Structural Characterization of the Lactobacillus Plantarum FlmC Protein Involved in Biofilm Formation.

Lactobacillus plantarum is one of the most predominant species in the human gut microbiota of healthy individuals. We have previously characterized some probiotic features of L. plantarum LM3, as the high resistance to different stress, the binding ability toward some extracellular matrix proteins and plasminogen and the immunomodulatory role of the surface expressed adhesin EnoA1. We have also identified the flmA, flmB and flmC genes, coding for putative proteins named FlmA, FlmB and FlmC, whose null mutations partially impaired biofilm development; the L. plantarum LM3⁻6 strain, carrying a deletion in flmC, showed a high rate of autolysis, supporting the hypothesis that FlmC might be involved in cell wall integrity. Here, we report the in-silico characterization of ΔTM-FlmC, a portion of the FlmC protein. The protein has been also expressed, purified and characterized by means of CD spectroscopy, ICP-mass and UHPLC-HRMS. The obtained experimental data validated the predicted model unveiling also the presence of a bound lipid molecule and of a Mg(II) ion. Overall, we provide strong evidences that ΔTM-FlmC belongs to the LytR-CpsA-Psr (LCP) family of domains and is involved in cell envelope biogenesis.

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.

Transcriptional analysis of exopolysaccharides biosynthesis gene clusters in Lactobacillus plantarum.

Exopolysaccharides (EPS) from lactic acid bacteria contribute to specific rheology and texture of fermented milk products and find applications also in non-dairy foods and in therapeutics. Recently, four clusters of genes (cps) associated with surface polysaccharide production have been identified in Lactobacillus plantarum WCFS1, a probiotic and food-associated lactobacillus. These clusters are involved in cell surface architecture and probably in release and/or exposure of immunomodulating bacterial molecules. Here we show a transcriptional analysis of these clusters. Indeed, RT-PCR experiments revealed that the cps loci are organized in five operons. Moreover, by reverse transcription-qPCR analysis performed on L. plantarum WCFS1 (wild type) and WCFS1-2 (ΔccpA), we demonstrated that expression of three cps clusters is under the control of the global regulator CcpA. These results, together with the identification of putative CcpA target sequences (catabolite responsive element CRE) in the regulatory region of four out of five transcriptional units, strongly suggest for the first time a role of the master regulator CcpA in EPS gene transcription among lactobacilli.

The Lactobacillus plantarum Eno A1 Enolase Is Involved in Immunostimulation of Caco-2 Cells and in Biofilm Development.

The role of probiotics in prevention and treatment of a variety of diseases is now well assessed. The presence of adhesive molecules on the cell surface of probiotics has been related to the ability to confer health benefit to the host. 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 cell surface-expressed and is involved in binding with human fibronectin and plasminogen. By means of comparative analysis between L. plantarum LM3 (wild type) and its isogenic LM3-CC1 (ΔenoA1) mutant strain, here we show that EnoA1 affects the ability of this bacterium to modulate immune response as determined by analysis of expression of immune system molecules in Caco-2 cells. Indeed, we observed induction of TLR2 expression in cells exposed to L. plantarum LM3, while no induction was detectable in cells exposed to LM3-CC1. This difference was much less consistent when expression of TLR4 was determined in cells exposed to the two strains. Pro-inflammatory (IL-6) and anti-inflammatory cytokines (IL-10, TGF-ß), and the antimicrobial peptide HBD-2 were induced in Caco-2 cells exposed to L. plantarum LM3, while lower levels of induction were detected in cells exposed to LM3-CC1. We also analyzed the ability to develop biofilm of the two strains, and observed a decrease of about 65 % in the development of mature biofilm in LM3-CC1 compared to the wild type.

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.

The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin.

Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile.

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.

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.

Effect of inactivation of ccpA and aerobic growth in Lactobacillus plantarum: A proteomic perspective.

Lactobacillus plantarum is a facultative heterofermentative lactic acid bacterium widely used in the production of most fermented food due to its ability to thrive in several environmental niches, including the human gut. In order to cope with different growth conditions, it has developed complex molecular response mechanisms, characterized by the induction of a large set of proteins mainly regulated by HrcA and CtsR repressors as well as by global regulators such as carbon catabolite control protein A (CcpA). In this study, the role of CcpA in the regulation of growth under anaerobiosis and aerobiosis, and the adaptation to aeration in L. plantarum WCFS1 were comprehensively investigated by differential proteomics. The inactivation of ccpA, in both growth conditions, significantly changed the expression level of 76 proteins, mainly associated with carbohydrate and energy metabolism, membrane transport, nucleotide metabolism, protein biosynthesis and folding. The role of CcpA as pleiotropic regulator was particularly evident at the shift from homolactic fermentation to mixed fermentation. Proteomic results also indicated that the mutant strain was more responsive to aerobic growth condition.

Expression of the Lactobacillus plantarum malE gene is regulated by CcpA and a MalR-like protein.

Lactobacillus plantarum is commonly used in the food industry as a starter in various fermentations, especially in vegetable fermentations, in which starch is a common substrate. This polysaccharide, which is obtained from potatoes or corn and is hydrolysed mainly to maltose and glucose by acids or enzymes, is commercially used for the production of lactate by lactic acid fermentation. In this study, we describe the regulation of malE gene expression in L. plantarum. This gene, located in a 7-gene cluster, probably organized in an operon, encodes a putative maltose/maltodextrin-binding protein. We studied the expression of malE in L. plantarum LM3 (wild type) and in LM3-2 (ccpA1), which carries a null mutation in the ccpA gene, encoding the catabolite control protein A (CcpA). In the presence of glucose, expression of the MalE protein was higher in the mutant strain as compared to that in the wild-type strain. Transcription of the malE gene was induced by maltose and regulated by a CcpA-mediated carbon catabolite repression. Further, we isolated strains carrying mutations in 2 genes, lp_0172 and lp_0173, whose deduced amino acid sequences share significant identity with MalR, a regulator of the maltose operon in several gram-positive bacteria. A double mutant exhibited glucose-insensitive malE transcription, while absence of the functional Lp_0172 open reading frame had no effect on malE expression.

Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein.

BACKGROUND: Lactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals. RESULTS: We show the identification of a Lactobacillus plantarum LM3 cell surface protein (48 kDa), which specifically binds to human fibronectin (Fn), an extracellular matrix protein. By means of mass spectrometric analysis this protein was identified as the product of the L. plantarum enoA1 gene, coding the EnoA1 alfa-enolase. Surface localization of EnoA1 was proved by immune electron microscopy. In the mutant strain LM3-CC1, carrying the enoA1 null mutation, the 48 kDa adhesin was not anymore detectable neither by anti-enolase Western blot nor by Fn-overlay immunoblotting assay. Moreover, by an adhesion assay we show that LM3-CC1 cells bind to fibronectin-coated surfaces less efficiently than wild type cells, thus demonstrating the significance of the surface displaced EnoA1 protein for the L. plantarum LM3 adhesion to fibronectin. CONCLUSION: Adhesion to host tissues represents a crucial early step in the colonization process of either pathogens or commensal bacteria. We demonstrated the involvement of the L. plantarum Eno A1 alfa-enolase in Fn-binding, by studying LM3 and LM3-CC1 surface proteins. Isolation of LM3-CC1 strain was possible for the presence of expressed enoA2 gene in the L. plantarum genome, giving the possibility, for the first time to our knowledge, to quantitatively compare adhesion of wild type and mutant strain, and to assess doubtless the role of L. plantarum Eno A1 as a fibronectin binding protein.