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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

Transcriptional analysis of the L. plantarum cggR operon. (A) Primer extension products were obtained by using oligonucleotide cgg4 and total RNA extracted from LM3 and LM3-CC1. As a reference, sequencing reactions were performed with the same primer. Lanes: 1, LM3; 2, LM3-CC1. Northern blot hybridization of total RNA extracted from LM3 and LM3-CC1 with cggR (B) or enoA1 (C) probes. Lanes: 1, LM3; 2, LM3-CC1.
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Figure 3: Transcriptional analysis of the L. plantarum cggR operon. (A) Primer extension products were obtained by using oligonucleotide cgg4 and total RNA extracted from LM3 and LM3-CC1. As a reference, sequencing reactions were performed with the same primer. Lanes: 1, LM3; 2, LM3-CC1. Northern blot hybridization of total RNA extracted from LM3 and LM3-CC1 with cggR (B) or enoA1 (C) probes. Lanes: 1, LM3; 2, LM3-CC1.

Mentions: In order to verify transcription of the cggR operon in the mutant strain, a primer extension analysis was performed on total transcripts extracted from LM3 and LM3-CC1 strains. No difference was detected in the two strains, as measured by PhosphorImager (Fig. 3, panel A), showing that replacement of the enoA1 gene with the ery antibiotic resistance cassette did not affect transcription of the operon. To further analyze the effect of the enoA1 gene deletion on transcription of the cggR operon, Northern blot analysis was performed on total RNA extracted from both strains. A 360-bp fragment internal to the cggR gene, the first gene of the pentacistronic operon, was used as a probe. A signal of about 5.7 kb, corresponding to transcripts from the entire cggR operon, was observed in both LM3 and LM3-CC1 strains (Fig. 3, panel B). The difference of calculated size between cggR transcripts from the wild-type and the mutant strain, less than 200 bp, is likely to be undetectable on a 1% agarose gel. When a different probe was used (320-bp fragment internal to the enoA1 gene), the 5.7 kb signal was observed in the LM3 strain, whereas no signal was detected in the LM3-CC1 strain (Fig. 3, panel C).


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)

Transcriptional analysis of the L. plantarum cggR operon. (A) Primer extension products were obtained by using oligonucleotide cgg4 and total RNA extracted from LM3 and LM3-CC1. As a reference, sequencing reactions were performed with the same primer. Lanes: 1, LM3; 2, LM3-CC1. Northern blot hybridization of total RNA extracted from LM3 and LM3-CC1 with cggR (B) or enoA1 (C) probes. Lanes: 1, LM3; 2, LM3-CC1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Transcriptional analysis of the L. plantarum cggR operon. (A) Primer extension products were obtained by using oligonucleotide cgg4 and total RNA extracted from LM3 and LM3-CC1. As a reference, sequencing reactions were performed with the same primer. Lanes: 1, LM3; 2, LM3-CC1. Northern blot hybridization of total RNA extracted from LM3 and LM3-CC1 with cggR (B) or enoA1 (C) probes. Lanes: 1, LM3; 2, LM3-CC1.
Mentions: In order to verify transcription of the cggR operon in the mutant strain, a primer extension analysis was performed on total transcripts extracted from LM3 and LM3-CC1 strains. No difference was detected in the two strains, as measured by PhosphorImager (Fig. 3, panel A), showing that replacement of the enoA1 gene with the ery antibiotic resistance cassette did not affect transcription of the operon. To further analyze the effect of the enoA1 gene deletion on transcription of the cggR operon, Northern blot analysis was performed on total RNA extracted from both strains. A 360-bp fragment internal to the cggR gene, the first gene of the pentacistronic operon, was used as a probe. A signal of about 5.7 kb, corresponding to transcripts from the entire cggR operon, was observed in both LM3 and LM3-CC1 strains (Fig. 3, panel B). The difference of calculated size between cggR transcripts from the wild-type and the mutant strain, less than 200 bp, is likely to be undetectable on a 1% agarose gel. When a different probe was used (320-bp fragment internal to the enoA1 gene), the 5.7 kb signal was observed in the LM3 strain, whereas no signal was detected in the LM3-CC1 strain (Fig. 3, panel C).

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