Specific Involvement of Pilus Type 2a in Biofilm Formation in Group B Streptococcus

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

Contribution of pilus 2a subunits and sortase A in biofilm formation.

<p>(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 <i>AP1</i> gene (<i>ΔAP1-2a</i>), for <i>BP</i> gene (<i>ΔBP-2a</i>), for SrtC1 gene (<i>ΔsrtC1</i>), for SrtC<i>-2</i> gene (<i>ΔsrtC-2</i>), for both sortases genes (<i>ΔsrtC-1/2</i>), for <i>AP2</i> gene (<i>ΔAP2-2a</i>) and for Sortase A gene (<i>ΔsrtA</i>). 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 (<i>ΔAP1-2a</i>, <i>ΔBP-2a</i>, <i>ΔAP2-2a</i>, <i>ΔsrtA</i>), complemented with an empty pAM401 expression vector alone and the corresponding complemented strains: <i>ΔAP1-2a+</i>, strain 515 <i>ΔAP1-2a</i> complemented by a plasmid containing the complete <i>AP1-2a</i> coding sequence; <i>ΔBP-2a+</i>, strain 515 <i>ΔBP-2a</i> complemented by a plasmid containing the <i>BP-2a</i> gene; <i>ΔAP2-2a+</i>, strain 515 Δ<i>AP2-2a</i> complemented by a plasmid containing the <i>AP2-2a</i> gene; <i>ΔsrtA</i>+, strain 515 <i>ΔsrtA</i> complemented by a plasmid containing the complete <i>srtA</i> 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.</p

Role of pilus type 1 and pilus type 2b in biofilm formation.

<p>(A) Schematic representation of Pilus Island 1 (PI-1) and Pilus Island 2b (PI-2b). Genes encoding the three LPXTG proteins are represented by black (BP-1, backbone protein of pilus 1; BP-2b, backbone protein of pilus 2b) and white (AP1-1 and AP2-1, ancillary protein 1 and ancillary protein 2 of pilus 1; AP1-2b and AP2-2b, ancillary protein 1 and ancillary protein 2 of pilus 2b) arrows. SrtC transpeptidase genes (SrtC1 and SrtC-2) are shown in gray. (B) Quantification of biofilm formation by crystal violet assay of GBS wild type strain CJB111 expressing pilus type 1 (WT, PI-1) and GBS wild type strain ABC020017623 expressing pilus type 2b (WT, PI-2b) in comparison with the corresponding deletion mutant strains lacking the pilus expression. <i>ΔBP-1</i>, strain CJB111 with in-frame deletion of the gene coding for the backbone protein of pilus 1; <i>ΔBP-2b</i>, strain ABC020017623 with in-frame deletion of the gene coding for the backbone protein of pilus 2b. Bacteria were grown under static conditions at 37°C for 18 h in 96-well uncoated polystyrene plates and surface-attached, crystal violet-stained 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.</p

Correlation between surface exposure of pilus type 2a and biofilm formation.

<p>(A) Determination by crystal violet (CV) assay of biofilm formation of 289 GBS clinical isolates grown in 96-well polystyrene plates in THB supplemented with 1% glucose at 37°C for 18 h. Adherent bacteria were stained with crystal violet (CV) and quantified by measuring the absorbance at 540 nm. The data represent the mean values of three experiments. Standard deviations (not shown) ranged from OD₅₄₀ values of 0.02 to 0.1. Strains were distributed into 3 groups according to the level of expression of pilus type 2a (PI-2a, Pilus Island 2a), measured by flow cytometry as the Fluorescence Fold Increase (FFI) of cells stained with anti-pilus 2a sera over cells stained with pre-immune sera. Group 1 (High pilus expression: PI-2a >5 FFI) including 171 strains; Group 2 (intermediate or low pilus expression: PI-2a <5 FFI) including 38 strains and Group 3, in which the PI-2a was absent, including 80 strains. Group 1 or Group 2 were also subdivided in strains expressing high levels of pilus type 1 (PI-1>5) and strains carrying only pilus 2a (PI-1 absent) or expressing pilus type 1 below the threshold level (PI-1<5). Group 3 was subdivided into strains expressing either PI-1 or PI-2b at high levels (FFI >5) and strains in which also PI-1 and PI-2b poorly expressed (FFI <5). The horizontal bar intersecting the value of absorbance 0.65 represents the cut-off established to discriminate between high-biofilm forming strains and low-biofilm forming strains. The cut-off was calculated as the value equidistant from the median of absorbance of groups 1, 2 and 3. The percentages are calculated considering the number of strains over or below the cut-off value. Circles indicates strains used for SEM analysis shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009216#pone-0009216-g003" target="_blank">Figure 3</a>. (B) Non parametric multi-comparison statistical analysis. The Kruskal-Wallis test was applied to test the equality of population median among the groups and confirmed that the three groups did not belong to the same population (p-value = 1.742 e⁻⁰⁸). The Behrens-Fisher test was applied to distinguish which of the groups were significantly different. Each of the lines connecting two groups is proportional to their comparison p-value and confirms a strong relation between Gr.2 and Gr.3, while the Gr.1 was found to be very different from the others.</p

Influence of glucose on biofilm formation in GBS strain 515.

<p>Determination of biofilm formation by crystal violet (CV) assay of GBS strain 515 grown in THB supplemented with different glucose concentrations in 96-well polystyrene plates under static conditions at 37°C, 5% CO₂ for 18 hours. Crystal violet stained, surface-attached cells were quantified by solubilizing the dye in 30% acetic acid and by measuring the absorbance at 540 nm. The mean values of three independent experiments and standard deviation are shown.</p

Preventing bacterial infections with pilus-based vaccines: The group B streptococcus paradigm

We recently described the presence of 3 pilus variants in the human pathogen group B streptococcus (GBS; also known as Streptococcus agalactiae), each encoded by a distinct pathogenicity island, as well as the ability of pilus components to elicit protection in mice against homologous challenge. To determine whether a vaccine containing a combination of proteins from the 3 pilus types could provide broad protection, we analyzed pili distribution and conservation in 289 clinical isolates. We found that pilus sequences in each island are conserved, all strains carried at least 1 of the 3 islands, and a combination of the 3 pilus components conferred protection against all tested GBS challenge strains. These data are the first to indicate that a vaccine exclusively constituted by pilus components can be effective in preventing infections caused by GBS, and they pave the way for the use of a similar approach against other pathogenic streptococci. \ua9 2008 by the Infectious Diseases Society of America. All rights reserved