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

Castaldo C, Vastano V, Siciliano RA, Candela M, Vici M, Muscariello L, Marasco R, Sacco M - Microb. Cell Fact. (2009)

Bottom Line: 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.In the mutant strain LM3-CC1, carrying the enoA1 mutation, the 48 kDa adhesin was not anymore detectable neither by anti-enolase Western blot nor by Fn-overlay immunoblotting assay.We demonstrated the involvement of the L. plantarum Eno A1 alfa-enolase in Fn-binding, by studying LM3 and LM3-CC1 surface proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Scienze Ambientali, Seconda Università di Napoli, via Vivaldi 43, 81100 Caserta, Italy. margherita.sacco@unina2.it.

ABSTRACT

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 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.

No MeSH data available.


Related in: MedlinePlus

Immunogold staining of enolase on L. plantarum LM3 thin sections. The enolase was detected on the bacterial surface by anti-Eno antibodies and 10 nm-colloidal gold-labeled secondary antibody. LM3 with (A) and without (B) primary antibody. The 10-nm gold particles are indicated by the arrows.
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Figure 2: Immunogold staining of enolase on L. plantarum LM3 thin sections. The enolase was detected on the bacterial surface by anti-Eno antibodies and 10 nm-colloidal gold-labeled secondary antibody. LM3 with (A) and without (B) primary antibody. The 10-nm gold particles are indicated by the arrows.

Mentions: To prove the surface localization of the EnoA1 protein, immune electron microscopy experiments were performed on whole L. plantarum LM3 cells, using an anti-enolase polyclonal antibody generated against the Streptococcus pneumoniae alfa-enolase, sharing 71% identity with the EnoA1 protein. Bacterial cells were incubated with anti-enolase antibody, then treated with secondary antibody conjugated with 10 nm-colloidal gold particles and embedded in Araldite M resin. The pellets were sliced using an ultramicrotome and observed by a scanning electron microscope. Electron microscopy observation revealed the presence of gold particles on the surface of L. plantarum LM3 cells, thus indicating the presence of a alfa-enolase on the cell wall of whole cells (Fig. 2).


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

Castaldo C, Vastano V, Siciliano RA, Candela M, Vici M, Muscariello L, Marasco R, Sacco M - Microb. Cell Fact. (2009)

Immunogold staining of enolase on L. plantarum LM3 thin sections. The enolase was detected on the bacterial surface by anti-Eno antibodies and 10 nm-colloidal gold-labeled secondary antibody. LM3 with (A) and without (B) primary antibody. The 10-nm gold particles are indicated by the arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654425&req=5

Figure 2: Immunogold staining of enolase on L. plantarum LM3 thin sections. The enolase was detected on the bacterial surface by anti-Eno antibodies and 10 nm-colloidal gold-labeled secondary antibody. LM3 with (A) and without (B) primary antibody. The 10-nm gold particles are indicated by the arrows.
Mentions: To prove the surface localization of the EnoA1 protein, immune electron microscopy experiments were performed on whole L. plantarum LM3 cells, using an anti-enolase polyclonal antibody generated against the Streptococcus pneumoniae alfa-enolase, sharing 71% identity with the EnoA1 protein. Bacterial cells were incubated with anti-enolase antibody, then treated with secondary antibody conjugated with 10 nm-colloidal gold particles and embedded in Araldite M resin. The pellets were sliced using an ultramicrotome and observed by a scanning electron microscope. Electron microscopy observation revealed the presence of gold particles on the surface of L. plantarum LM3 cells, thus indicating the presence of a alfa-enolase on the cell wall of whole cells (Fig. 2).

Bottom Line: 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.In the mutant strain LM3-CC1, carrying the enoA1 mutation, the 48 kDa adhesin was not anymore detectable neither by anti-enolase Western blot nor by Fn-overlay immunoblotting assay.We demonstrated the involvement of the L. plantarum Eno A1 alfa-enolase in Fn-binding, by studying LM3 and LM3-CC1 surface proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Scienze Ambientali, Seconda Università di Napoli, via Vivaldi 43, 81100 Caserta, Italy. margherita.sacco@unina2.it.

ABSTRACT

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 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.

No MeSH data available.


Related in: MedlinePlus