Limits...
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

Binding of LM3 and LM3-CC1 to fibronectin immobilized on microtiter plate wells. Adhesion of both strains was evaluated by Real-time PCR. Adhesion is given as number of bacteria bound to fibronectin at different values of pH. Error bars represent ± standard deviation of the mean values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2654425&req=5

Figure 6: Binding of LM3 and LM3-CC1 to fibronectin immobilized on microtiter plate wells. Adhesion of both strains was evaluated by Real-time PCR. Adhesion is given as number of bacteria bound to fibronectin at different values of pH. Error bars represent ± standard deviation of the mean values.

Mentions: In order to prove the significance of the 48 kDa surface protein for L. plantarum LM3 adhesion to fibronectin, the extent of binding of LM3 and LM3-CC1 cells was analyzed on Fn-coated microtiter plates. To verify if pH could affect the binding, experiments were repeated with bacteria cells pre-treated in Tris buffers at pH ranging from 5.0 to 7.0. Bacterial adhesion was evaluated by quantification of Fn-bound cells by Real-time PCR, amplifying 16S ribosomal DNA with species-specific primers [27]. A significant difference in adherence (P < 0.05) was observed, with about ten times more wild type than mutant cells adhering to fibronectin (Fig. 6). Nevertheless, no significant difference in adherence was found upon pre-treatment of both strains in different pH buffers.


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)

Binding of LM3 and LM3-CC1 to fibronectin immobilized on microtiter plate wells. Adhesion of both strains was evaluated by Real-time PCR. Adhesion is given as number of bacteria bound to fibronectin at different values of pH. Error bars represent ± standard deviation of the mean values.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Binding of LM3 and LM3-CC1 to fibronectin immobilized on microtiter plate wells. Adhesion of both strains was evaluated by Real-time PCR. Adhesion is given as number of bacteria bound to fibronectin at different values of pH. Error bars represent ± standard deviation of the mean values.
Mentions: In order to prove the significance of the 48 kDa surface protein for L. plantarum LM3 adhesion to fibronectin, the extent of binding of LM3 and LM3-CC1 cells was analyzed on Fn-coated microtiter plates. To verify if pH could affect the binding, experiments were repeated with bacteria cells pre-treated in Tris buffers at pH ranging from 5.0 to 7.0. Bacterial adhesion was evaluated by quantification of Fn-bound cells by Real-time PCR, amplifying 16S ribosomal DNA with species-specific primers [27]. A significant difference in adherence (P < 0.05) was observed, with about ten times more wild type than mutant cells adhering to fibronectin (Fig. 6). Nevertheless, no significant difference in adherence was found upon pre-treatment of both strains in different pH buffers.

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