ABSTRACTStreptococcus pneumoniae(Spn) is an asymptomatic colonizer of the human nasopharynx but can also cause invasive diseases in the inner ear, meninges, lung and blood. Although various mechanisms contribute to the effective clearance ofSpn, opsonophagocytosis by neutrophils is perhaps most critical. Upon phagocytosis,Spnis exposed to various degradative molecules, including a family of neutrophil serine proteases (NSPs) that are stored within intracellular granules. Despite the critical importance of NSPs in killingSpn, the bacterial proteins that are degraded by NSPs leading toSpndeath are still unknown. In this report, we identify a 90kDa protein in a purified cell wall (CW) preparation, aminopeptidase N (PepN) that is degraded by the NSP, neutrophil elastase (NE). Since PepN lacked a canonical signal sequence or LPxTG motif, we created a mutant expressing a FLAG tagged version of the protein and confirmed its localization to the CW compartment. We determined that not only is PepN abona fideCW protein, but also is a substrate of NE in the context of intactSpncells. Furthermore, in comparison to wild-type TIGR4Spn, a mutant strain lacking PepN demonstrated a significant hyper-resistance phenotypein vitroin the presence of purified NE as well as in opsonophagocytic assays with purified human neutrophilsex vivo. Taken together, this is the first study to demonstrate that PepN is a CW-localized protein and a substrate of NE that contributes to the effective killing ofSpnby NSPs and human neutrophils.IMPORTANCENeutrophils are innate immune cells needed to effectively clearStreptococcus pneumoniae(Spn). Neutrophil serine proteases (NSPs) are important for killing phagocytosedSpn, however, the identity of theSpnproteins that are degraded by NSPs are unknown. This study identifies aSpncell wall protein, aminopeptidase N (PepN) that is degraded by the NSP, neutrophil elastase (NE). We demonstrate that PepN is abona fidecell wall protein and mutants lacking PepN are significantly more resistant than wild-type to killing by purified NE and human neutrophils. This study demonstrates that PepN is a NE substrate and its degradation contributes to effectiveSpnkilling. By better understanding how neutrophils killSpn, we aim to inform the development of improved therapeutic interventions.