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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

Role of the three pilus islands in biofilm formation.(A) Western Blot analysis of total protein extracts from GBS strain JM9130013 expressing both PI-1 and PI-2b, strain 515 expressing only PI-2a, the deletion strain 515ΔPI-2a (no pilus expression) and the strain 515ΔPI-2a, complemented with each of the three pilus islands: pilus island 1 (ΔPI-2a/pAM-PI-1), pilus island 2a (ΔPI-2a/pAM-PI-2a) and pilus island 2b (ΔPI-2a/pAM-PI-2b). The membranes were probed with antisera specific for the backbone proteins of pilus 1 (α -BP-1), pilus 2a (α -BP-2a) and pilus 2b (α -BP-2b). High molecular weight polymers, which indicate incorporation of the protein into pilus-like structures were detected. (B) Flow cytometry analysis of 515ΔPI-2a (no pilus expression), complemented with an empty pAM plasmid and with each of the three pilus islands, 515ΔPI-2a/pAM-PI-1, 515ΔPI-2a/pAM-PI-2a and 515ΔPI-2a/pAM-PI-2b, probed with antisera specific for pilus 1 (α -BP-1), for pilus 2a (α -BP-2a) and for pilus 2b (α -BP-2b). (C) Quantification of biofilm formation by crystal violet assay of GBS wild type strain 515 (515 WT), GBS 515ΔPI-2a and the complemented strains 515 ΔPI-2a/pAM-PI-1, ΔPI-2a/pAM-PI-2a and ΔPI-2a/pAM-PI-2b. Bacteria were grown under static conditions at 37°C for 18 h in 96-well uncoated polystyrene plates and on plates coated with a purified Extracellular Matrix (ECM) derived from human placenta. Crystal violet-stained, surface-attached cells were quantified by solubilizing the dye in 30% acetic acid and determining the absorbance at 540 nm. The mean values of three independent experiments and standard deviation are shown.
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pone-0009216-g004: Role of the three pilus islands in biofilm formation.(A) Western Blot analysis of total protein extracts from GBS strain JM9130013 expressing both PI-1 and PI-2b, strain 515 expressing only PI-2a, the deletion strain 515ΔPI-2a (no pilus expression) and the strain 515ΔPI-2a, complemented with each of the three pilus islands: pilus island 1 (ΔPI-2a/pAM-PI-1), pilus island 2a (ΔPI-2a/pAM-PI-2a) and pilus island 2b (ΔPI-2a/pAM-PI-2b). The membranes were probed with antisera specific for the backbone proteins of pilus 1 (α -BP-1), pilus 2a (α -BP-2a) and pilus 2b (α -BP-2b). High molecular weight polymers, which indicate incorporation of the protein into pilus-like structures were detected. (B) Flow cytometry analysis of 515ΔPI-2a (no pilus expression), complemented with an empty pAM plasmid and with each of the three pilus islands, 515ΔPI-2a/pAM-PI-1, 515ΔPI-2a/pAM-PI-2a and 515ΔPI-2a/pAM-PI-2b, probed with antisera specific for pilus 1 (α -BP-1), for pilus 2a (α -BP-2a) and for pilus 2b (α -BP-2b). (C) Quantification of biofilm formation by crystal violet assay of GBS wild type strain 515 (515 WT), GBS 515ΔPI-2a and the complemented strains 515 ΔPI-2a/pAM-PI-1, ΔPI-2a/pAM-PI-2a and ΔPI-2a/pAM-PI-2b. Bacteria were grown under static conditions at 37°C for 18 h in 96-well uncoated polystyrene plates and on plates coated with a purified Extracellular Matrix (ECM) derived from human placenta. Crystal violet-stained, surface-attached cells were quantified by solubilizing the dye in 30% acetic acid and determining the absorbance at 540 nm. The mean values of three independent experiments and standard deviation are shown.

Mentions: The data shown above suggest that only pilus type 2a and not the other pilus types plays a role in biofilm formation. To support this finding, we generated an isogenic knock-out (KO) mutant strain 515ΔPI-2a in which the whole pilus island 2a was deleted and we complemented this non piliated strain with each of the three pilus islands identified in GBS. Complementation was carried out by transforming the mutant 515ΔPI-2a with recombinant pAM401 plasmids carrying the whole pilus islands. Western Blotting and FACS analyses using antibodies specific for each pilus type confirmed the absence of pili in 515ΔPI-2a and the expression of the corresponding pilus type in the complemented strains 515ΔPI-2a/pAM-PI-1, 515ΔPI-2a/pAM-PI-2a and 515ΔPI-2a/pAM-PI-2b (Figure 4A and 4B). The deletion mutant strain 515ΔPI-2a and its complemented strains expressing a single pilus type were then analyzed for their capacity to form biofilms on abiotic surfaces. As shown in Figure 4C, deletion of the whole pilus island 2a abolished the ability of bacteria to adhere to the plates and only the complemented strain expressing pilus 2a, and not those expressing pilus 1 and 2b, was able to significantly adhere to plates. Similar data were obtained when plates were coated with a purified Extracellular Matrix derived from human placenta, enriched of laminin, collagen type IV and heparan sulfate proteoglycan (Figure 4C). To further confirm the specific involvement of only pilus 2a in biofilm formation we investigated the ability to adhere to polystyrene plates of biofilm-forming strains expressing the other two pilus types (pilus 1 or pilus 2b), in which we knocked out the corresponding backbone protein essential for pilus polymerization (Figure 5A). These deletion mutant strains were not able to express pili on their surface (data not shown), yet the lack of pilus expression did not impact their ability to form biofilms on polystyrene plates (Figure 5B).


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)

Role of the three pilus islands in biofilm formation.(A) Western Blot analysis of total protein extracts from GBS strain JM9130013 expressing both PI-1 and PI-2b, strain 515 expressing only PI-2a, the deletion strain 515ΔPI-2a (no pilus expression) and the strain 515ΔPI-2a, complemented with each of the three pilus islands: pilus island 1 (ΔPI-2a/pAM-PI-1), pilus island 2a (ΔPI-2a/pAM-PI-2a) and pilus island 2b (ΔPI-2a/pAM-PI-2b). The membranes were probed with antisera specific for the backbone proteins of pilus 1 (α -BP-1), pilus 2a (α -BP-2a) and pilus 2b (α -BP-2b). High molecular weight polymers, which indicate incorporation of the protein into pilus-like structures were detected. (B) Flow cytometry analysis of 515ΔPI-2a (no pilus expression), complemented with an empty pAM plasmid and with each of the three pilus islands, 515ΔPI-2a/pAM-PI-1, 515ΔPI-2a/pAM-PI-2a and 515ΔPI-2a/pAM-PI-2b, probed with antisera specific for pilus 1 (α -BP-1), for pilus 2a (α -BP-2a) and for pilus 2b (α -BP-2b). (C) Quantification of biofilm formation by crystal violet assay of GBS wild type strain 515 (515 WT), GBS 515ΔPI-2a and the complemented strains 515 ΔPI-2a/pAM-PI-1, ΔPI-2a/pAM-PI-2a and ΔPI-2a/pAM-PI-2b. Bacteria were grown under static conditions at 37°C for 18 h in 96-well uncoated polystyrene plates and on plates coated with a purified Extracellular Matrix (ECM) derived from human placenta. Crystal violet-stained, surface-attached cells were quantified by solubilizing the dye in 30% acetic acid and determining the absorbance at 540 nm. The mean values of three independent experiments and standard deviation are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009216-g004: Role of the three pilus islands in biofilm formation.(A) Western Blot analysis of total protein extracts from GBS strain JM9130013 expressing both PI-1 and PI-2b, strain 515 expressing only PI-2a, the deletion strain 515ΔPI-2a (no pilus expression) and the strain 515ΔPI-2a, complemented with each of the three pilus islands: pilus island 1 (ΔPI-2a/pAM-PI-1), pilus island 2a (ΔPI-2a/pAM-PI-2a) and pilus island 2b (ΔPI-2a/pAM-PI-2b). The membranes were probed with antisera specific for the backbone proteins of pilus 1 (α -BP-1), pilus 2a (α -BP-2a) and pilus 2b (α -BP-2b). High molecular weight polymers, which indicate incorporation of the protein into pilus-like structures were detected. (B) Flow cytometry analysis of 515ΔPI-2a (no pilus expression), complemented with an empty pAM plasmid and with each of the three pilus islands, 515ΔPI-2a/pAM-PI-1, 515ΔPI-2a/pAM-PI-2a and 515ΔPI-2a/pAM-PI-2b, probed with antisera specific for pilus 1 (α -BP-1), for pilus 2a (α -BP-2a) and for pilus 2b (α -BP-2b). (C) Quantification of biofilm formation by crystal violet assay of GBS wild type strain 515 (515 WT), GBS 515ΔPI-2a and the complemented strains 515 ΔPI-2a/pAM-PI-1, ΔPI-2a/pAM-PI-2a and ΔPI-2a/pAM-PI-2b. Bacteria were grown under static conditions at 37°C for 18 h in 96-well uncoated polystyrene plates and on plates coated with a purified Extracellular Matrix (ECM) derived from human placenta. Crystal violet-stained, surface-attached cells were quantified by solubilizing the dye in 30% acetic acid and determining the absorbance at 540 nm. The mean values of three independent experiments and standard deviation are shown.
Mentions: The data shown above suggest that only pilus type 2a and not the other pilus types plays a role in biofilm formation. To support this finding, we generated an isogenic knock-out (KO) mutant strain 515ΔPI-2a in which the whole pilus island 2a was deleted and we complemented this non piliated strain with each of the three pilus islands identified in GBS. Complementation was carried out by transforming the mutant 515ΔPI-2a with recombinant pAM401 plasmids carrying the whole pilus islands. Western Blotting and FACS analyses using antibodies specific for each pilus type confirmed the absence of pili in 515ΔPI-2a and the expression of the corresponding pilus type in the complemented strains 515ΔPI-2a/pAM-PI-1, 515ΔPI-2a/pAM-PI-2a and 515ΔPI-2a/pAM-PI-2b (Figure 4A and 4B). The deletion mutant strain 515ΔPI-2a and its complemented strains expressing a single pilus type were then analyzed for their capacity to form biofilms on abiotic surfaces. As shown in Figure 4C, deletion of the whole pilus island 2a abolished the ability of bacteria to adhere to the plates and only the complemented strain expressing pilus 2a, and not those expressing pilus 1 and 2b, was able to significantly adhere to plates. Similar data were obtained when plates were coated with a purified Extracellular Matrix derived from human placenta, enriched of laminin, collagen type IV and heparan sulfate proteoglycan (Figure 4C). To further confirm the specific involvement of only pilus 2a in biofilm formation we investigated the ability to adhere to polystyrene plates of biofilm-forming strains expressing the other two pilus types (pilus 1 or pilus 2b), in which we knocked out the corresponding backbone protein essential for pilus polymerization (Figure 5A). These deletion mutant strains were not able to express pili on their surface (data not shown), yet the lack of pilus expression did not impact their ability to form biofilms on polystyrene plates (Figure 5B).

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