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Enhanced expression of lmb gene encoding laminin-binding protein in Streptococcus agalactiae strains harboring IS1548 in scpB-lmb intergenic region.

Al Safadi R, Amor S, Hery-Arnaud G, Spellerberg B, Lanotte P, Mereghetti L, Gannier F, Quentin R, Rosenau A - PLoS ONE (2010)

Bottom Line: IS1548 was mostly found (72.2%) in CC19 serotype III strains recovered more specifically (92.3%) from neonatal meningitis.No MGE was found in most strains of the other CCs (76.0%), notably CC23, CC10 and CC1.Deletion of the IS1548 sequence between scpB and lmb genes in a CC19 serotype III GBS strain substantially reduced the transcription of lmb gene (13.5-fold), the binding ability to laminin (6.2-fold), and the expression of Lmb protein (5.0-fold).

View Article: PubMed Central - PubMed

Affiliation: Equipe d'Accueil 3854 Bactéries et Risque Materno-Foetal, Institut Fédératif de Recherche 136 Agents Transmissibles et Infectiologie, UFR Médecine, Université François Rabelais de Tours, Tours, France.

ABSTRACT
Group B streptococcus (GBS) is the main cause of neonatal sepsis and meningitis. Bacterial surface proteins play a major role in GBS binding to and invasion of different host surfaces. The scpB and lmb genes, coding for fibronectin-binding and laminin-binding surface proteins, are present in almost all human GBS isolates. The scpB-lmb intergenic region is a hot spot for integration of two mobile genetic elements (MGEs): the insertion element IS1548 or the group II intron GBSi1. We studied the structure of scpB-lmb intergenic region in 111 GBS isolates belonging to the intraspecies major clonal complexes (CCs). IS1548 was mostly found (72.2%) in CC19 serotype III strains recovered more specifically (92.3%) from neonatal meningitis. GBSi1 was principally found (70.6%) in CC17 strains, mostly (94.4%) of serotype III, but also (15.7%) in CC19 strains, mostly (87.5%) of serotype II. No MGE was found in most strains of the other CCs (76.0%), notably CC23, CC10 and CC1. Twenty-six strains representing these three genetic configurations were selected to investigate the transcription and expression levels of scpB and lmb genes. Quantitative RT-PCR showed that lmb transcripts were 5.0- to 9.6-fold higher in the group of strains with IS1548 than in the other two groups of strains (P<0.001). Accordingly, the binding ability to laminin was 3.8- to 6.6-fold higher in these strains (P< or =0.001). Moreover, Lmb amount expressed on the cell surface was 2.4- to 2.7-fold greater in these strains (P<0.001). By contrast, scpB transcript levels and fibronectin binding ability were similar in the three groups of strains. Deletion of the IS1548 sequence between scpB and lmb genes in a CC19 serotype III GBS strain substantially reduced the transcription of lmb gene (13.5-fold), the binding ability to laminin (6.2-fold), and the expression of Lmb protein (5.0-fold). These data highlight the importance of MGEs in bacterial virulence and demonstrate the up-regulation of lmb gene by IS1548; the increased lmb gene expression observed in CC19 serotype III strains with IS1548 may play a role in their ability to cause neonatal meningitis and endocarditis.

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Detection of Lmb protein by Western blot in L29 wild type GBS strain and isogenic IS1548 deletion mutants.10 µg of total bacterial cell proteins isolated from strains 1: NEM316 (no MGE), 2: ΔscpB-lmb NEM316 mutant, 3: L29 wild type (IS1548), 4 to 7: four independent L29ΔIS1548 mutant strains were separated by SDS PAGE and transferred to an Immobilon P polyvinylidene difluoride membrane. The blots were probed with rabbit anti-Lmb antibodies that were detected with a horseradish peroxidase labeled secondary anti-rabbit antibody that was then visualized by the ECL system (Amersham Biosciences). Molecular sizes in kDa are depicted at the left.
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pone-0010794-g005: Detection of Lmb protein by Western blot in L29 wild type GBS strain and isogenic IS1548 deletion mutants.10 µg of total bacterial cell proteins isolated from strains 1: NEM316 (no MGE), 2: ΔscpB-lmb NEM316 mutant, 3: L29 wild type (IS1548), 4 to 7: four independent L29ΔIS1548 mutant strains were separated by SDS PAGE and transferred to an Immobilon P polyvinylidene difluoride membrane. The blots were probed with rabbit anti-Lmb antibodies that were detected with a horseradish peroxidase labeled secondary anti-rabbit antibody that was then visualized by the ECL system (Amersham Biosciences). Molecular sizes in kDa are depicted at the left.

Mentions: In order to determine whether the up-regulation of the lmb gene is caused by the upstream copy of IS1548, we deleted the IS1548 sequence between scpB and lmb genes in the genome of ST-19 serotype III L29 strain. The successful deletion was confirmed by DNA sequencing of a 1.2 kb-PCR product obtained with scpB2987/rlmb497 primer set (data not shown). L29 wild type (WT) strain and the isogenic mutant (ΔIS1548) were subsequently tested for transcription of lmb gene by quantitative RT-PCR, expression of Lmb protein on cell surface by ELISA and Western blot, and binding ability to laminin. As depicted in Fig. 4, the isogenic mutant showed a 13.5-fold decreased level of lmb transcripts, a 6.2-fold decreased ability to bind to laminin, and a 5.0-fold decreased Lmb protein amount as determined by ELISA, as compared to the WT strain. Similarly, detection of Lmb protein by Western blotting (Fig. 5) showed a decreased expression in L29ΔIS1548 as compared to the WT strain.


Enhanced expression of lmb gene encoding laminin-binding protein in Streptococcus agalactiae strains harboring IS1548 in scpB-lmb intergenic region.

Al Safadi R, Amor S, Hery-Arnaud G, Spellerberg B, Lanotte P, Mereghetti L, Gannier F, Quentin R, Rosenau A - PLoS ONE (2010)

Detection of Lmb protein by Western blot in L29 wild type GBS strain and isogenic IS1548 deletion mutants.10 µg of total bacterial cell proteins isolated from strains 1: NEM316 (no MGE), 2: ΔscpB-lmb NEM316 mutant, 3: L29 wild type (IS1548), 4 to 7: four independent L29ΔIS1548 mutant strains were separated by SDS PAGE and transferred to an Immobilon P polyvinylidene difluoride membrane. The blots were probed with rabbit anti-Lmb antibodies that were detected with a horseradish peroxidase labeled secondary anti-rabbit antibody that was then visualized by the ECL system (Amersham Biosciences). Molecular sizes in kDa are depicted at the left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2875397&req=5

pone-0010794-g005: Detection of Lmb protein by Western blot in L29 wild type GBS strain and isogenic IS1548 deletion mutants.10 µg of total bacterial cell proteins isolated from strains 1: NEM316 (no MGE), 2: ΔscpB-lmb NEM316 mutant, 3: L29 wild type (IS1548), 4 to 7: four independent L29ΔIS1548 mutant strains were separated by SDS PAGE and transferred to an Immobilon P polyvinylidene difluoride membrane. The blots were probed with rabbit anti-Lmb antibodies that were detected with a horseradish peroxidase labeled secondary anti-rabbit antibody that was then visualized by the ECL system (Amersham Biosciences). Molecular sizes in kDa are depicted at the left.
Mentions: In order to determine whether the up-regulation of the lmb gene is caused by the upstream copy of IS1548, we deleted the IS1548 sequence between scpB and lmb genes in the genome of ST-19 serotype III L29 strain. The successful deletion was confirmed by DNA sequencing of a 1.2 kb-PCR product obtained with scpB2987/rlmb497 primer set (data not shown). L29 wild type (WT) strain and the isogenic mutant (ΔIS1548) were subsequently tested for transcription of lmb gene by quantitative RT-PCR, expression of Lmb protein on cell surface by ELISA and Western blot, and binding ability to laminin. As depicted in Fig. 4, the isogenic mutant showed a 13.5-fold decreased level of lmb transcripts, a 6.2-fold decreased ability to bind to laminin, and a 5.0-fold decreased Lmb protein amount as determined by ELISA, as compared to the WT strain. Similarly, detection of Lmb protein by Western blotting (Fig. 5) showed a decreased expression in L29ΔIS1548 as compared to the WT strain.

Bottom Line: IS1548 was mostly found (72.2%) in CC19 serotype III strains recovered more specifically (92.3%) from neonatal meningitis.No MGE was found in most strains of the other CCs (76.0%), notably CC23, CC10 and CC1.Deletion of the IS1548 sequence between scpB and lmb genes in a CC19 serotype III GBS strain substantially reduced the transcription of lmb gene (13.5-fold), the binding ability to laminin (6.2-fold), and the expression of Lmb protein (5.0-fold).

View Article: PubMed Central - PubMed

Affiliation: Equipe d'Accueil 3854 Bactéries et Risque Materno-Foetal, Institut Fédératif de Recherche 136 Agents Transmissibles et Infectiologie, UFR Médecine, Université François Rabelais de Tours, Tours, France.

ABSTRACT
Group B streptococcus (GBS) is the main cause of neonatal sepsis and meningitis. Bacterial surface proteins play a major role in GBS binding to and invasion of different host surfaces. The scpB and lmb genes, coding for fibronectin-binding and laminin-binding surface proteins, are present in almost all human GBS isolates. The scpB-lmb intergenic region is a hot spot for integration of two mobile genetic elements (MGEs): the insertion element IS1548 or the group II intron GBSi1. We studied the structure of scpB-lmb intergenic region in 111 GBS isolates belonging to the intraspecies major clonal complexes (CCs). IS1548 was mostly found (72.2%) in CC19 serotype III strains recovered more specifically (92.3%) from neonatal meningitis. GBSi1 was principally found (70.6%) in CC17 strains, mostly (94.4%) of serotype III, but also (15.7%) in CC19 strains, mostly (87.5%) of serotype II. No MGE was found in most strains of the other CCs (76.0%), notably CC23, CC10 and CC1. Twenty-six strains representing these three genetic configurations were selected to investigate the transcription and expression levels of scpB and lmb genes. Quantitative RT-PCR showed that lmb transcripts were 5.0- to 9.6-fold higher in the group of strains with IS1548 than in the other two groups of strains (P<0.001). Accordingly, the binding ability to laminin was 3.8- to 6.6-fold higher in these strains (P< or =0.001). Moreover, Lmb amount expressed on the cell surface was 2.4- to 2.7-fold greater in these strains (P<0.001). By contrast, scpB transcript levels and fibronectin binding ability were similar in the three groups of strains. Deletion of the IS1548 sequence between scpB and lmb genes in a CC19 serotype III GBS strain substantially reduced the transcription of lmb gene (13.5-fold), the binding ability to laminin (6.2-fold), and the expression of Lmb protein (5.0-fold). These data highlight the importance of MGEs in bacterial virulence and demonstrate the up-regulation of lmb gene by IS1548; the increased lmb gene expression observed in CC19 serotype III strains with IS1548 may play a role in their ability to cause neonatal meningitis and endocarditis.

Show MeSH
Related in: MedlinePlus