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

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.

Figure 1. Genomic organization of the scpB-lmb intergenic region.

Dark grey box represents the noncoding spacer region. Primer set scpB3382/rlmb51 was used to determine the structure of the scpB-lmb intergenic region. The presence of the insertion sequence IS1548 and of the group II intron GBSi1 in the spacer was then confirmed using primer sets scpB3382/r1S1548, and scpB3382/rGBSi143.

Figure 2. Properties of 26 GBS isolates in relation to the scpB-lmb intergenic region structure (8 strains without MGE, 9 strains with GBSi1 and 9 strains with IS1548).

(A) Relative transcription levels of scpB (filled boxes) and lmb (open boxes) genes. The amount of transcripts of each gene was normalized to the amount of gyrA transcripts and expressed relative to the level of transcription in L19 GBS isolate that has no MGE. Boxes are means and bars are standard deviation of the means of the relative level of transcription in each group of strains. ** The mean transcription level of lmb gene was significantly higher in the group of strains with IS1548 than in the other two groups of strains (P<0.001). (B) Binding ability of GBS isolates to immobilized human fibronectin (filled boxes) and to immobilized human laminin (open boxes). Boxes are means and bars are standard deviation of the means of the fibronectin and laminin binding percentages in each group of strains. ** The mean binding ability to laminin was significantly higher in the group of strains with IS1548 than in the other two groups of strains (P≤0.001).

Figure 3. Expression of Lmb protein on GBS cells in relation to the scpB-lmb intergenic region structure.

Lmb protein was quantified in an ELISA-type assay. GBS cells (8 strains without MGE, 9 strains with GBSi1, 9 strains with IS1548), were immobilized in microtiter wells and incubated with rabbit anti-Lmb antibodies. Binding of antibodies to the Lmb protein was quantified by addition of peroxidase-conjugated goat anti-rabbit IgG, and subsequent addition of OPD peroxidase substrate. Boxes are means and bars are standard deviation of the means of the absorbance at 492 nm (A₄₉₂) per adhering bacteria in each group of strains. ** The amount of Lmb protein was significantly higher in the group of strains with IS1548 as compared with the other two groups of strains (P<0.001).

Figure 4. Properties of L29 wild type GBS strain and isogenic IS1548 deletion mutant.

Relative transcription levels of lmb gene, binding ability of GBS cells to immobilized human laminin, and expression of Lmb protein on GBS cells as determined by ELISA, in an isogenic mutant deleted for IS1548 sequence between scpB and lmb genes (L29ΔIS1548) (striped boxes) as compared to the parent strain (L29 WT) (open boxes). Each experiment was performed at least three times. Boxes are means and bars are standard deviation of the means.

Figure 5. 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.