24Streptococcus pneumoniae (Spn) is an asymptomatic colonizer of the human nasopharynx 25 but can also cause invasive diseases in the inner ear, meninges, lung and blood. Although various 26 mechanisms contribute to the effective clearance of Spn, opsonophagocytosis by neutrophils is 27 perhaps most critical. Upon phagocytosis, Spn is exposed to various degradative molecules, 28 including a family of neutrophil serine proteases (NSPs) that are stored within intracellular 29 granules. Despite the critical importance of NSPs in killing Spn, the bacterial proteins that are 30 degraded by NSPs leading to Spn death are still unknown. In this report, we identify a 90kDa 31 protein in a purified cell wall (CW) preparation, aminopeptidase N (PepN) that is degraded by 32 the NSP, neutrophil elastase (NE). Since PepN lacked a canonical signal sequence or LPxTG 33 motif, we created a mutant expressing a FLAG tagged version of the protein and confirmed its 34 localization to the CW compartment. We determined that not only is PepN a bona fide CW 35 protein, but also is a substrate of NE in the context of intact Spn cells. Furthermore, in 36 comparison to wild-type TIGR4 Spn, a mutant strain lacking PepN demonstrated a significant 37 hyper-resistance phenotype in vitro in the presence of purified NE as well as in 38 opsonophagocytic assays with purified human neutrophils ex vivo. Taken together, this is the 39 first study to demonstrate that PepN is a CW-localized protein and a substrate of NE that 40 contributes to the effective killing of Spn by NSPs and human neutrophils. 41 42 43 44 45 46 3 IMPORTANCE 47 Neutrophils are innate immune cells needed to effectively clear Streptococcus 48 pneumoniae (Spn). Neutrophil serine proteases (NSPs) are important for killing phagocytosed 49 Spn, however, the identity of the Spn proteins that are degraded by NSPs are unknown. This 50 study identifies a Spn cell wall protein, aminopeptidase N (PepN) that is degraded by the NSP, 51 neutrophil elastase (NE). We demonstrate that PepN is a bona fide cell wall protein and mutants 52 lacking PepN are significantly more resistant than wild-type to killing by purified NE and human 53 neutrophils. This study demonstrates that PepN is a NE substrate and its degradation contributes 54 to effective Spn killing. By better understanding how neutrophils kill Spn, we aim to inform the 55 development of improved therapeutic interventions.