Identification of binding sites of Lactobacillus plantarum enolase involved in the interaction with human plasminogen

Abstract

The enolase EnoA1 of Lactobacillus plantarum is here shown to interact with human plasminogen (Plg). By sequence alignment of EnoA1 with Streptococcus pneumoniae and Bifidobacterium lactis enolases, we identified BS1 and BS2 Plg-binding sites. A structure prediction of EnoA1 showed lysine residues in position 255 (BS2), and 422 (BS1) exposed on protein surface. A lysine residue in position 259 was as well identified as surface-exposed amino acid. The enoA1 gene was site directed-mutagenized to generate four mutated proteins, carrying K255A, K259A, K422A and K259A/K422A substitutions. The functional role of these lysine residues was assessed evaluating specific Plg-binding activity of the mutated proteins. While the binding activity of the mutated proteins was drastically reduced, the residual enzymatic activity was more than 50% of EnoA1. Our results show that L. plantarum EnoA1 exhibits the Plg-BS1, and the Plg-BS2 extending up to the lysine residue in position 259, therefore consisting of 12-aa residues instead of 9-aa residues described in S. pneumoniae. A test performed on whole cells of L. plantarum, demonstrated that after inducing conversion of the cell-bound plasminogen to plasmin, this was released into the medium, unlike the mechanism reported for most pathogens, that retained plasmin bound to the cell surface.

Dancing to Another Tune—Adhesive Moonlighting Proteins in Bacteria

“…Several pathogenic species seem to retain moonlighting proteins on cell surface at neutral pH [38,39,41,42,45,50,53,60], whereas this is not the case for several lactobacilli [16,28,32,47,92]. The latter is illustrated for L. crispatus in Figure 3.…”

“…, enolase, GAPDH, GS, and GPI of L. crispatus ST1 are detached from cell surface at neutral or basic pH but remain surface-bound at pH 4 (Figure 2A) [17,28,92]. This seems to also hold for several other species of Lactobacillus [17,47]. These proteins have isoelectric points around 5 and thus a positive net charge at pH values below 5, a condition where purified enolase and GAPDH of L. crispatus ST1 bind in vitro to negatively charged lipoteichoic acids as well as to the L. crispatus ST1 cell surface [28,92].…”

“…The well characterized enolase of the severe pathogen Streptococcus pneumoniae has multiple adhesive properties which include binding of the human circulating protease precursor, plasminogen [39]. Plasminogen harbors lysine-binding kringle domains, and the interaction with pneumococcal enolase apparently takes place via two pairs of lysines in enolase, whose mutations influence plasminogen binding [47,74,75]. The two lysines in the sequence 248 FYNKDDHKY are exposed on the surface of the enolase octamer [76], and the mutations affect the enzymatic activity only partially or not at all, which suggests that the enzymatic and the adhesive functions do not involve overlapping regions.…”

Effects of the Peptide Pheromone Plantaricin A and Cocultivation with Lactobacillus sanfranciscensis DPPMA174 on the Exoproteome and the Adhesion Capacity of Lactobacillus plantarum DC400

“…Cell surface-associated PKI of L. johnsonii interacts with mucin and intestinal cells (56). Alpha-enolase of L. plantarum shows activity for binding to plasminogen and fibronectin (10,66). Fibronectin is responsible for the bacterium-endothelial cell interaction (10).…”

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

“…Structurally, it would seem much less is known about enolases' interactions with fibronectin. Though in silico methods and homology models have been effectively employed of late [13,24], it is hoped that structural information will aid in the investigation of this moonlighting function and potential relevance of the catalytic loops' conformation. Here we report the first enolase structure from a Lactobacillus.…”