Host-to-Host Transmission of Streptococcus pneumoniae Is Driven by Its Inflammatory Toxin, Pneumolysin

Abstract

Host-to-host transmission is a critical step for infection. Here we studied transmission of the opportunistic pathogen Streptococcus pneumoniae in an infant mouse model. Transmission from nasally colonized pups required high levels of bacterial shedding in nasal secretions and was temporally correlated with, and dependent upon, the acute inflammatory response. Pneumolysin, a pore-forming cytotoxin and major virulence determinant, was both necessary and sufficient to promote inflammation, which increased shedding and allowed for intralitter transmission. Direct contact between pups was not required for transmission indicating the importance of an environmental reservoir. An additional in vivo effect of pneumolysin was to enhance bacterial survival outside of the host. Our findings provide experimental evidence of a microbial strategy for transit to new hosts and explain why an organism expresses a toxin that damages the host upon which it depends.

Keywords: bacteria; colonization; infection; pneumococcus; pneumolysin; shedding; toxin; transmission.

Figure 1. Pneumococcal Shedding Correlates with Upper Respiratory Tract Inflammation

(A) 4-day-old pups were intranasally (IN) inoculated with ~2,000 CFUs S. pneumoniae strain T4S. (B) Daily shedding was collected and quantified from nasal secretions on the days shown with median values indicated and each symbol representing the CFU observed from a single pup. Statistical analysis compares shedding on ages 6 and 9 days. Dashed line represents the 300 CFU threshold level described in the Results. (C) Colonization density for T4S in cultures of upper respiratory tract (URT) lavages obtained from pups at the age indicated with the median value shown. (D) Numbers of neutrophils as determined by flow cytometry (CD45⁺, CD11b⁺, and Ly6G⁺ events) in URT lavages obtained at the age indicated in colonized pups. Values are ±SEM (n = 5–11). (E) The URT was lavaged with RLT RNA lysis buffer to isolate RNA from the epithelium to make cDNA from T4S colonized pups. Gene expression relative to PBS (mock)-inoculated mice was measured by qRT-PCR for the chemokine/cytokine shown. Values are ±SEM (n = 5–8). (F) IL-1β measured by ELISA in URT lavages obtained at age 7 days for T4S or PBS (mock)-colonized pups. Values are ±SEM (n = 6–11). *p < 0.05, **p < 0.01.

Figure 2. Pneumococcal Shedding Requires URT Inflammation

(A–C) Pups colonized at age 4 days with T4S were treated IN twice daily with dexamethasone (Dex) or PBS-vehicle control. Daily shedding quantified in secretions from ages 5 to 9 days from nasal secretions with median values indicated and each symbol representing the CFU observed from a single pup on a single day (A). Colonization density in URT lavages obtained from Dex- or PBS-treated pups at age 9 days with the median value shown (B). Gene expression for the Dex-treated relative to PBS-treated pups at age 9 days measured by qRT-PCR for the chemokine/cytokine shown (C). Values are ±SEM (n = 5–7). (D–F) WT or congenic pafr^−/− pups were colonized at age 4 days with T4S. Daily shedding quantified in secretions from ages 5 to 9 days from nasal secretions with median values indicated and each symbol representing the CFU observed from a single pup (D). Colonization density in URT lavages obtained at age 9 days with the median value shown (E). Gene expression in colonized pafr^−/− pups at age 9 days relative to PBS (mock)-inoculated pups measured by qRT-PCR for the chemokine/cytokine shown (F). Values are ±SEM (n = 7–9). Dashed line represents the 300 CFU threshold level described in the Results. **p < 0.01. ****p < 0.0001.

Figure 3. Recognition of Pneumococci and Pneumococcal Products Increases URT Inflammation and Promotes Shedding

(A–D) WT or congenic tlr2^−/− pups were colonized at age 4 days with T4S. Daily shedding quantified in secretions from ages 5 to 9 days from nasal secretions with median values indicated and each symbol representing the CFU observed from a single pup on a single day (A). Dashed line represents the 300 CFU threshold level described in the Results. Colonization density in URT lavages obtained at age 9 days with the median value shown (B). Numbers of neutrophils as determined by flow cytometry (CD45⁺, CD11b⁺, and Ly6G⁺ events) in URT lavages obtained from tlr2^−/− or PBS (mock)-inoculated pups at age 7 days (C). Values are ±SEM (n = 8–11). Gene expression in colonized tlr2^−/− pups relative to PBS (mock)-inoculated pups at age 7 days measured by qRT-PCR for the chemokine/cytokine shown (D). Values are ±SEM (n = 8). (E–H) WT pups were colonized with the ply− strain at age 4 days. From ages 4 to 8 days, pups were given a daily IN dose (100 ng/pup) of purified pneumolysin (Ply), a non-pore-forming toxoid with the W433F point mutation (PdB), or PBS-vehicle control. Daily shedding quantified in secretions from ages 5 to 9 days from nasal secretions with median values indicated and each symbol representing the CFU observed from a single pup (E). Colonization density in URT lavages obtained at age 9 days with the median value shown (F). Numbers of neutrophils as determined by flow cytometry (CD45⁺, CD11b⁺, and Ly6G⁺ events) in URT lavages at age 9 days (G). Values are ±SEM (n = 8). Gene expression relative to PBS (mock)-inoculated pups measured by qRT-PCR for the chemokine/cytokine shown at age 9 days (H). Values are ±SEM (n = 10–18). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. Pneumolysin Is Required for Robust Inflammation and Shedding

WT pups were colonized at age 4 days with T4S, ply−, a point mutant deficient in pore formation (ply_W433F), or a corrected mutant (ply+) and monitored over the period of peak inflammation until age 7 days. (A) Daily shedding quantified in secretions from nasal secretions with median values indicated and each symbol representing the CFU observed from a single pup on a single day. Horizontal dashed line represents the 300 CFU threshold level described in the Results. T4S and ply− were tested among littermates and the proportion of shedding events >300 CFUs compared for significance using the Fisher’s exact test. (B) Colonization density in URT lavages obtained with the median value shown. (C) Numbers of neutrophils as determined by flow cytometry (CD45⁺, CD11b⁺, and Ly6G⁺ events) in URT lavages. (Data for T4S is also shown in Figure 1C and provided here for comparison.) Values are ±SEM (n = 12–19). (D) Gene expression of IL-1β relative to PBS (mock)-inoculated pups measured by qRT-PCR. Values are ±SEM (n = 10–19). *p< 0.05, **p < 0.01, ***p < 0.001.

Figure 5. Pneumolysin Increases Host-to-Host Transmission

(A) At age 4 days, half the pups in a litter were colonized with the strain indicated (index pups, I) and the other half left uncolonized (contact pups, C). At age 14 days, nasal lavages were obtained to quantify colonization density with each pup, and median values are shown. Intralitter transmission is shown by acquisition of the strain by contact pups. (B) Index pups colonized with the ply− strain were treated daily from age 4 days with either purified Ply (100 ng/dose) or PBS-vehicle control. For IAV co-infection, both index and contact pups were infected with strain x31 at age 8 days. (C) Summary of transmission data for the strain indicated. Unless otherwise indicated, the pups were C57/Bl6 (WT). The p value was calculated using the Fisher’s exact test. * in comparison to ply− without IAV. ns, non-significant.

Figure 6. Experiments with IAV Co-infection Showing Increased Shedding with Pneumolysin Expression and the Route of Pup-to-Pup Transmission

(A) WT pups were colonized at age 4 days with the T4S or ply− strain and at age 8 days co-infected IN with IAV virus strain x31. (B) Daily shedding was quantified in secretions from ages 9 to 14 days from nasal secretions with median values indicated and each symbol representing the CFU observed from a single pup on a single day. Dashed line represents the 300 CFU threshold level described in the Results. *p < 0.05. (C) Colonization density in URT lavages obtained with the median value shown. (D–F) WT index pups in one cage were colonized at age 4 days with T4S. At age 8 days, both index pups and contact pups (housed in a separate cage) were inoculated with IAV. Colonization was measured in cultures of nasal lavages at age 14 days after index and contact pups were switched between cages 2–3 times/day (D), cages containing the bedding material were switched 3 times/day (E), or the dams only were switched between cages (F). Horizontal line indicates limit of detection.

Figure 7. Pneumolysin Expression Promotes Bacterial Survival and Infectivity outside of the Host

(A–C) To compare ex vivo survival, we incubated pneumococci in PBS and viable counts determined at the times indicated. Prior to incubation in PBS, bacteria were obtained from nasal lavages obtained at age 9 days from mice colonized at age 4 days with the strain indicated (A), broth culture in nutrient medium (B), or nasal lavages at age 9 days from mice colonized at age 4 days with ply− and then treated daily with an IN dose of Ply or PdB (100 ng/day) or PBS control (C). Statistical comparisons are to the group(s) without pneumolysin. *p < 0.05, **p < 0.01. (D) Nasal lavages obtained from T4S or ply− colonized mice were diluted to the desired density in PBS and incubated for 4 hr at 30° C. Aliquots then used to compare the infectious dose by IN inoculation of 4-day-old pups. Colonization density at age 6 days is shown relative to the inoculum size.