Respiratory syncytial virus increases the virulence of Streptococcus pneumoniae by binding to penicillin binding protein 1a. A new paradigm in respiratory infection

Abstract

Rationale: Respiratory syncytial virus (RSV) and Streptococcus pneumoniae are major respiratory pathogens. Coinfection with RSV and S. pneumoniae is associated with severe and often fatal pneumonia but the molecular basis for this remains unclear.

Objectives: To determine if interaction between RSV and pneumococci enhances pneumococcal virulence.

Methods: We used confocal microscopy and Western blot to identify the receptors involved in direct binding of RSV and pneumococci, the effects of which were studied in both in vivo and in vitro models of infection. Human ciliated respiratory epithelial cell cultures were infected with RSV for 72 hours and then challenged with pneumococci. Pneumococci were collected after 2 hours exposure and changes in gene expression determined using quantitative real-time polymerase chain reaction.

Measurements and main results: Following incubation with RSV or purified G protein, pneumococci demonstrated a significant increase in the inflammatory response and bacterial adherence to human ciliated epithelial cultures and markedly increased virulence in a pneumonia model in mice. This was associated with extensive changes in the pneumococcal transcriptome and significant up-regulation in the expression of key pneumococcal virulence genes, including the gene for the pneumococcal toxin, pneumolysin. We show that mechanistically this is caused by RSV G glycoprotein binding penicillin binding protein 1a.

Conclusions: The direct interaction between a respiratory virus protein and the pneumococcus resulting in increased bacterial virulence and worsening disease outcome is a new paradigm in respiratory infection.

Keywords: G protein; cilia; pneumococcus; respiratory syncytial virus; virulence.

Figures

Figure 1.

The effect of respiratory syncytial virus (RSV) and Streptococcus pneumoniae alone and together on ciliated epithelial cells. (A) The adherence of pneumococci after 1 hour to ciliated epithelial cells previously mock-infected or infected with RSV for 72 hours. (B) The mean (SEM) level of ciliated cells with dyskinetic cilia expressed as a percentage of the total motile ciliated cells after 1 hour. Data were collected from seven independent experiments. Cells were infected with RSV, mock infected followed by pneumococci, or infected with RSV followed by pneumococci. Control cells were treated with bovine serum albumin alone. (C and D) Relative quantification of pneumococcal adherence (C) or toxicity-associated gene expression (D) when exposed to naive (green bars) or RSV-infected (blue bars) human ciliated epithelial cells. The fold changes in gene expression were measured by quantitative real-time polymerase chain reaction and calculated using the 2–∆∆CT method (27). The internal control gene was gyrB and the reference condition was pneumococci exposed to naive respiratory epithelial cells obtained from the same donor. Data were analyzed using a parametric paired t test. A P value of less than 0.05 was statistically significant. n = 5 individual experiments. *P < 0.05; **P < 0.001. (SeeVideos 1A and 1B.)

Figure 2.

Respiratory syncytial virus (RSV) directly binds to Streptococcus pneumoniae. (A) ELISA showing inhibition of pneumococcus binding to an RSV-coated plate by prior treatment of the plate with an anti-RSV G antibody. Bound pneumococci were detected using an anticapsular polysaccharide antibody. (B) Growth curve of pneumococci exposed to bovine serum albumin (BSA; empty squares), RSV (filled circles), or purified RSV G protein (empty circles). (C) Confocal images of pneumococci exposed to BSA, RSV, or RSV G protein. Bacterial preparations were stained with an antibody specific for RSV (green) and an antipneumococcal capsule antibody (red). Areas of antigen colocalization are shown in yellow (and indicated by arrows). Scale bar = 1 µm. (D) Western blot of pneumococcal cell membrane extract incubated with purified RSV G protein. The bound G protein was detected using a monoclonal antibody. The band excised for mass spectrometry identification is indicated by the red box. (E) Peptide fragment alignment of MALDI-time of flight mass spectrometry of the excised band from the Western blot of S. pneumoniae RSV G protein-binding protein obtained after digestion with trypsin. The data show the identification of S. pneumoniae RSV G protein binding protein. The protein sequencing was performed as described in Methods in the online supplement from two individual sequencings, which resulted in 29 peptide matches. Database searching showed that the peptides covered 43% of a 79.7-kD protein that showed a greater than 99% match probability to the S. pneumoniae PBP1A protein. Normal font, no highlight = not sequenced; bold font and yellow highlight = 100% sequence match to PBP1a; bold font and green highlight = random amino acid mismatch. (F) Summary of changes in the transcriptome of S. pneumoniae following incubation with RSV. The data indicate the number of genes in each category and the proportion showing increased (red) or decreased (green). ***Statistically significant change (P < 0.0001).

Figure 2.

Respiratory syncytial virus (RSV) directly binds to Streptococcus pneumoniae. (A) ELISA showing inhibition of pneumococcus binding to an RSV-coated plate by prior treatment of the plate with an anti-RSV G antibody. Bound pneumococci were detected using an anticapsular polysaccharide antibody. (B) Growth curve of pneumococci exposed to bovine serum albumin (BSA; empty squares), RSV (filled circles), or purified RSV G protein (empty circles). (C) Confocal images of pneumococci exposed to BSA, RSV, or RSV G protein. Bacterial preparations were stained with an antibody specific for RSV (green) and an antipneumococcal capsule antibody (red). Areas of antigen colocalization are shown in yellow (and indicated by arrows). Scale bar = 1 µm. (D) Western blot of pneumococcal cell membrane extract incubated with purified RSV G protein. The bound G protein was detected using a monoclonal antibody. The band excised for mass spectrometry identification is indicated by the red box. (E) Peptide fragment alignment of MALDI-time of flight mass spectrometry of the excised band from the Western blot of S. pneumoniae RSV G protein-binding protein obtained after digestion with trypsin. The data show the identification of S. pneumoniae RSV G protein binding protein. The protein sequencing was performed as described in Methods in the online supplement from two individual sequencings, which resulted in 29 peptide matches. Database searching showed that the peptides covered 43% of a 79.7-kD protein that showed a greater than 99% match probability to the S. pneumoniae PBP1A protein. Normal font, no highlight = not sequenced; bold font and yellow highlight = 100% sequence match to PBP1a; bold font and green highlight = random amino acid mismatch. (F) Summary of changes in the transcriptome of S. pneumoniae following incubation with RSV. The data indicate the number of genes in each category and the proportion showing increased (red) or decreased (green). ***Statistically significant change (P < 0.0001).

Figure 3.

Table showing in detail the exact genes that are up-regulated (red) and down-regulated (green) in the transcriptome of S. pneumoniae. SeeFigure 2 for further details.

Figure 3.

Table showing in detail the exact genes that are up-regulated (red) and down-regulated (green) in the transcriptome of S. pneumoniae. SeeFigure 2 for further details.

Figure 4.

Prior exposure to respiratory syncytial virus (RSV) increases pneumococcal virulence in vivo. (A) The survival curve of mice challenged intranasally with 1 × 105 cfu Streptococcus pneumoniae D39 previously exposed to RSV (dark blue), exposed to RSV alone (light blue), or exposed to bovine serum albumin (BSA) alone. The RSV and BSA alone data overlap completely (pale blue). (B) The condition score of MF1 mice 6 hours after challenge with either wild-type S. pneumoniae previously exposed to RSV, exposed to RSV alone, or exposed to pneumococci alone for 2 hours. **Statistically significant change (P < 0.001). (C) Viable counts of S. pneumoniae D39 recovered from the lungs of mice 6 hours after challenge with 1 × 105 cfu S. pneumoniae D39 previously exposed to either BSA (Control Pneumococci) or RSV (RSV+Pneumococci) for 2 hours. nd = no significant difference (P > 0.05); n = 3.