Biofilm antigens drive natural immunity to Streptococcus pneumoniae. - PROJECT SUMMARY Streptococcus pneumoniae (Spn) is a Gram-positive bacterium that resides in the nasopharynx. Colonization is the critical first step in the pathogenesis of this pathobiont, during which Spn forms biofilms that protect against desiccation and host defense mechanisms. Studies on Spn colonization of naïve animals describe damage to the epithelia, sloughing of host cells, exposure of the basement membrane, and alarmin release. Spn takes advantage of this by binding to dying mucosal epithelial cells via Pneumococcal surface protein A (PspA), allowing the bacterium to benefit from released nutrients and, when discharged as respiratory mucous, to avoid death by desiccation. Importantly, most work done to understand how the host immunological response to colonization by Spn develops has been done using naïve animals and antigens/cell lysates from planktonic grown pneumococci. These studies have not taken into consideration the biofilm phenotype, which is the biological state Spn is present in the nasopharynx, and the impact of repeated colonization events. Our preliminary data suggests repeated colonization events result in a robust humoral immune response to proteins produced by biofilm Spn that far exceed their planktonic counterpart. Our hypothesis therefore states that: repetitive Spn colonization results in the generation of a humoral adaptive immune response against biofilm- associated proteins of Spn which influence subsequent colonization events and disease pathogenesis. This proposal will expand our understanding of Spn colonization, host immunity, and has the potential to identify novel candidates for immunization against colonization, the first step of pneumococcal pathogenesis and disease. To test this, Aim 1 will elucidate how successive colonization events with different Spn impact mucosal host- pathogen interactions and immunity. Using a representative murine model of successive colonization involving three distinct Spn strains, we will assess the pathological and morphological differences between successive Spn colonization events using scanning electron microscopy to image mucosal epithelial cells in the nasopharynx of sequentially colonized mice, histological examination of sectioned nares, and immunofluorescent microscopy. Pathological results will be correlated to bacterial burden in nasal elutes. We will determine the changes in the immune response to planktonic versus biofilm Spn by quantitating infiltrating immune cells, measuring antibody titers to biofilm-specific antigens and pneumococcal proteins, and determining which factors generate the strongest antibody response. Aim 2 will determine how antibody production and immunization impedes Spn colonization following repeated exposure events. Our preliminary data indicates that sufficient generation of antibodies to biofilm antigens confers protection against colonization. We will determine how immunization against PspA and Spn biofilm whole-cell lysates protect the host mucosal epithelia from proinflammatory damage and drive the humoral immune response to colonization. Human nasal organoids will be used to validate protection following antibody treatment prior to using our murine repeated Spn colonization model.
