Type I Interferon Signaling Is a Common Factor Driving Streptococcus pneumoniae and Influenza A Virus Shedding and Transmission

Abstract

The dynamics underlying respiratory contagion (the transmission of infectious agents from the airways) are poorly understood. We investigated host factors involved in the transmission of the leading respiratory pathogen Streptococcus pneumoniae Using an infant mouse model, we examined whether S. pneumoniae triggers inflammatory pathways shared by influenza A virus (IAV) to promote nasal secretions and shedding from the upper respiratory tract to facilitate transit to new hosts. Here, we show that amplification of the type I interferon (IFN-I) response is a critical host factor in this process, as shedding and transmission by both IAV and S. pneumoniae were decreased in pups lacking the common IFN-I receptor (Ifnar1^-/- mice). Additionally, providing exogenous recombinant IFN-I to S. pneumoniae-infected pups was sufficient to increase bacterial shedding. The expression of IFN-stimulated genes (ISGs) was upregulated in S. pneumoniae-infected wild-type (WT) but not Ifnar1^-/- mice, including genes involved in mucin type O-glycan biosynthesis; this correlated with an increase in secretions in S. pneumoniae- and IAV-infected WT compared to Ifnar1^-/- pups. S. pneumoniae stimulation of ISGs was largely dependent on its pore-forming toxin, pneumolysin, and coinfection with IAV and S. pneumoniae resulted in synergistic increases in ISG expression. We conclude that the induction of IFN-I signaling appears to be a common factor driving viral and bacterial respiratory contagion.IMPORTANCE Respiratory tract infections are a leading cause of childhood mortality and, globally, Streptococcus pneumoniae is the leading cause of mortality due to pneumonia. Transmission of S. pneumoniae primarily occurs through direct contact with respiratory secretions, although the host and bacterial factors underlying transmission are poorly understood. We examined transmission dynamics of S. pneumoniae in an infant mouse model and here show that S. pneumoniae colonization of the upper respiratory tract stimulates host inflammatory pathways commonly associated with viral infections. Amplification of this response was shown to be a critical host factor driving shedding and transmission of both S. pneumoniae and influenza A virus, with infection stimulating expression of a wide variety of genes, including those involved in the biosynthesis of mucin, a major component of respiratory secretions. Our findings suggest a mechanism facilitating S. pneumoniae contagion that is shared by viral infection.

Keywords: influenza virus; interferon; pneumococcus; transmission.

FIG 1

Type I interferon is necessary for and promotes high-level shedding of S. pneumoniae and IAV. WT and Ifnar1^−/− pups were infected i.n. with 10³ CFU S. pneumoniae (Spn), 250 PFU IAV-x31, or S. pneumoniae plus IAVx31. (A and B) Ifnar1^−/− pups shed significantly fewer bacteria than WT pups (A), and Ifnar1^−/− pups shed significantly less IAV than WT pups (B). (C) WT and Ifnar1^−/− pups first received IAV and then S. pneumoniae. Ifnar1^−/− pups shed significantly fewer bacteria than WT pups. (D and E) Exogenous recombinant IFN-α2 or IFN-β increase pneumococcal shedding from WT but not Ifnar1^−/− mice. WT and Ifnar1^−/− pups were infected i.n. with 10³ CFU S. pneumoniae and daily received either 1,000 IU of recombinant mouse IFN-α2 or 1,000 to 5,000 IU of recombinant mouse IFN-β or vehicle control (0.1% BSA-PBS) by i.n. instillation. Treatment of WT pups with rIFN-α2 (D) or rIFN-β (E) was sufficient to increase pneumococcal shedding over the baseline. Shedding data are shown as a Tukey box-and-whisker plot, with outliers shown as symbols. Each symbol represents the value from an individual pup on a single day. n ≥ 8 pups/group. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Mann-Whitney test).

FIG 2

Pneumococci are shed and stimulate IFN-I at 21 days p.i. in a pneumolysin-dependent manner. (A and B) WT pups were infected i.n. with 10³ CFU S. pneumoniae; mock-treated mice received PBS. RNA was isolated from URT lavages on day 21 p.i. and analyzed by RNA-seq. Shown is a heat map of 543 genes identified as the interferome, comparing S. pneumoniae to mock infection (A) or S. pneumoniae to the S. pneumoniae ply mutant strain (B), demonstrating relative gene expression as log₂ fold change, with increased expression in red and decreased expression in blue. n = 5 mice per group. (C) RNA was isolated from URT lavages on day 21 p.i. and analyzed by qRT-PCR. At 21 days p.i., S. pneumoniae-infected mice showed significantly increased expression of the ISGs Ifit2, Mx1, and Oasl2 over mock-infected mice. This increased expression was seen only in WT and not Ifnar1^−/− mice. (D to F) WT pups were infected i.n. with 10³ CFU S. pneumoniae T4 or T23F (D), or WT and Ifnar1^−/− pups were infected i.n. with 10³ CFU S. pneumoniae T4 or an isogenic T4 ply mutant (E and F). (D) T4 and T23F are shed 21 days p.i., which correlates with gene expression data. (E) At 21 days p.i., there are more high-shedding events (>200) in S. pneumoniae-infected mice than mice infected with the isogenic S. pneumoniae ply mutant. (F) At 21 days p.i., there is no difference in the number of high-shedding events between S. pneumoniae and S. pneumoniae ply mutant strains in Ifnar1^−/− mice. Gene expression data are log₁₀ transformed; each symbol represents the value from an individual pup, and the line represents the mean. Comparisons (Mann-Whitney test) are to mock-infected mice of the respective genotypes. Shedding data are shown as Tukey box-and-whisker plots, with outliers shown as symbols. Each symbol represents the value from an individual pup on a single day. n ≥ 8 pups/group. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Mann-Whitney test); #, P = 0.03 (one-tailed Fisher’s exact test).

FIG 3

S. pneumoniae and IAV stimulate IFN-I signaling at 48 h p.i. WT and Ifnar1^−/− pups were infected i.n. with 10³ CFU the S. pneumoniae T4 or T4Δply mutant, 250 PFU IAV, or T4 plus IAV; mock-infected mice received PBS. RNA was isolated from URT lavages 48 h p.i. and analyzed by qRT-PCR. S. pneumoniae-infected mice showed significantly increased expression of the ISGs Ifit2, Mx1, and Oasl2 compared to that of mock-infected mice. This increased expression was seen only in WT and not Ifnar1^−/− mice. The stimulation of ISGs by S. pneumoniae was ply dependent. Gene expression data are log₁₀ transformed; each symbol represents the value from an individual pup. Comparisons (Mann-Whitney test) are to mock-infected mice of the respective genotype. n ≥ 5 pups/group. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 4

S. pneumoniae colonization increases nasal secretions of pups. (A and B) WT and Ifnar1^−/− pups were infected i.n. with 10³ CFU S. pneumoniae or 250 PFU IAV; mock-infected mice received PBS. RNA was isolated from URT lavages 48 h (A) or 21 days (B) p.i. and analyzed by qRT-PCR. S. pneumoniae-infected mice showed significantly increased expression of the ISG sialyltransferase St3gal1 over mock-infected mice. This increased expression was seen only in WT and not Ifnar1^−/− mice. The stimulation of ISGs by S. pneumoniae was also ply dependent. Gene expression data are log₁₀ transformed; each symbol represents the value from an individual pup, and the line represents the mean. Comparisons (Mann-Whitney test) are to mock-infected mice of the respective genotype. n ≥ 5 pups/group. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Pups infected with IAV or S. pneumoniae secreted more sialic acid from their nares than pups that received PBS. IAV comparisons are days 14 to 18 p.i. to matched PBS controls; S. pneumoniae comparisons are of days 9 to 12 p.i. The increase in nasal secretions from S. pneumoniae-infected pups is IFN-I dependent, as there was no difference detected in sialic acid shed from infected Ifnar1^−/− pups compared to PBS controls (days 9 to 12 p.i.). WT and Ifnar1^−/− pups were infected i.n. with either 10³ CFU S. pneumoniae T23F P2636 (neuraminidase mutant) or 250 PFU IAV-x31 or received PBS. Band integrated pixel density data are for individual pups, with the bar indicating the median. Each symbol represents the value from an individual pup on a single day. n ≥ 3 pups/group. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Mann-Whitney test).

FIG 5

Lack of IFN-I signaling reduces transmission of IAV and S. pneumoniae. (A) WT and Ifnar1^−/− index pups were infected i.n. with 10³ CFU S. pneumoniae at a ratio of 6 index to 20 contact in WT litters or 5 index to 18 contact in Ifnar1^−/− mice in three separate litters. The S. pneumoniae transmission rate and bacterial burden were higher in WT than Ifnar1^−/− contact mice. (B) WT and Ifnar1^−/− index pups were infected i.n. with 250 PFU IAV at a ratio of 12 index to 22 contact in WT litters or 10 index to 28 contact in Ifnar1^−/− mice in five separate litters. The IAV titer was higher in WT than Ifnar1^−/− contact mice. Colonization and titer data are for individual pups, with the line indicating the geometric mean. Each symbol represents the value from an individual pup on a single day. n ≥ 5 pups/group. *, P < 0.05; ***, P < 0.001 (Mann-Whitney test). The dotted line shows the limit of detection.