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1.
Probiotics Antimicrob Proteins ; 7(3): 193-202, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25917402

ABSTRACT

Enolases are generally thought of as cytoplasmic enzymes involved in glycolysis and gluconeogenesis. However, several bacteria have active forms of enolase associated with the cell surface and these proteins are utilized for functions other than central metabolism. Recently, a surface-associated protein produced by Lactobacillus gasseri ATCC 33323 with homology to enolase was found to inhibit the adherence of the sexually transmitted pathogen, Neisseria gonorrhoeae, to epithelial cells in culture. Here, we show that the protein is an active enolase in vitro. A recombinantly expressed, C-terminal His-tagged version of the protein, His6-Eno3, inhibited gonococcal adherence. Assays utilizing inhibitors of enolase enzymatic activity showed that this inhibitory activity required the substrate-binding site to be in an open conformation; however, the enolase enzymatic activity of the protein was not necessary for inhibition of gonococcal adherence. An L. gasseri strain carrying an insertional mutation in eno3 was viable, indicating that eno3 is not an essential gene in L. gasseri 33323. This observation, along with the results of the enzyme assays, is consistent with reports that this strain encodes more than one enolase. Here we show that the three L. gasseri genes annotated as encoding an enolase are expressed. The L. gasseri eno3 mutant exhibited reduced, but not abolished, inhibition of gonococcal adherence, which supports the hypothesis that L. gasseri inhibition of gonococcal adherence is a multifactorial process.


Subject(s)
Bacterial Proteins/metabolism , Epithelial Cells/microbiology , Lactobacillus/enzymology , Neisseria gonorrhoeae/growth & development , Phosphopyruvate Hydratase/metabolism , Bacterial Adhesion , Bacterial Proteins/genetics , Cell Line, Tumor , Cells, Cultured , Gonorrhea/therapy , Humans , Lactobacillus/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutagenesis, Insertional , Phosphopyruvate Hydratase/genetics , RNA, Bacterial/genetics , Sequence Analysis, RNA
2.
FEBS Lett ; 588(14): 2212-6, 2014 Jun 27.
Article in English | MEDLINE | ID: mdl-24859038

ABSTRACT

Enolases are highly conserved metalloenzymes ubiquitous to cellular metabolism. While these enzymes share a large degree of sequence and structural similarity, they have been shown to possess a wide range of moonlighting functions. Recent studies showed that an enolase from Lactobacillus gasseri impedes the ability of Neisseria gonorrhoeae to adhere to epithelial cells. We present the crystal structure of this enolase, the first from Lactobacillus, with one of its Mg(2+) cofactors. Determined using molecular replacement to 2.08Å, the structure has a flexible and surface exposed catalytic loop containing lysines, and may play a role in the inhibitory function.


Subject(s)
Bacterial Proteins/chemistry , Lactobacillus/enzymology , Phosphopyruvate Hydratase/chemistry , Catalytic Domain , Crystallography, X-Ray , Magnesium/chemistry , Models, Molecular , Neisseria gonorrhoeae , Protein Structure, Quaternary , Protein Structure, Secondary , Structural Homology, Protein
3.
Biochim Biophys Acta ; 1844(2): 406-15, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24316251

ABSTRACT

The type II secretion complex exports folded proteins from the periplasm to the extracellular milieu. It is used by the pathogenic bacterium Vibrio cholerae to export several proteins, including its major virulence factor, cholera toxin. The pseudopilus is an essential component of the type II secretion system and likely acts as a piston to push the folded proteins across the outer membrane through the secretin pore. The pseudopilus is composed of the major pseudopilin, EpsG, and four minor pseudopilins, EpsH, EpsI, EpsJ and EpsK. We determined the x-ray crystal structure of the head domain of EpsH at 1.59Å resolution using molecular replacement with the previously reported EpsH structure, 2qv8, as the template. Three additional N-terminal amino acids present in our construct prevent an artifactual conformation of residues 160-166, present in one of the two monomers of the 2qv8 structure. Additional crystal contacts stabilize a long flexible loop comprised of residues 104-135 that is more disordered in the 2qv8 structure but is partially observed in our structure in very different positions for the two EpsH monomers in the asymmetric unit. In one of the conformations the loop is highly extended. Modeling suggests the highly charged loop is capable of contacting EpsG and possibly secreted protein substrates, suggesting a role in specificity of pseudopilus assembly or secretion function.


Subject(s)
Fimbriae Proteins/chemistry , Vibrio cholerae/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Fimbriae Proteins/genetics , Models, Molecular , Molecular Docking Simulation , Molecular Sequence Data , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid
4.
Article in English | MEDLINE | ID: mdl-20693674

ABSTRACT

Recombinant Lactobacillus jensenii enolase fused to a C-terminal noncleavable His tag was expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 3.25 A resolution. The crystals belonged to space group I4, with unit-cell parameters a=b=145.31, c=99.79 A. There were two protein subunits in the asymmetric unit, which gave a Matthews coefficient VM of 2.8 A3 Da(-1), corresponding to 55.2% solvent content.


Subject(s)
Lactobacillus/enzymology , Phosphopyruvate Hydratase/chemistry , Crystallization , Crystallography, X-Ray , Gene Expression , Phosphopyruvate Hydratase/genetics , Phosphopyruvate Hydratase/isolation & purification
5.
Article in English | MEDLINE | ID: mdl-20445273

ABSTRACT

Protein crystallization screens frequently yield salt crystals as well as protein crystals. A simple method for determining whether a crystal is composed of salt or macromolecules is suggested. A drop containing one or more crystals is transferred to a glass cover slip and the cover slip is then passed through the flame of a Bunsen burner. Macromolecule crystals are destroyed by this treatment, while salt crystals generally remain. The test can be performed after other commonly used tests such as crushing and staining.


Subject(s)
Proteins/analysis , Crystallization , Salts/analysis
6.
Article in English | MEDLINE | ID: mdl-19574644

ABSTRACT

EpsH is a minor pseudopilin protein of the Vibrio cholerae type II secretion system. A truncated form of EpsH with a C-terminal noncleavable His tag was constructed and expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 1.71 A resolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.39, b = 71.11, c = 84.64 A. There were two protein molecules in the asymmetric unit, which gave a Matthews coefficient V(M) of 2.1 A(3) Da(-1), corresponding to 41.5% solvent content.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Vibrio cholerae/chemistry , Crystallization , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel
7.
Article in English | MEDLINE | ID: mdl-19478449

ABSTRACT

EpsG is the major pseudopilin protein of the Vibrio cholerae type II secretion system. An expression plasmid that encodes an N-terminally truncated form of EpsG with a C-terminal noncleavable His tag was constructed. Recombinant EpsG was expressed in Escherichia coli; the truncated protein was purified and crystallized by hanging-drop vapor diffusion against a reservoir containing 6 mM zinc sulfate, 60 mM MES pH 6.5, 15% PEG MME 550. The crystals diffracted X-rays to a resolution of 2.26 A and belonged to space group P2(1), with unit-cell parameters a = 88.61, b = 70.02, c = 131.54 A.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Vibrio cholerae/genetics , Vibrio cholerae/isolation & purification , X-Ray Diffraction , Amino Acid Substitution , Crystallization , Data Collection , Escherichia coli/genetics , Gene Expression , Genes, Bacterial , Histidine/chemistry , Hydrogen-Ion Concentration , Methionine/metabolism , Molecular Weight , Plasmids , Recombinant Proteins/isolation & purification , Statistics as Topic , Temperature
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