Enolase occurs as a cytoplasmic and a surface-associated protein in bacteria. Enolases of the bacterial pathogens Streptococcus pyogenes, Streptococcus pneumoniae and Staphylococcus aureus, as well as of the commensal lactic acid bacteria, Lactobacillus crispatus and Lactobacillus johnsonii, were purified as His(6)-fusion proteins from recombinant Escherichia coli. The fusion proteins were compared for putative virulence-associated functions, i.e., binding of human plasminogen, enhancement of plasminogen activation by human plasminogen activators, as well as binding to immobilized laminin, fibronectin and collagens. The individual enolases showed varying efficiencies in these functions. In particular, highly and equally effective interactions with plasminogen and laminin were seen with lactobacillar and staphylococcal enolases.
The plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase of Lactobacillus crispatus were localized on the cell surface at pH 5 but released into the medium at an alkaline pH. These proteins bound to lipoteichoic acids at a pH below their isoelectric point. The results indicate that lactobacilli rapidly modify their surface properties in response to changes in pH.The glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are found on the surface of several gram-positive bacteria, where they are involved in pathogen-host interactions (3-6, 17, 18, 20, 21). Commensal Lactobacillus crispatus and several other species of the Acidophilus group of Lactobacillus have enolase and GAPDH as major constituents of their extracellular proteome at neutral pHs (12). The secretion and anchoring mechanisms of enolase and GAPDH on the bacterial surface have not been characterized. Lactobacillus species are strongly fermentative and secrete lactic acid as a primary metabolite, which rapidly reduces the pH of the environment to 4. Here, we assessed the role of pH in the surface localization of enolase and GAPDH in L. crispatus.Association of enolase and GAPDH with the cell wall depends on pH. L. crispatus ST1 (9, 12) was cultivated overnight in De Man, Rogosa, and Sharpe (MRS) broth (Difco), the cells were collected by centrifugation, suspended without washing at 10 10 bacteria/ml in 50 mM Tris-HCl at either pH 5 or pH 8, and incubated at 37°C for 1 h, during which time the pHs of the suspensions decreased to 4.5 and to 7.5. The presence of enolase and GAPDH, as well as of an unrelated surface layer (S-layer) protein, on the cells was analyzed by use of indirect immunofluorescence. The cells were used to coat glass slides and fixed with 3.5% (wt/vol) paraformaldehyde prior to detection with anti-His 6 -GAPDH (12), anti-His 6 -enolase (12), or anti-S-layer protein (2) immunoglobulins as primary antibodies and tetramethylrhodamine isothiocyanate-labeled antibodies (Dako) as detailed previously (19). Enolase and GAPDH were present on the surface of the cells from the pH 5 suspension, whereas the cells from the pH 8 suspension showed only weak fluorescence (Fig. 1A). In contrast, no change in cellbound S-layer protein was detected (Fig. 1A). Next, the cells from an overnight culture were incubated for 1 h at pH 5 or pH 8, the cell and the supernatant fractions were separated, and the supernatant was filtered through a 0.2-m-pore-size membrane (12). Surface-attached proteins were extracted by boiling the cell pellet in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (8) for 1 min. Enolase and GAPDH were detected by Western blotting in the supernatant from the pH 8 suspension, but not from the pH 5 suspension, and more of these proteins were found on the surfaces of cells from the pH 5 suspension than from the pH 8 suspension (Fig. 1B). Small amounts of surface-associated enolase and GAPDH were detectable by Western blotting of samples from t...
Lactobacillus crispatus is a common member of the beneficial microbiota present in the vertebrate gastrointestinal and human genitourinary tracts. Here, we report the genome sequence of L. crispatus ST1, a chicken isolate displaying strong adherence to vaginal epithelial cells.Lactobacillus crispatus can persist in the vertebrate gastrointestinal tract and is among the most prevalent species of the Lactobacillus-dominated human vaginal microbiota (2,9,13,14). It belongs to the so-called acidophilus group (3), which has attracted interest because some of its species are important factors in the production of fermented foods (12) and some can, at least transiently, colonize the human host (2,9,13,14). Moreover, some specific strains, mainly L. acidophilus NCFM and L. johnsonii NCC 533, have received prominence as intestinal-health-promoting microbes (4). Although the genomes of seven members of the acidophilus complex have been sequenced to date (12), the genome sequences of L. crispatus and other predominant lactobacillar species in the urogenital flora have mostly remained obscure. Vaginal lactobacilli can have an important role in controlling the health of the host (2, 14). They can, for example, positively influence and stabilize the host's vaginal microbiota via the production of compounds that are acidic or exert a direct inhibiting action toward pathogenic bacteria (2,14). In addition to the antimicrobial compounds, the competitive exclusion of pathogens is another mechanism by which the host's microbiota can be balanced (2). L. crispatus ST1 was originally isolated from the crop of a chicken, and PCR profiling of L. crispatus isolates has verified it to be an abundant colonizer of the chicken crop (6,8). It also displays a strong protein-dependent adhesion to the epithelial cells of the human vagina and has been shown to inhibit the adhesion of avian pathogenic Escherichia coli (6, 7).The genome was sequenced (18ϫ coverage) using a 454 pyrosequencer with GS FLX chemistry (Roche). The contig order was confirmed and gaps were filled by sequencing PCR fragments from the genomic DNA template using ABI 3730 and Big Dye chemistry (Applied Biosystems). Genomic data were processed using the Staden Package (11) and gsAssembler (Roche). Coding sequences (CDSs) were predicted using Glimmer3 (5) followed by manual curation of the start sites. The remaining intergenic regions were reanalyzed for missed CDSs by using BlastX (1). Annotation transfer was performed based on a BlastP search, followed by Blannotator analysis using default settings (http://ekhidna.biocenter.helsinki.fi/poxo /blannotator) and manual verification. Orthologous groups between the different lactobacillar proteomes were identified using OrthoMCL (10).The genome of L. crispatus ST1 consists of a single circular chromosome 2.04 Mbp in size, with an overall GϩC content of 37%, without any plasmids. There are 64 tRNA genes, 4 rRNA operons, and 2 CRISPR loci. Out of the 2,024 predicted CDSs, a putative function was assigned to 77%, whereas 10% of the CD...
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