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Specific involvement of pilus type 2a in biofilm formation in group B Streptococcus.

Rinaudo CD, Rosini R, Galeotti CL, Berti F, Necchi F, Reguzzi V, Ghezzo C, Telford JL, Grandi G, Maione D - PLoS ONE (2010)

Bottom Line: Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands.We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype.Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Novartis Vaccines and Diagnostics, Siena, Italy.

ABSTRACT
Streptococcus agalactiae is the primary colonizer of the anogenital mucosa of up to 30% of healthy women and can infect newborns during delivery and cause severe sepsis and meningitis. Persistent colonization usually involves the formation of biofilm and increasing evidences indicate that in pathogenic streptococci biofilm formation is mediated by pili. Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands. Here we have investigated the capacity of these strains to form biofilms. We have found that most of the biofilm-formers carry pilus 2a, and using insertion and deletion mutants we have confirmed that pilus type 2a, but not pilus types 1 and 2b, confers biofilm-forming phenotype. We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype. Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells.

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Related in: MedlinePlus

Contribution of pilus 2a subunits and sortase A in biofilm formation.(A) Schematic representation of pilus island 2a in GBS strain 515. Genes encoding the three LPXTG proteins are represented by black (backbone protein, BP-2a) and white (ancillary proteins 1 and 2, AP1-2a and AP2-2a) arrows. SrtC transpeptidase genes (SrtC1 and SrtC-2) are shown in gray. (B) Comparison of biofilm formation by crystal violet assay between GBS wild type strain 515 (515 WT), and the deletion mutant strains 515 for the whole pilus island 2a (515ΔPI-2a), for AP1 gene (ΔAP1-2a), for BP gene (ΔBP-2a), for SrtC1 gene (ΔsrtC1), for SrtC-2 gene (ΔsrtC-2), for both sortases genes (ΔsrtC-1/2), for AP2 gene (ΔAP2-2a) and for Sortase A gene (ΔsrtA). Cells were grown in polystyrene plates at 37°C for 18 hours. Adherent bacteria were stained with crystal violet and quantified by measuring the absorbance at 540 nm. (C) Comparison of biofilm formation by crystal violet assay between the 515 deletion mutant strains (ΔAP1-2a, ΔBP-2a, ΔAP2-2a, ΔsrtA), complemented with an empty pAM401 expression vector alone and the corresponding complemented strains: ΔAP1-2a+, strain 515 ΔAP1-2a complemented by a plasmid containing the complete AP1-2a coding sequence; ΔBP-2a+, strain 515 ΔBP-2a complemented by a plasmid containing the BP-2a gene; ΔAP2-2a+, strain 515 ΔAP2-2a complemented by a plasmid containing the AP2-2a gene; ΔsrtA+, strain 515 ΔsrtA complemented by a plasmid containing the complete srtA coding sequence. Bacteria were grown under static conditions in polystyrene plates at 37°C for 48 h. Crystal violet-stained, surface-attached cells were quantified by solubilizing the dye in 30% acid acetic and determining the absorbance at 540 nm. The mean values of three independent experiments and standard deviations are shown.
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pone-0009216-g006: Contribution of pilus 2a subunits and sortase A in biofilm formation.(A) Schematic representation of pilus island 2a in GBS strain 515. Genes encoding the three LPXTG proteins are represented by black (backbone protein, BP-2a) and white (ancillary proteins 1 and 2, AP1-2a and AP2-2a) arrows. SrtC transpeptidase genes (SrtC1 and SrtC-2) are shown in gray. (B) Comparison of biofilm formation by crystal violet assay between GBS wild type strain 515 (515 WT), and the deletion mutant strains 515 for the whole pilus island 2a (515ΔPI-2a), for AP1 gene (ΔAP1-2a), for BP gene (ΔBP-2a), for SrtC1 gene (ΔsrtC1), for SrtC-2 gene (ΔsrtC-2), for both sortases genes (ΔsrtC-1/2), for AP2 gene (ΔAP2-2a) and for Sortase A gene (ΔsrtA). Cells were grown in polystyrene plates at 37°C for 18 hours. Adherent bacteria were stained with crystal violet and quantified by measuring the absorbance at 540 nm. (C) Comparison of biofilm formation by crystal violet assay between the 515 deletion mutant strains (ΔAP1-2a, ΔBP-2a, ΔAP2-2a, ΔsrtA), complemented with an empty pAM401 expression vector alone and the corresponding complemented strains: ΔAP1-2a+, strain 515 ΔAP1-2a complemented by a plasmid containing the complete AP1-2a coding sequence; ΔBP-2a+, strain 515 ΔBP-2a complemented by a plasmid containing the BP-2a gene; ΔAP2-2a+, strain 515 ΔAP2-2a complemented by a plasmid containing the AP2-2a gene; ΔsrtA+, strain 515 ΔsrtA complemented by a plasmid containing the complete srtA coding sequence. Bacteria were grown under static conditions in polystyrene plates at 37°C for 48 h. Crystal violet-stained, surface-attached cells were quantified by solubilizing the dye in 30% acid acetic and determining the absorbance at 540 nm. The mean values of three independent experiments and standard deviations are shown.

Mentions: To investigate the role of each gene of pilus island 2a (Figure 6A) in biofilm formation, we also analyzed biofilm formation in the mutant strains, generated in strain 515, lacking either one of the three structural LPXTG proteins (BP, AP1 and AP2) or the two SrtC-like sortases SrtC-1 and SrtC-2 [22]. As published elsewhere, inactivation of either the backbone protein BP or both sortases results in the loss of pilus whereas pilus assembly still occurs in mutants lacking the two ancillary proteins (AP1 and AP2) and one of the two sortases [22]. Consistently with these data, biofilm formation in mutants lacking pili was abolished (Figure 6B). By contrast, deletion of the AP1 gene (ΔAP1), or deletion of the sortase SrtC-2 (ΔsrtC-2) involved in the incorporation of AP1 protein into the pilus structure, did not affect biofilm formation. Finally, the mutant lacking the AP2 gene (ΔAP2-2a), as well as inactivation of sortase SrtC-1 (ΔsrtC-1), essential for AP2 incorporation into the pilus, reduced the ability of GBS to form biofilms (Figure 6B). Complementation of all biofilm-defective mutant strains restored biofilm formation to a level comparable with that of the wild-type strain (Figure 6C). Identical results were obtained both on abiotic and biotic surfaces (data not shown). The fact that mutant strains impaired in AP2 synthesis (ΔAP2) or AP2 covalent binding to the backbone protein BP (ΔsrtC-1) are unable to form biofilms is consistent with the role of the minor ancillary as pilus anchoring protein [28]. In terms of biofilm formation these mutants, which tend to release polymerized pili into the culture supernatants, behave like a non piliated strain. Also in line with this is the phenotype of ΔsrtA mutant. The constitutive SrtA catalyzes the covalent binding of the minor ancillary AP2 to peptidoglycan and since its inactivation leads to pilus secretion [28], the 515 ΔsrtA mutant is incapable of forming biofilms according to data obtained in GBS NEM316 ΔsrtA mutant strain (Figure 6B and [9]). The wild type phenotype was completely restored when the 515 ΔsrtA mutant strain was complemented with a plasmid expressing the complete srtA gene (ΔsrtA+) (Figure 6C).


Specific involvement of pilus type 2a in biofilm formation in group B Streptococcus.

Rinaudo CD, Rosini R, Galeotti CL, Berti F, Necchi F, Reguzzi V, Ghezzo C, Telford JL, Grandi G, Maione D - PLoS ONE (2010)

Contribution of pilus 2a subunits and sortase A in biofilm formation.(A) Schematic representation of pilus island 2a in GBS strain 515. Genes encoding the three LPXTG proteins are represented by black (backbone protein, BP-2a) and white (ancillary proteins 1 and 2, AP1-2a and AP2-2a) arrows. SrtC transpeptidase genes (SrtC1 and SrtC-2) are shown in gray. (B) Comparison of biofilm formation by crystal violet assay between GBS wild type strain 515 (515 WT), and the deletion mutant strains 515 for the whole pilus island 2a (515ΔPI-2a), for AP1 gene (ΔAP1-2a), for BP gene (ΔBP-2a), for SrtC1 gene (ΔsrtC1), for SrtC-2 gene (ΔsrtC-2), for both sortases genes (ΔsrtC-1/2), for AP2 gene (ΔAP2-2a) and for Sortase A gene (ΔsrtA). Cells were grown in polystyrene plates at 37°C for 18 hours. Adherent bacteria were stained with crystal violet and quantified by measuring the absorbance at 540 nm. (C) Comparison of biofilm formation by crystal violet assay between the 515 deletion mutant strains (ΔAP1-2a, ΔBP-2a, ΔAP2-2a, ΔsrtA), complemented with an empty pAM401 expression vector alone and the corresponding complemented strains: ΔAP1-2a+, strain 515 ΔAP1-2a complemented by a plasmid containing the complete AP1-2a coding sequence; ΔBP-2a+, strain 515 ΔBP-2a complemented by a plasmid containing the BP-2a gene; ΔAP2-2a+, strain 515 ΔAP2-2a complemented by a plasmid containing the AP2-2a gene; ΔsrtA+, strain 515 ΔsrtA complemented by a plasmid containing the complete srtA coding sequence. Bacteria were grown under static conditions in polystyrene plates at 37°C for 48 h. Crystal violet-stained, surface-attached cells were quantified by solubilizing the dye in 30% acid acetic and determining the absorbance at 540 nm. The mean values of three independent experiments and standard deviations are shown.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2821406&req=5

pone-0009216-g006: Contribution of pilus 2a subunits and sortase A in biofilm formation.(A) Schematic representation of pilus island 2a in GBS strain 515. Genes encoding the three LPXTG proteins are represented by black (backbone protein, BP-2a) and white (ancillary proteins 1 and 2, AP1-2a and AP2-2a) arrows. SrtC transpeptidase genes (SrtC1 and SrtC-2) are shown in gray. (B) Comparison of biofilm formation by crystal violet assay between GBS wild type strain 515 (515 WT), and the deletion mutant strains 515 for the whole pilus island 2a (515ΔPI-2a), for AP1 gene (ΔAP1-2a), for BP gene (ΔBP-2a), for SrtC1 gene (ΔsrtC1), for SrtC-2 gene (ΔsrtC-2), for both sortases genes (ΔsrtC-1/2), for AP2 gene (ΔAP2-2a) and for Sortase A gene (ΔsrtA). Cells were grown in polystyrene plates at 37°C for 18 hours. Adherent bacteria were stained with crystal violet and quantified by measuring the absorbance at 540 nm. (C) Comparison of biofilm formation by crystal violet assay between the 515 deletion mutant strains (ΔAP1-2a, ΔBP-2a, ΔAP2-2a, ΔsrtA), complemented with an empty pAM401 expression vector alone and the corresponding complemented strains: ΔAP1-2a+, strain 515 ΔAP1-2a complemented by a plasmid containing the complete AP1-2a coding sequence; ΔBP-2a+, strain 515 ΔBP-2a complemented by a plasmid containing the BP-2a gene; ΔAP2-2a+, strain 515 ΔAP2-2a complemented by a plasmid containing the AP2-2a gene; ΔsrtA+, strain 515 ΔsrtA complemented by a plasmid containing the complete srtA coding sequence. Bacteria were grown under static conditions in polystyrene plates at 37°C for 48 h. Crystal violet-stained, surface-attached cells were quantified by solubilizing the dye in 30% acid acetic and determining the absorbance at 540 nm. The mean values of three independent experiments and standard deviations are shown.
Mentions: To investigate the role of each gene of pilus island 2a (Figure 6A) in biofilm formation, we also analyzed biofilm formation in the mutant strains, generated in strain 515, lacking either one of the three structural LPXTG proteins (BP, AP1 and AP2) or the two SrtC-like sortases SrtC-1 and SrtC-2 [22]. As published elsewhere, inactivation of either the backbone protein BP or both sortases results in the loss of pilus whereas pilus assembly still occurs in mutants lacking the two ancillary proteins (AP1 and AP2) and one of the two sortases [22]. Consistently with these data, biofilm formation in mutants lacking pili was abolished (Figure 6B). By contrast, deletion of the AP1 gene (ΔAP1), or deletion of the sortase SrtC-2 (ΔsrtC-2) involved in the incorporation of AP1 protein into the pilus structure, did not affect biofilm formation. Finally, the mutant lacking the AP2 gene (ΔAP2-2a), as well as inactivation of sortase SrtC-1 (ΔsrtC-1), essential for AP2 incorporation into the pilus, reduced the ability of GBS to form biofilms (Figure 6B). Complementation of all biofilm-defective mutant strains restored biofilm formation to a level comparable with that of the wild-type strain (Figure 6C). Identical results were obtained both on abiotic and biotic surfaces (data not shown). The fact that mutant strains impaired in AP2 synthesis (ΔAP2) or AP2 covalent binding to the backbone protein BP (ΔsrtC-1) are unable to form biofilms is consistent with the role of the minor ancillary as pilus anchoring protein [28]. In terms of biofilm formation these mutants, which tend to release polymerized pili into the culture supernatants, behave like a non piliated strain. Also in line with this is the phenotype of ΔsrtA mutant. The constitutive SrtA catalyzes the covalent binding of the minor ancillary AP2 to peptidoglycan and since its inactivation leads to pilus secretion [28], the 515 ΔsrtA mutant is incapable of forming biofilms according to data obtained in GBS NEM316 ΔsrtA mutant strain (Figure 6B and [9]). The wild type phenotype was completely restored when the 515 ΔsrtA mutant strain was complemented with a plasmid expressing the complete srtA gene (ΔsrtA+) (Figure 6C).

Bottom Line: Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands.We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype.Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Novartis Vaccines and Diagnostics, Siena, Italy.

ABSTRACT
Streptococcus agalactiae is the primary colonizer of the anogenital mucosa of up to 30% of healthy women and can infect newborns during delivery and cause severe sepsis and meningitis. Persistent colonization usually involves the formation of biofilm and increasing evidences indicate that in pathogenic streptococci biofilm formation is mediated by pili. Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands. Here we have investigated the capacity of these strains to form biofilms. We have found that most of the biofilm-formers carry pilus 2a, and using insertion and deletion mutants we have confirmed that pilus type 2a, but not pilus types 1 and 2b, confers biofilm-forming phenotype. We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype. Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells.

Show MeSH
Related in: MedlinePlus