s)
Chemotaxis/physiology , Escherichia coli Proteins , Escherichia coli/physiology , Membrane Proteins/metabolism , Peptide Fragments/metabolism , Protein Kinases/metabolism , Receptors, Cell Surface/metabolism , Bacterial Proteins/metabolism , Escherichia coli/enzymology , Flagella/physiology , Histidine Kinase , Membrane Proteins/chemistry , Membrane Proteins/isolation & purification , Methyl-Accepting Chemotaxis Proteins , Phosphorylation , Protein Kinases/chemistry , Protein Kinases/isolation & purification , Recombinant Fusion Proteins , Sensory Receptor Cells , Signal Transduction/physiology
6.
J Immunol ; 158(10): 4838-45, 1997 May 15.
Article in English | MEDLINE | ID: mdl-9144499

ABSTRACT

Lyme disease is caused by infection with the spirochete Borrelia burgdorferi and is characterized by bacterial persistence and inflammation of many host tissues. B. burgdorferi express outer surface lipoproteins, including OspA, with inflammatory properties that could contribute to the localized tissue inflammation. Neutrophils are the predominant infiltrate into the inflamed arthritic joints, and are crucial for controlling the spirochete infection. They may also contribute to the joint pathology associated with Lyme arthritis. This study examines the effect of OspA on the activities of the neutrophil. Picomolar concentrations of OspA induce surface markers associated with neutrophil activation: increased CD10 and CD11b expression; decreased CD62-L expression; and an increased adherence to extracellular matrix. These events were similar in kinetics and magnitude to those induced by the strong activators LPS and FMLP. Like LPS, OspA could prime neutrophils for FMLP-induced release of lysosomal granules and production of superoxide. Thus, models of Lyme arthritis should include the possible contribution of direct activation of neutrophils to both defense and disease.


Subject(s)
Antigens, Surface/immunology , Bacterial Outer Membrane Proteins/immunology , Borrelia burgdorferi Group/immunology , Lipoproteins , Neutrophils/immunology , Bacterial Vaccines , Cell Adhesion/drug effects , Extracellular Matrix , Humans , Interleukin-8/biosynthesis , L-Selectin/metabolism , Leukocyte Elastase/metabolism , Lipopolysaccharides/pharmacology , Macrophage-1 Antigen/metabolism , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neprilysin/metabolism , Respiratory Burst , Superoxides/metabolism , Time Factors
7.
Biochemistry ; 34(42): 13871-80, 1995 Oct 24.
Article in English | MEDLINE | ID: mdl-7577981

ABSTRACT

CheA is the histidine autokinase in the Escherichia coli chemotaxis signal transduction pathway responsible for coupling of signals received by transmembrane receptors to the response regulators CheY and CheB. Here NMR spectroscopy is used to study a 14 kDa fragment of CheA, residues 124-257, that binds the response regulator CheY. Backbone atom resonance assignments were obtained by analysis of 3D HNCACB, 3D CBCA(CO)NH, and HNCO spectra, whereas side-chain assignments were obtained primarily by analysis of 3D H(CCO)NH, 3D C(CO)NH, 3D HCCH-TOCSY, and 3D 1H, 15N TOCSY-HSMQC spectra. NOE cross peak patterns and intensities as well as torsion angle restraints were used to determine the secondary structure, and a low-resolution structure was calculated by hybrid distance-geometry simulated annealing methods. The CheA124-257 fragment consists of four antiparallel beta strands and two helices, arranged in an "open-faced beta-sandwich" motif, as well as two unstructured ends that correspond to domain linkers in the full-length protein. The 15N-1H correlation spectrum of 15N-labeled CheA124-257 bound to unlabeled CheY shows specific localized changes that may correspond to a CheY-binding face on CheA.


Subject(s)
Bacterial Proteins , Chemotaxis , Escherichia coli/chemistry , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Protein Kinases/chemistry , Amino Acid Sequence , Carrier Proteins , Computer Graphics , Escherichia coli/enzymology , Escherichia coli Proteins , Histidine Kinase , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Methyl-Accepting Chemotaxis Proteins , Models, Molecular , Molecular Sequence Data , Protein Folding , Protein Kinases/metabolism , Protein Structure, Secondary
8.
J Bacteriol ; 177(10): 2713-20, 1995 May.
Article in English | MEDLINE | ID: mdl-7751280

ABSTRACT

The cheA locus of Escherichia coli encodes two similar proteins, CheAL (654 amino acids) and CheAS (557 amino acids), which are made by initiating translation from different in-frame start sites [start(L) and start(S)]. CheAL plays an essential role in chemotactic signaling. It autophosphorylates at a histidine residue (His-48) and then donates this phosphate to response regulator proteins that modulate flagellar rotation and sensory adaptation. CheAS lacks the first 97 amino acids of CheAL, including the phosphorylation site at His-48. Although it is unable to autophosphorylate, CheAS can form heterodimers with mutant CheAL subunits to restore kinase function and chemoreceptor control of autophosphorylation activity. To determine whether these or other activities of CheAS are important for chemotaxis, we constructed cheA lesions that abrogated CheAS expression. Mutants in which the CheAS start codon was changed from methionine to isoleucine (M98I) or glutamine (M98Q) retained chemotactic ability, ranging from 50% (M98Q) to 80% (M98I) of wild-type function. These partial defects could not be alleviated by supplying CheAS from a specialized transducing phage, indicating that the lesions in CheAL--not the lack of CheAS--were responsible for the reduced chemotactic ability. In other respects, the behavior of the M98I mutant was essentially normal. Its flagellar rotation pattern was indistinguishable from wild type, and it exhibited wild-type detection thresholds and peak positions in capillary chemotaxis assays. The lack of any substantive defect in this start(S) mutant argues that CheAS makes a negligible contribution to chemotactic ability in the laboratory. Whether it has functional significance in other settings remains to be seen.


Subject(s)
Bacterial Proteins , Chemotaxis/genetics , Escherichia coli/genetics , Genes, Bacterial/genetics , Membrane Proteins/genetics , Base Sequence , Chromosomes, Bacterial/genetics , Escherichia coli/drug effects , Escherichia coli Proteins , Flagella/physiology , Histidine Kinase , Methyl-Accepting Chemotaxis Proteins , Molecular Sequence Data , Mutagenesis , Plasmids/genetics , Reading Frames/genetics , Serine/pharmacology , Signal Transduction , Structure-Activity Relationship
9.
Biotechniques ; 17(5): 922-6, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7840974

ABSTRACT

A protein phosphorylation cascade involved in chemotactic signaling in Escherichia coli was investigated with purified components in vitro. CheA, an auto-phosphorylating histidine kinase, was mixed with [gamma-32P]ATP, and the labeled protein was purified for use as a reagent in the assays. CheY, a response regulator protein, can acquire phosphate groups from CheA but then undergoes rapid hydrolysis, which releases inorganic phosphate. To follow the kinetics of the CheA-CheY phospho-transfer reaction and the subsequent dephosphorylation of phospho-CheY, we separated the reaction components by polyacrylamide gel electrophoresis and measured the amount of 32P label in the CheA. CheY and inorganic phosphate bands with phosphor storage screens. By reducing the time needed to separate and quantify the reaction products, we minimized diffusive spreading of the low molecular weight inorganic phosphate, which enabled us to measure it accurately on the same gel with the much larger proteins. In principle, any radiolabeled molecules that can be separated by relatively rapid means, such as acrylamide gel electrophoresis, and that are detectable with a phosphor storage screen, should be amenable to this technique.


Subject(s)
Bacterial Proteins , Electrophoresis, Polyacrylamide Gel , Membrane Proteins/analysis , Phosphorus Radioisotopes/analysis , Adenosine Triphosphate , Escherichia coli/chemistry , Escherichia coli Proteins , Histidine Kinase , Membrane Proteins/isolation & purification , Membrane Proteins/metabolism , Methyl-Accepting Chemotaxis Proteins , Phosphates/analysis , Phosphorylation , Plasmids , Protein Kinases/metabolism
10.
Proc Natl Acad Sci U S A ; 91(12): 5485-9, 1994 Jun 07.
Article in English | MEDLINE | ID: mdl-8202513

ABSTRACT

The CheA protein of Escherichia coli is a histidine autokinase that donates its phosphate groups to two target proteins, CheY and CheB, to regulate flagellar rotation and sensory adaptation during chemotactic responses. The amino-terminal third of CheA contains the autophosphorylation site, determinants needed to interact with the catalytic center of the molecule, and determinants needed for specific recognition of its phosphorylation targets. To understand the structural basis for these activities, we examined the domain organization of the CheA phosphotransfer region by using DNA sequence analysis, limited proteolytic digestion, and a genetic technique called domain liberation. Comparison of the functionally interchangeable CheA proteins of E. coli and Salmonella typhimurium revealed two extensively mismatched segments within the phosphotransfer region, 22 and 25 aa long, with sequences characteristic of domain linkers. Both segments were readily susceptible to proteases, implying that they have an extended, flexible structure. In contrast, the intervening segments of the phosphotransfer region, designated P1 and P2 (roughly 140 and 65 aa, respectively), were relatively insensitive, suggesting they correspond to more compactly folded structural domains. Their functional properties were explored by identifying portions of the cheA coding region capable of interfering with chemotactic behavior when "liberated" and expressed as polypeptides. P1 fragments were not inhibitory, but P2 fragments blocked the interaction of CheY with the rotational switch at the flagellar motor, leading to incessant forward swimming. These results suggest that P2 contains CheY-binding determinants which are normally responsible for phosphotransfer specificity. Domain-liberation approaches should prove generally useful for analyzing multidomain proteins and their interaction targets.


Subject(s)
Bacterial Proteins , Membrane Proteins/metabolism , Protein Kinases/chemistry , Amino Acid Sequence , Chemotaxis , Escherichia coli/enzymology , Escherichia coli Proteins , Histidine Kinase , Methyl-Accepting Chemotaxis Proteins , Molecular Sequence Data , Phosphorylation , Salmonella typhimurium , Signal Transduction , Structure-Activity Relationship
11.
Nature ; 338(6211): 127-32, 1989 Mar 09.
Article in English | MEDLINE | ID: mdl-2465497

ABSTRACT

The effects of amino-acid replacements on the disulphide-coupled folding pathway of bovine pancreatic trypsin inhibitor have been examined. Replacements at three sites destabilize the native protein relative to the unfolded state, but have different effects on the relative stabilities of the disulphide-bonded folding intermediates, thus allowing the roles of the altered residues during folding to be distinguished.


Subject(s)
Aprotinin , Protein Conformation , Amino Acid Sequence , Kinetics , Models, Molecular , Mutation
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