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1.
Figure 2

Figure 2. PsrP promotes the formation of bacterial aggregates in vivo.. From: The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms.

SEM images of bronchial and alveolar epithelial cells following infection with TIGR4 (WT) and T4 ΔpsrP (ΔpsrP). White arrows point at attached bacteria. Note the presence of WT bacteria in large aggregates of various sizes.

Carlos J. Sanchez, et al. PLoS Pathog. 2010 August;6(8):e1001044.
2.
Figure 1

Figure 1. Hypothetical model of PsrP on the surface of S. pneumoniae.. From: The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms.

A) Domain structure of PsrP: N-terminal signal peptide (S); serine-rich repeat motif 1 (SAS[A/E/V]SAST X 11) (SRR1); basic region (BR); serine-rich repeat motif 2 (SRR2); and the cell wall anchoring domain (CW) at the C-terminus. B) Illustration of PsrP on the bacterial surface. Based on the structural organization of PsrP and studies demonstrating that the BR domain binds to K10 on lung cells [13], we propose that the CWAD attaches the protein to the cell wall, while the long glycosylated SRR2 domain serves to extend BR through the capsular polysaccharide to mediate interactions.

Carlos J. Sanchez, et al. PLoS Pathog. 2010 August;6(8):e1001044.
3.
Figure 7

Figure 7. Recombinant BR interacts with pneumococci that carry amino acids 122–166 of PsrP.. From: The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms.

A) The designated recombinant PsrP constructs were expressed and purified from E. coli. B) Micrographs of FITC-labeled bacteria following their incubation with CY3-labeled rBR or the designated truncated versions. Note that only Cy3-labeled rBR and rBR.A bound to TIGR4. Moreover, neither bound to T4 ΔpsrP. This suggests that recombinant BR binds to PsrP on the bacteria surface. C) Far Western blot examining the interaction of Gst-BR with cell lysates from E. coli expressing assorted rBR constructs spotted on a membrane. D) Co-immunoprecipitation of Gst-BR (65 kDa) from spiked E. coli lysates expressing His-tagged rBR constructs. E) Far Western blot examining the interaction of Gst-BR with purified rBR constructs and a synthesized peptide corresponding to AA 122–166. F) Far Western blot examining the interaction of a glycosylated PsrP construct purified from S. pneumoniae, PsrPSRR2(33)-HIS with glycosylated PsrP constructs expressed in TIGR4.

Carlos J. Sanchez, et al. PLoS Pathog. 2010 August;6(8):e1001044.
4.
Figure 8

Figure 8. Incubation of bacteria with AA 122–166 impairs bacterial aggregation but not adhesion to cells.. From: The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms.

A) Adhesion of TIGR4 following pre-incubation of A549 cells with media containing 1.0 µM of the designated rBR constructs. All values were normalized against cells incubated with BSA (163±4 bacteria/106 cells). TIGR4 in media containing 1 µM BR.C or the synthesized peptide 122–126 was used to inoculate and grow biofilms in the line biofilm model. After 3 days, biofilms within the lines were extruded. B) Representative photograph of the exudate suspension immediately following its collection and staining with CV. Images are representative of at least 3 experiments. C) Optical density (OD540) of bacterial exudates. D) Levels of protein in bacteria line exudates. Statistical analyses were performed using a Student's t-test. Error bars denote standard error.

Carlos J. Sanchez, et al. PLoS Pathog. 2010 August;6(8):e1001044.
5.
Figure 5

Figure 5. PsrP promotes bacterial aggregation in a line biofilm model.. From: The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms.

Mid-logarithmic growth phase TIGR4 (WT) and T4 ΔpsrP (ΔpsrP) were used to inoculate 1 meter of a 0.8 mm diameter silicone-lined plastic tubing. After 3 days, biofilms within the lines were extruded. A) Representative photograph of the exudate suspension immediately following its collection. B) Optical density (OD540) of bacterial exudates. C). Microscopic images of CV stained bacteria extracted from the lines. Note the formation of aggregates by TIGR4 but not T4 ΔpsrP. D) Levels of protein in bacteria line exudates as determined by bicinchoninic acid assay (BCA) following detergent lysis of the bacteria. Images are representative of at least 3 experiments. Statistical analyses were performed using a two-tailed Student's t-test. Error bars denote standard error.

Carlos J. Sanchez, et al. PLoS Pathog. 2010 August;6(8):e1001044.
6.
Figure 3

Figure 3. Frequency of bacterial aggregates in the nasopharynx and lungs.. From: The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms.

A) Micrographs of TIGR4 (WT) and T4 ΔpsrP (ΔpsrP) Gram-stained bacteria from either BAL or nasal lavage (IN) elutes. Images were taken at 400× magnification. Images are not representative of the total bacteria population, but instead are shown to demonstrate the typical pneumococcal aggregate containing at least 10 or more individual diplococci (10+). B) Actual percentages of pneumococcal aggregate based on size in the nasopharynx and C) lungs following counting of >100 randomly selected CFUs per biological replicate. Note that TIGR4 had significantly greater levels of 2–9 and 10+ aggregates compared to T4 ΔpsrP. Furthermore, while 10+ aggregates were observed in mice infected with T4 ΔpsrP, albeit infrequently, the largest of these aggregates were not comparable in size to those formed by TIGR4. Statistical analyses were performed using a Student's t-test.

Carlos J. Sanchez, et al. PLoS Pathog. 2010 August;6(8):e1001044.
7.
Figure 9

Figure 9. Antibodies to the BR domain but not the serine-rich motif block intra-species bacterial interactions.. From: The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms.

THB was supplemented with either a 1∶1000 dilution of naïve rabbit serum (control), rabbit antiserum from rabbits immunized with a SASASASTSASASAST peptide designed after the SRR motif, or rabbit antiserum to recombinant BR. A) Micrographs of CV stained bacteria extruded from the biofilm lines. B) Optical density of bacterial exudates. C) Levels of protein in bacterial line exudates as determined by BCA analysis. Images are representative of at least 3 independent experiments. Statistical analyses were performed using a two-tailed Student's t-test. Number sign denotes statistical significance versus whole serum. Asterisks denote statistical significance versus anti-SRR serum. Error bars denote standard error.

Carlos J. Sanchez, et al. PLoS Pathog. 2010 August;6(8):e1001044.
8.
Figure 4

Figure 4. Deletion of psrP alters bacterial interactions in mature biofilms but not during early biofilm attachment.. From: The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms.

A) Attachment of TIGR4 (WT) and T4 ΔpsrP (ΔpsrP) to the bottom of 96-well polystyrene microtiter plate in an early biofilm model. Biofilm biomass was determined using crystal violet (CV540) stain as described in the methods. B) Micrographs of mature TIGR4 and T4 ΔpsrP biofilms grown in a flow cell under once-through flow conditions for 3 days. Bacteria were visualized with Live/Dead BacLight stain using an inverted confocal laser scanning microscope at 400× magnification. C) Quantitative analysis of the biofilms was performed using COMSTAT image analysis software. All experiments were performed in triplicate. Statistical analyses were performed using a two-tailed Student's t-test. For panel C error bars denote standard error.

Carlos J. Sanchez, et al. PLoS Pathog. 2010 August;6(8):e1001044.
9.
Figure 10

Figure 10. The SRRPs of S. gordonii and S. aureus promote bacterial aggregation.. From: The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms.

Light microscopic images of CV stained A) S. gordonii M99 and B) S. aureus ISP479C and their respective isogenic SRRP mutants following 24 hours of growth in a 96-well polystyrene microtiter plate early biofilm model. B) Average biomass of early biofilms as determined by CV540 analyses. Error bars denote standard deviation. C) Microscopic images of bacteria extruded from the biofilm lines after 3 days for S. gordonii and 1 day for S. aureus. D) Measurement of optical density of bacterial exudates collected from the biofilm lines. Error bars denote standard deviation. E) Far-western examining the intra- and inter-species specificity of the NR domains of S. pneumoniae, S. gordonii and S. aureus. Whole cell lysates from S. gordonii, S. aureus, and their respective isogenic SRRP mutants were spotted onto nitrocellulose membranes and probed with these GST-tagged proteins. Images are representative of three individual experiments. For panels B) and D) statistical analyses were performed using a Student's t-test.

Carlos J. Sanchez, et al. PLoS Pathog. 2010 August;6(8):e1001044.
10.
Figure 6

Figure 6. The BR domain of PsrP mediates intra-species bacterial interactions.. From: The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms.

Encapsulated and unencapsulated mutants of TIGR4, lacking PsrP (T4 ΩpsrP, T4R ΩpsrP, respectively), were complemented with plasmids expressing: a truncated version of PsrP having only 33 SRR2 repeats (PsrP(33)), the truncated version of PsrP missing the BR domain (PsrP(33)-BR), or with the empty expression vector (pNE1). A) Microscopic images of CV stained bacteria isolated from the line biofilm model. B) Optical density (OD620) of biofilm line exudates. C) Biomass of the biofilms as determined by protein levels using the BCA assay. D) Far Western analyses of recombinant BR interactions with membrane-bound truncated versions of PsrP expressed in S. pneumoniae. All images are representative of at least 3 independent experiments. Statistical analyses were performed using 1-Way ANOVA analysis. Error bars denote standard error. For panel B and C asterisks denote statistical significance versus WT; hash sign denotes statistical significance versus the empty vector control.

Carlos J. Sanchez, et al. PLoS Pathog. 2010 August;6(8):e1001044.

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