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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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The pneumococcal pilus is expressed in the absence of RrgA. Production of polymeric high-molecular-weight extracellular pili in isogenic sets of pneumococci was evaluated by preparation of cell wall-associated proteins and immunoblotting for pilus subunits RrgA (A), RrgB (B) and RrgC (C). In all cases, cell wall proteins were separated by 4–12% gradient SDS-PAGE, electrotransferred to a PVDF membrane, and probed for pilus subunits. Approximate molecular weight in kDa, based on marker proteins, are indicated on left. A. Immunoblotting for RrgA in wild-type T4, T4ΔrrgA (‘ΔrrgA’), T4ΔrrgBC (‘ΔrrgBC’), and T4Δ(rrgA-srtD) (‘rrgA-srtD’). B. Immunoblotting for RrgB in wild-type piliated T4, T4ΔrrgA, T4ΔrrgBC and T4Δ(rrgA-srtD), labelled as in A. C. Immunoblotting for RrgC in wild-type piliated T4, T4ΔrrgA, T4ΔrrgBC, and T4Δ(rrgA-srtD), labelled as in A. These data demonstrate that extracellular RrgB-positive, RrgC-positive pili are formed in the absence of rrgA expression, but not in the absence of expression of rrgB and rrgC. In the absence of RrgB and RrgC, RrgA is found as a 100 kDa monomer.
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fig03: The pneumococcal pilus is expressed in the absence of RrgA. Production of polymeric high-molecular-weight extracellular pili in isogenic sets of pneumococci was evaluated by preparation of cell wall-associated proteins and immunoblotting for pilus subunits RrgA (A), RrgB (B) and RrgC (C). In all cases, cell wall proteins were separated by 4–12% gradient SDS-PAGE, electrotransferred to a PVDF membrane, and probed for pilus subunits. Approximate molecular weight in kDa, based on marker proteins, are indicated on left. A. Immunoblotting for RrgA in wild-type T4, T4ΔrrgA (‘ΔrrgA’), T4ΔrrgBC (‘ΔrrgBC’), and T4Δ(rrgA-srtD) (‘rrgA-srtD’). B. Immunoblotting for RrgB in wild-type piliated T4, T4ΔrrgA, T4ΔrrgBC and T4Δ(rrgA-srtD), labelled as in A. C. Immunoblotting for RrgC in wild-type piliated T4, T4ΔrrgA, T4ΔrrgBC, and T4Δ(rrgA-srtD), labelled as in A. These data demonstrate that extracellular RrgB-positive, RrgC-positive pili are formed in the absence of rrgA expression, but not in the absence of expression of rrgB and rrgC. In the absence of RrgB and RrgC, RrgA is found as a 100 kDa monomer.

Mentions: Given that RrgA is required for pilus-mediated adherence, we sought to determine the form of RrgA on the bacterial surface in the presence and absence of RrgB and RrgC expression. Cell wall-associated material was prepared by digestion with the muramidase mutanolysin, and separated by SDS-PAGE for Western blot analysis. In wild-type organisms, RrgA was incorporated into high-molecular-weight ladders consistent with pilus production, as well as in a 100 kDa monomer (Fig. 3A, lane 1). No RrgA was detected in material derived from the T4ΔrrgA (Fig. 3A, lane 2) and the T4ΔrrgA-srtD negative control (Fig. 3A, lane 4). In organisms that lack both of the other two structural subunits of the pilus, RrgB and RrgC, RrgA was found predominantly in the 100 kDa monomeric form (Fig. 3A, lane 3).


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)

The pneumococcal pilus is expressed in the absence of RrgA. Production of polymeric high-molecular-weight extracellular pili in isogenic sets of pneumococci was evaluated by preparation of cell wall-associated proteins and immunoblotting for pilus subunits RrgA (A), RrgB (B) and RrgC (C). In all cases, cell wall proteins were separated by 4–12% gradient SDS-PAGE, electrotransferred to a PVDF membrane, and probed for pilus subunits. Approximate molecular weight in kDa, based on marker proteins, are indicated on left. A. Immunoblotting for RrgA in wild-type T4, T4ΔrrgA (‘ΔrrgA’), T4ΔrrgBC (‘ΔrrgBC’), and T4Δ(rrgA-srtD) (‘rrgA-srtD’). B. Immunoblotting for RrgB in wild-type piliated T4, T4ΔrrgA, T4ΔrrgBC and T4Δ(rrgA-srtD), labelled as in A. C. Immunoblotting for RrgC in wild-type piliated T4, T4ΔrrgA, T4ΔrrgBC, and T4Δ(rrgA-srtD), labelled as in A. These data demonstrate that extracellular RrgB-positive, RrgC-positive pili are formed in the absence of rrgA expression, but not in the absence of expression of rrgB and rrgC. In the absence of RrgB and RrgC, RrgA is found as a 100 kDa monomer.
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fig03: The pneumococcal pilus is expressed in the absence of RrgA. Production of polymeric high-molecular-weight extracellular pili in isogenic sets of pneumococci was evaluated by preparation of cell wall-associated proteins and immunoblotting for pilus subunits RrgA (A), RrgB (B) and RrgC (C). In all cases, cell wall proteins were separated by 4–12% gradient SDS-PAGE, electrotransferred to a PVDF membrane, and probed for pilus subunits. Approximate molecular weight in kDa, based on marker proteins, are indicated on left. A. Immunoblotting for RrgA in wild-type T4, T4ΔrrgA (‘ΔrrgA’), T4ΔrrgBC (‘ΔrrgBC’), and T4Δ(rrgA-srtD) (‘rrgA-srtD’). B. Immunoblotting for RrgB in wild-type piliated T4, T4ΔrrgA, T4ΔrrgBC and T4Δ(rrgA-srtD), labelled as in A. C. Immunoblotting for RrgC in wild-type piliated T4, T4ΔrrgA, T4ΔrrgBC, and T4Δ(rrgA-srtD), labelled as in A. These data demonstrate that extracellular RrgB-positive, RrgC-positive pili are formed in the absence of rrgA expression, but not in the absence of expression of rrgB and rrgC. In the absence of RrgB and RrgC, RrgA is found as a 100 kDa monomer.
Mentions: Given that RrgA is required for pilus-mediated adherence, we sought to determine the form of RrgA on the bacterial surface in the presence and absence of RrgB and RrgC expression. Cell wall-associated material was prepared by digestion with the muramidase mutanolysin, and separated by SDS-PAGE for Western blot analysis. In wild-type organisms, RrgA was incorporated into high-molecular-weight ladders consistent with pilus production, as well as in a 100 kDa monomer (Fig. 3A, lane 1). No RrgA was detected in material derived from the T4ΔrrgA (Fig. 3A, lane 2) and the T4ΔrrgA-srtD negative control (Fig. 3A, lane 4). In organisms that lack both of the other two structural subunits of the pilus, RrgB and RrgC, RrgA was found predominantly in the 100 kDa monomeric form (Fig. 3A, lane 3).

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