Streptococcus pneumoniae modulates reassortment of Influenza A Virus in a Pneumolysin dependent manner

Influenza A viruses (IAV) can undergo rapid evolution by acquisition of new genes through reassortment between IAV strains leading to immune evasion, antiviral resistance, and change in host range. Understanding of the factors that can enhance or reduce reassortment frequency therefore has implications for protection of individual and public health. Reassortment requires co-infection by two or more viral particles to the same host cell. Prior studies had identified the potential for IAV particles to aggregate on the bacterial surface of Streptococcus pneumoniae and other respiratory pathobionts, suggesting that bacterial cells could serve as a method to aggregate IAV particles and potentially facilitate reassortment. To test this hypothesis, a temperature sensitive, oseltamivir resistant viral strain was generated and used in in vivo and in vitro co-infection experiments with wild type virus in the presence of live and killed bacterial cells. While killed pneumococcal cells can enhance IAV reassortment frequency in a density-dependent manner, live pneumococci cannot, suggesting a product produced by viable pneumococci inhibits viral reassortment. Genetic deletion of the pneumococcal cholesterol-dependent cytolysin, Pneumolysin (Ply), in combination with inhibition of inflammatory signaling induced by Ply, restored the enhancement of reassortment to the levels seen with killed pneumococci. The results of this work suggest that the bacterial cells that colonize the human upper respiratory tract can have a complex role in modulating IAV reassortment frequency. Bacterial cells are capable of facilitating enhancement of viral reassortment, however, bacterial products can negate these effects. This work demonstrates the role of one such interaction with Ply inhibiting the enhanced reassortment otherwise conferred by S. pneumoniae cells. Overall, this suggests a new model whereby the human nasopharyngeal microbial community which differs from individual to individual and across the lifespan can impact IAV evolutionary dynamics, with some bacterial cells and metabolites increasing and others decreasing IAV reassortment frequency. SummaryInfluenza A virus can evolve rapidly by exchanging genes between viral strains, leading to vaccine failure, spread of antiviral resistance, and potential pandemic emergence. Bacterial cells may be able to increase the frequency of viral genetic exchange through concentration of viral particles on the bacterial cell surface. Prior findings had shown that Streptococcus pneumoniae, a frequent colonizer of the human upper respiratory tract, can directly bind to IAV particles. This work showed in cell culture and animal models that killed S. pneumoniae cells can increase viral genetic exchange. However, live bacteria capable of producing the bacterial toxin Pneumolysin are unable to promote viral genetic exchange. This suggests that bacterial modulation of viral evolutionary dynamics is a complex interaction whereby bacterial cells and products can both enhance and reduce viral genetic exchange, and the human nasopharyngeal microbial community which differs from person to person and on human age could have a role in Influenza A virus evolutionary dynamics.