Streptococcus pneumoniae (the “pneumococcus”) is an important human commensal pathogen. A key determinant of pneumococcal fitness and virulence is its ability to produce a protective polysaccharide (PS) capsule which can take the form of >100 biochemically distinct “serotypes”. Pneumococcal capsule PS conjugate vaccines (PCVs) induce protective antibodies that mediate opsonophagocytic killing (OPK) of pneumococci and have effectively reduced the global burden of disease caused by serotypes included in vaccines. Despite this success, immunized people occasionally experience breakthrough infections by PCV serotypes, and the cause for these cases of “vaccine failure” remains unclear. Investigation of vaccine failure has largely focused on host factors and ineffective antibody response, while microbiological aspects have gone largely unaddressed. Furthermore, closely-related serotypes 19A and 19F are the serotypes most commonly implicated in vaccine failure cases, but few studies have evaluated their role in this phenomenon. Appreciating the breadth of capsule malleability and its impact on clinical outcomes, we are examining a potential link between serotype 19A/19F capsule variants, evasion of anti-capsule immune responses, and vaccine failure. Preliminary analyses identified multiple candidate mechanisms through which polymorphisms in the 19A/19F capsule polymerase Wzy can mediate considerable capsule variability while preserving most capsule features. We also found a strain that was serotyped as “19F” by conventional methods, but in fact produces a novel capsule PS structure, herein called 19x. Thus, the full diversity of 19A/19F-like capsule types is yet to be defined. As even small changes in capsule structure can abrogate cross-protective immunity, it is possible that some variants, which are indistinguishable from 19A/19F pneumococci in conventional serotyping methods, can nonetheless evade OPK by anti-19A/19F capsule antibodies in vaccinated individuals and, thus, spur the vaccine failure attributed to these serotypes. In this R21 proposal, we will perform directed mutagenesis to test the impact of Wzy polymorphisms on 19A/19F capsules structure and test the effect of these putative capsule changes on evading anti-capsule antibody-mediated OPK in vitro (Aim 1). We will also structurally/genetically/antigenically characterize the novel 19x capsule type and perform bioinformatics analysis of expansive genomic databases with the goal of identifying other putative capsule variants found among immunized populations (Aim 2). Importantly, tools and concepts developed here will fuel future investigation of the impact capsule PS malleability has in additional pneumococcal serotypes and other medically important bacterial pathogens that employ similar pathways for glycan synthesis. Independent of glycobiological advances, elucidation of the molecular basis of 19A/19F Wzy enzymatic specificity would immediately improve the precision of the molecular ...