A Moonlighting Enolase from Lactobacillus gasseri does not Require Enzymatic Activity to Inhibit Neisseria gonorrhoeae Adherence to Epithelial Cells.

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.

Crystal structure of an efficacious gonococcal adherence inhibitor: an enolase from Lactobacillus gasseri.

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.

The 1.59Å resolution structure of the minor pseudopilin EpsH of Vibrio cholerae reveals a long flexible loop.

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.

Expression, purification, crystallization and preliminary X-ray studies of Lactobacillus jensenii enolase.

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.

Trial by fire: are the crystals macromolecules?

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.

Expression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae pseudopilin EpsH.

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.

Overexpression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae EpsG.

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.