Contributions of pneumococcal virulence factors to secondary Streptococcus pneumoniae infection following influenza infection

Abstract

Influenza infection increases susceptibility to secondary infection with Streptococcus pneumoniae resulting in significantly increased morbidity and mortality. Whereas viral contributions to this synergism have been explored, little is known concerning contributions of the bacterium, specifically those provided through bacterial virulence factors. To assess the contributions of the known pneumococcal virulence factors hyaluronidase (Hyl), neuraminidase (NanA) and pneumococcal surface protein A (PspA) to secondary S. pneumoniae infection following influenza infection, mutants lacking these proteins were administered with wildtype pneumococci in a competitive growth model. Whereas mutants lacking the Hyl and NanA proteins did not exhibit attenuation, mutants lacking PspA were severely attenuated in mice without influenza infection and significantly more so in mice with a prior influenza infection. Additionally, mice received intranasal immunization with recombinant PspA protein and subsequently received primary and secondary challenges with serotypes 2, 3 and 4 pneumococci. Immunization with PspA significantly reduced bacterial burdens of all three challenge serotypes in primary and secondary pneumococcal infection and significantly reduced lung damage markers in mice receiving secondary pneumococcal challenges. In addition to known virulence factors, two surface-exposed proteins, Spr0075 and Spr1345, were assessed for virulence contributions to primary and secondary pneumococcal infections. Mutants lacking Spr0075 or Spr1345 were found to be severely attenuated in both primary and secondary pneumococcal challenges. Whereas immunization with either recombinant Spr0075 or Spr1345 significantly reduced primary pneumococcal burdens, only immunization with Spr0075 significantly reduced secondary pneumococcal burdens. Together these results indicate virulence contributions to both primary and secondary pneumococcal challenges for the PspA, Spr0075 and Spr1345 proteins. However, whereas immunization with PspA and Spr0075 significantly reduced both primary and secondary pneumococcal burdens, immunization with Spr1345 did not significantly impact secondary pneumococcal burdens. This result illustrates that a virulence contribution and/or an ability to protect against primary infection does not necessarily translate into a protein's capacity to protect against secondary infection. The results presented here are the first experimental evidence demonstrating virulence roles for the Spr0075 and Spr1345 proteins and are the first reports of immunization with pneumococcal proteins, specifically PspA and Spr0075, providing protection against secondary pneumococcal infection following influenza.