I. PROJECT SUMMARY Streptococcus pneumoniae (Spn) colonizes the human nasopharynx during early childhood. This opportunistic pathogen causes ~15 million cases of invasive pneumococcal disease (IPD) and ~500,000 deaths in children worldwide each year. While pneumococcal conjugate vaccine (PCV) development has impacted disease incidence, the emergence and spread of antimicrobial resistance in Spn, an NIAID priority, has complicated IPD treatment regimens. Consequently, the CDC and WHO list Spn amongst the antibiotic-resistant priority pathogens. In particular, antibiotic resistance in Spn is increasingly due to acquisition of >20 kb, Tn916-like integrative and conjugative elements (ICEs), most notably Tn2009, Tn6002, and Tn2010. The specific mechanisms facilitating the wide spread of these large ICEs among Spn have not been elucidated. In other bacteria, ICEs are transferred via transposition with the formation of circular intermediates and a conjugative type 4 secretion system. Although conjugative genes are conserved within Spn ICEs, their roles in Spn ICE transference have not been established. ICEs are unusually large elements for efficient in vitro transformation of naturally competent Spn, which typically acquire ~2-6 kb DNA fragments. Accordingly, we confirmed that competent Spn strains did not uptake ICE DNA (<10-9) under in vitro conditions. Yet, when two Spn strains were co-inoculated in an ex vivo bioreactor system, forming a mixed biofilm on human nasopharyngeal cells, the transference of the entire ICE occurred at frequencies up to 10-4, which likely recapitulates the scenario during in vivo host colonization. In Aim 1, I propose to use the bioreactor system and a collection of various mutant strains to decipher the mechanism(s) responsible for this five-orders of magnitude increase in dissemination of large ICEs among naturally competent Spn strains. Specifically, we will assess the roles of ICE-encoded conjugation genes, competence factors, and the DNA uptake apparatus. Conjugative and competence gene expression will be measured via qRT-PCR and ICE circular intermediates, if present, will also be quantified using qPCR. Conjugative protein expression will be investigated by Western blots. The role of extracellular membrane vesicles (EVs) in the delivery of >20 kb ICE DNA will also be evaluated. Whether EVs carry ICE DNA will be examined by ICE-targeting qPCR and purified biofilm-derived EVs will be tested via in vitro transformations. In Aim 2, genomic characteristics of multi-drug resistance-conferring ICE elements will be defined in the global Spn population. Using innovative bioinformatics tools and approaches, publicly available whole genome sequences from global Spn isolates will be examined to identify new ICEs and novel ICE insertion sites in addition to previously defined sites from a regional isolate collection. Analyses will be based on pre- and post-vaccine periods, vaccine or non-vaccine serotypes, and clonal complexes. Pr...