Pyruvate dehydrogenase subunit ß of Lactobacillus plantarum is a collagen adhesin involved in biofilm formation.

Multi-functional surface proteins have been observed in a variety of pathogenic bacteria, where they mediate host cell adhesion and invasion, as well as in commensal bacterial species, were they mediate positive interaction with the host. Among these proteins, some glycolytic enzymes, expressed on the bacterial cell surface, can bind human extracellular matrix components (ECM). A major target for them is collagen, an abundant glycoprotein of connective tissues. We have previously shown that the enolase EnoA1 of Lactobacillus plantarum, one of the most predominant species in the gut microbiota of healthy individuals, is involved in binding with collagen type I (CnI). In this study, we found that PDHB, a component of the pyruvate dehydrogenase complex, contributes to the L. plantarum LM3 adhesion to CnI. By a cellular adhesion assay to immobilized CnI, we show that LM3-B1 cells, carrying a null mutation in the pdhB gene, bind to CnI - coated surfaces less efficiently than wild-type cells. Moreover, we show that the PDHB-CnI interaction requires a native state for PDHB. We also analyzed the ability to develop biofilm in wild-type and mutant strains and we found that the lack of the PDHB on cell surface generates cells partially impaired in biofilm development.

Identification and characterization of enolase as a collagen-binding protein in Lactobacillus plantarum.

Collagen is a target of pathogens for adhesion, colonization, and invasion of host tissue. Probiotic bacteria can mimic the same mechanism as used by the pathogens in the colonization process, expressing cell surface proteins that specifically interact with extracellular matrix component proteins. The capability to bind collagen is expressed by several Lactobacillus isolates, including some Lactobacillus plantarum strains. In this study we report the involvement of the L. plantarum EnoA1 alfa-enolase in type I collagen (CnI) binding. By adhesion assays, we show that the mutant strain LM3-CC1, carrying a null mutation in the enoA1 gene, binds to immobilized collagen less efficiently than wild type strain. CnI overlay assay and Elisa tests, performed on the purified EnoA1, show that this protein can bind collagen both under denaturing and native conditions. By using truncated recombinant enolase proteins, we also show that the region spanning from 73rd to the 140th amino acid residues is involved in CnI binding.

CcpA and three newly identified proteins are involved in biofilm development in Lactobacillus plantarum.

The aim of this study was to identify genes involved in biofilm development in the probiotic lactic acid bacterium Lactobacillus plantarum. The ability of L. plantarum LM3 and of some derivative mutant strains to form biofilm has been investigated. Biofilm microtitre plate assays showed that L. plantarum LM3-2, carrying a null mutation in the ccpA gene, coding the CcpA master regulator, was partially impaired in biofilm production compared to wild type (LM3). Moreover, we found three genes in the L. plantarum genome, hereby named flmA, flmB, and flmC, whose deduced amino acid sequences show significant identity with the Streptococcus mutans BrpA (biofilm regulatory protein A). We investigated the role of FlmA, FlmB, and FlmC in biofilm formation by isolating strains carrying null mutations in the corresponding genes. Our results suggest involvement of the Flm proteins in biofilm development. Moreover, transcriptional studies show that expression of flmA, flmB, and flmC is under the control of CcpA. These results, together with the reduced ability of LM3-2 (ccpA1) to form biofilm, strongly suggest a positive role of the master regulator CcpA in biofilm development.

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

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.