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.
