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RrgA is a pilus-associated adhesin in Streptococcus pneumoniae.

Nelson AL, Ries J, Bagnoli F, Dahlberg S, Fälker S, Rounioja S, Tschöp J, Morfeldt E, Ferlenghi I, Hilleringmann M, Holden DW, Rappuoli R, Normark S, Barocchi MA, Henriques-Normark B - Mol. Microbiol. (2007)

Bottom Line: Non-adherent RrgA-negative, RrgB- and RrgC-positive organisms produce pili, suggesting that pilus-mediated adherence is due to expression of RrgA, rather than the pilus backbone itself.In contrast, RrgA-positive strains with disrupted rrgB and rrgC genes exhibit wild-type adherence despite failure to produce pili by Western blot or immunoelectron microscopy.These data suggest that RrgA is central in pilus-mediated adherence and disease, even in the absence of polymeric pilus production.

View Article: PubMed Central - PubMed

Affiliation: Swedish Institute for Infectious Disease Control and Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
Adherence to host cells is important in microbial colonization of a mucosal surface, and Streptococcus pneumoniae adherence was significantly enhanced by expression of an extracellular pilus composed of three subunits, RrgA, RrgB and RrgC. We sought to determine which subunit(s) confers adherence. Bacteria deficient in RrgA are significantly less adherent than wild-type organisms, while overexpression of RrgA enhances adherence. Recombinant monomeric RrgA binds to respiratory cells, as does RrgC with less affinity, and pre-incubation of epithelial cells with RrgA reduces adherence of wild-type piliated pneumococci. Non-adherent RrgA-negative, RrgB- and RrgC-positive organisms produce pili, suggesting that pilus-mediated adherence is due to expression of RrgA, rather than the pilus backbone itself. In contrast, RrgA-positive strains with disrupted rrgB and rrgC genes exhibit wild-type adherence despite failure to produce pili by Western blot or immunoelectron microscopy. The density of bacteria colonizing the upper respiratory tract of mice inoculated with piliated RrgA-negative pneumococci was significantly less compared with wild-type; in contrast, non-piliated pneumococci expressing non-polymeric RrgA had similar numbers of bacteria in the nasopharynx as piliated wild-type bacteria. These data suggest that RrgA is central in pilus-mediated adherence and disease, even in the absence of polymeric pilus production.

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Recombinant RrgA and RrgC proteins bind to human respiratory epithelial cells. A–C. Recombinant RrgA binds directly to A549 respiratory epithelial cells. Cells were incubated with 100 μg ml−1 of purified RrgA (A1 and A2, ‘+RrgA’) or GFP (B1 and B2, ‘+GFP’) in DMEM culture medium, or medium alone (A3–4, ‘-RrgA’; B3–4, ‘-GFP’) for 2 h at 4°C. Cells were fixed and stained with anti-RrgA and anti-GFP antibodies (A1–4, ‘αRrgA’; B1–4, ‘αGFP’) and phalloidin, which served as a control to demonstrate the presence of cells (A2, A4, B2 and B4). Imaging was performed with a confocal microscope. Scale bar is 20 μm. C. RrgA and RrgC bind to A549 cells in a dose-dependent manner. A549 cells were mixed in suspension with either medium alone (0 μg ml−1) or three concentrations (5, 50 and 100 μg ml−1) of pilus subunits RrgA (squares with solid black lines), RrgB (triangles with dashed grey lines), RrgC (upside-down triangles with dashed black lines), or GFP protein (diamond with solid grey lines), incubated for 2 h at 4°C, stained with antisera specific to each protein, and detected with Alexa Fluor 488-conjugated secondaries. Cells were analysed with a FACS-Calibur flow cytometer, and the net mean fluorescence intensity for each population was calculated from three independent experiments. Significant differences were detected by repeated-measure anova (P< 0.0001), and both RrgA and RrgC binding was significantly different from RrgB and GFP binding at 50 and 100 μg ml−1 by post hoc Bonferroni analysis (*P < 0.001). D–G. Pre-incubation of A549 cells with purified RrgA protein inhibits pilus-mediated adherence. A549 cells were pre-incubated with media alone (D), media containing 100 μg ml−1 of RrgA (E), or 100 μg ml−1 of GFP (F). After pre-incubation, A549 monolayers were infected with S. pneumoniae strain T4. Cells were stained with phalloidin (red) anti-S. pneumoniae capsule antibody (green), and imaged with a confocal microscope. RrgA pre-incubation inhibits the adherence of strain T4 to the A549 cells (E versus D), while the negative control GFP protein does not (F versus D). Scale bar is 20 μm. Adherent bacteria were counted, and the number of bacteria adherent to 100 A549 cells is shown in G (n = 6 fields, *P= 0.0002, **P= 0.8).
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fig02: Recombinant RrgA and RrgC proteins bind to human respiratory epithelial cells. A–C. Recombinant RrgA binds directly to A549 respiratory epithelial cells. Cells were incubated with 100 μg ml−1 of purified RrgA (A1 and A2, ‘+RrgA’) or GFP (B1 and B2, ‘+GFP’) in DMEM culture medium, or medium alone (A3–4, ‘-RrgA’; B3–4, ‘-GFP’) for 2 h at 4°C. Cells were fixed and stained with anti-RrgA and anti-GFP antibodies (A1–4, ‘αRrgA’; B1–4, ‘αGFP’) and phalloidin, which served as a control to demonstrate the presence of cells (A2, A4, B2 and B4). Imaging was performed with a confocal microscope. Scale bar is 20 μm. C. RrgA and RrgC bind to A549 cells in a dose-dependent manner. A549 cells were mixed in suspension with either medium alone (0 μg ml−1) or three concentrations (5, 50 and 100 μg ml−1) of pilus subunits RrgA (squares with solid black lines), RrgB (triangles with dashed grey lines), RrgC (upside-down triangles with dashed black lines), or GFP protein (diamond with solid grey lines), incubated for 2 h at 4°C, stained with antisera specific to each protein, and detected with Alexa Fluor 488-conjugated secondaries. Cells were analysed with a FACS-Calibur flow cytometer, and the net mean fluorescence intensity for each population was calculated from three independent experiments. Significant differences were detected by repeated-measure anova (P< 0.0001), and both RrgA and RrgC binding was significantly different from RrgB and GFP binding at 50 and 100 μg ml−1 by post hoc Bonferroni analysis (*P < 0.001). D–G. Pre-incubation of A549 cells with purified RrgA protein inhibits pilus-mediated adherence. A549 cells were pre-incubated with media alone (D), media containing 100 μg ml−1 of RrgA (E), or 100 μg ml−1 of GFP (F). After pre-incubation, A549 monolayers were infected with S. pneumoniae strain T4. Cells were stained with phalloidin (red) anti-S. pneumoniae capsule antibody (green), and imaged with a confocal microscope. RrgA pre-incubation inhibits the adherence of strain T4 to the A549 cells (E versus D), while the negative control GFP protein does not (F versus D). Scale bar is 20 μm. Adherent bacteria were counted, and the number of bacteria adherent to 100 A549 cells is shown in G (n = 6 fields, *P= 0.0002, **P= 0.8).

Mentions: As rrgA expression is required for wild-type pilus-mediated adherence to A549 cell monolayers, we sought to characterize the binding of isolated His-tagged RrgA protein. Adherent A549 cell monolayers were incubated with RrgA in media in the absence of serum, the cells were washed, and the protein was subsequently detected immunologically. Abundant RrgA was detected on the surface of monolayers, suggesting that the recombinant subunit had bound to the adherent cells (Fig. 2A1). Further, it appeared that RrgA binding occurred in small patches or microdomains on the monolayer surface. Fluorescence was not due to binding of the anti-RrgA antibody to the A549 cell surface, as exclusion of the RrgA protein abolished fluorescent signal (Fig. 2A3), although cells were present (Fig. 2A2 and A4). Green fluorescence protein (GFP) was used as a negative control, and did not bind to A549 monolayers at an equivalent dose used for RrgA (Fig. 2B1).


RrgA is a pilus-associated adhesin in Streptococcus pneumoniae.

Nelson AL, Ries J, Bagnoli F, Dahlberg S, Fälker S, Rounioja S, Tschöp J, Morfeldt E, Ferlenghi I, Hilleringmann M, Holden DW, Rappuoli R, Normark S, Barocchi MA, Henriques-Normark B - Mol. Microbiol. (2007)

Recombinant RrgA and RrgC proteins bind to human respiratory epithelial cells. A–C. Recombinant RrgA binds directly to A549 respiratory epithelial cells. Cells were incubated with 100 μg ml−1 of purified RrgA (A1 and A2, ‘+RrgA’) or GFP (B1 and B2, ‘+GFP’) in DMEM culture medium, or medium alone (A3–4, ‘-RrgA’; B3–4, ‘-GFP’) for 2 h at 4°C. Cells were fixed and stained with anti-RrgA and anti-GFP antibodies (A1–4, ‘αRrgA’; B1–4, ‘αGFP’) and phalloidin, which served as a control to demonstrate the presence of cells (A2, A4, B2 and B4). Imaging was performed with a confocal microscope. Scale bar is 20 μm. C. RrgA and RrgC bind to A549 cells in a dose-dependent manner. A549 cells were mixed in suspension with either medium alone (0 μg ml−1) or three concentrations (5, 50 and 100 μg ml−1) of pilus subunits RrgA (squares with solid black lines), RrgB (triangles with dashed grey lines), RrgC (upside-down triangles with dashed black lines), or GFP protein (diamond with solid grey lines), incubated for 2 h at 4°C, stained with antisera specific to each protein, and detected with Alexa Fluor 488-conjugated secondaries. Cells were analysed with a FACS-Calibur flow cytometer, and the net mean fluorescence intensity for each population was calculated from three independent experiments. Significant differences were detected by repeated-measure anova (P< 0.0001), and both RrgA and RrgC binding was significantly different from RrgB and GFP binding at 50 and 100 μg ml−1 by post hoc Bonferroni analysis (*P < 0.001). D–G. Pre-incubation of A549 cells with purified RrgA protein inhibits pilus-mediated adherence. A549 cells were pre-incubated with media alone (D), media containing 100 μg ml−1 of RrgA (E), or 100 μg ml−1 of GFP (F). After pre-incubation, A549 monolayers were infected with S. pneumoniae strain T4. Cells were stained with phalloidin (red) anti-S. pneumoniae capsule antibody (green), and imaged with a confocal microscope. RrgA pre-incubation inhibits the adherence of strain T4 to the A549 cells (E versus D), while the negative control GFP protein does not (F versus D). Scale bar is 20 μm. Adherent bacteria were counted, and the number of bacteria adherent to 100 A549 cells is shown in G (n = 6 fields, *P= 0.0002, **P= 0.8).
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fig02: Recombinant RrgA and RrgC proteins bind to human respiratory epithelial cells. A–C. Recombinant RrgA binds directly to A549 respiratory epithelial cells. Cells were incubated with 100 μg ml−1 of purified RrgA (A1 and A2, ‘+RrgA’) or GFP (B1 and B2, ‘+GFP’) in DMEM culture medium, or medium alone (A3–4, ‘-RrgA’; B3–4, ‘-GFP’) for 2 h at 4°C. Cells were fixed and stained with anti-RrgA and anti-GFP antibodies (A1–4, ‘αRrgA’; B1–4, ‘αGFP’) and phalloidin, which served as a control to demonstrate the presence of cells (A2, A4, B2 and B4). Imaging was performed with a confocal microscope. Scale bar is 20 μm. C. RrgA and RrgC bind to A549 cells in a dose-dependent manner. A549 cells were mixed in suspension with either medium alone (0 μg ml−1) or three concentrations (5, 50 and 100 μg ml−1) of pilus subunits RrgA (squares with solid black lines), RrgB (triangles with dashed grey lines), RrgC (upside-down triangles with dashed black lines), or GFP protein (diamond with solid grey lines), incubated for 2 h at 4°C, stained with antisera specific to each protein, and detected with Alexa Fluor 488-conjugated secondaries. Cells were analysed with a FACS-Calibur flow cytometer, and the net mean fluorescence intensity for each population was calculated from three independent experiments. Significant differences were detected by repeated-measure anova (P< 0.0001), and both RrgA and RrgC binding was significantly different from RrgB and GFP binding at 50 and 100 μg ml−1 by post hoc Bonferroni analysis (*P < 0.001). D–G. Pre-incubation of A549 cells with purified RrgA protein inhibits pilus-mediated adherence. A549 cells were pre-incubated with media alone (D), media containing 100 μg ml−1 of RrgA (E), or 100 μg ml−1 of GFP (F). After pre-incubation, A549 monolayers were infected with S. pneumoniae strain T4. Cells were stained with phalloidin (red) anti-S. pneumoniae capsule antibody (green), and imaged with a confocal microscope. RrgA pre-incubation inhibits the adherence of strain T4 to the A549 cells (E versus D), while the negative control GFP protein does not (F versus D). Scale bar is 20 μm. Adherent bacteria were counted, and the number of bacteria adherent to 100 A549 cells is shown in G (n = 6 fields, *P= 0.0002, **P= 0.8).
Mentions: As rrgA expression is required for wild-type pilus-mediated adherence to A549 cell monolayers, we sought to characterize the binding of isolated His-tagged RrgA protein. Adherent A549 cell monolayers were incubated with RrgA in media in the absence of serum, the cells were washed, and the protein was subsequently detected immunologically. Abundant RrgA was detected on the surface of monolayers, suggesting that the recombinant subunit had bound to the adherent cells (Fig. 2A1). Further, it appeared that RrgA binding occurred in small patches or microdomains on the monolayer surface. Fluorescence was not due to binding of the anti-RrgA antibody to the A549 cell surface, as exclusion of the RrgA protein abolished fluorescent signal (Fig. 2A3), although cells were present (Fig. 2A2 and A4). Green fluorescence protein (GFP) was used as a negative control, and did not bind to A549 monolayers at an equivalent dose used for RrgA (Fig. 2B1).

Bottom Line: Non-adherent RrgA-negative, RrgB- and RrgC-positive organisms produce pili, suggesting that pilus-mediated adherence is due to expression of RrgA, rather than the pilus backbone itself.In contrast, RrgA-positive strains with disrupted rrgB and rrgC genes exhibit wild-type adherence despite failure to produce pili by Western blot or immunoelectron microscopy.These data suggest that RrgA is central in pilus-mediated adherence and disease, even in the absence of polymeric pilus production.

View Article: PubMed Central - PubMed

Affiliation: Swedish Institute for Infectious Disease Control and Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
Adherence to host cells is important in microbial colonization of a mucosal surface, and Streptococcus pneumoniae adherence was significantly enhanced by expression of an extracellular pilus composed of three subunits, RrgA, RrgB and RrgC. We sought to determine which subunit(s) confers adherence. Bacteria deficient in RrgA are significantly less adherent than wild-type organisms, while overexpression of RrgA enhances adherence. Recombinant monomeric RrgA binds to respiratory cells, as does RrgC with less affinity, and pre-incubation of epithelial cells with RrgA reduces adherence of wild-type piliated pneumococci. Non-adherent RrgA-negative, RrgB- and RrgC-positive organisms produce pili, suggesting that pilus-mediated adherence is due to expression of RrgA, rather than the pilus backbone itself. In contrast, RrgA-positive strains with disrupted rrgB and rrgC genes exhibit wild-type adherence despite failure to produce pili by Western blot or immunoelectron microscopy. The density of bacteria colonizing the upper respiratory tract of mice inoculated with piliated RrgA-negative pneumococci was significantly less compared with wild-type; in contrast, non-piliated pneumococci expressing non-polymeric RrgA had similar numbers of bacteria in the nasopharynx as piliated wild-type bacteria. These data suggest that RrgA is central in pilus-mediated adherence and disease, even in the absence of polymeric pilus production.

Show MeSH
Related in: MedlinePlus