PepN is a non-essential, cell wall-localized protein that contributes to neutrophil elastase-mediated killing of Streptococcus pneumoniae

Streptococcus 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 of Spn, opsonophagocytosis by neutrophils is perhaps most critical. Upon phagocytosis, Spn is 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 killing Spn, the bacterial proteins that are degraded by NSPs leading to Spn death 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 a bona fide CW protein, but also is a substrate of NE in the context of intact Spn cells. Furthermore, in comparison to wild-type TIGR4 Spn, a mutant strain lacking PepN demonstrated a significant hyper-resistance phenotype in vitro in the presence of purified NE as well as in opsonophagocytic assays with purified human neutrophils ex 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 of Spn by NSPs and human neutrophils.\n\nIMPORTANCENeutrophils are innate immune cells needed to effectively clear Streptococcus pneumoniae (Spn). Neutrophil serine proteases (NSPs) are important for killing phagocytosed Spn, however, the identity of the Spn proteins that are degraded by NSPs are unknown. This study identifies a Spn cell wall protein, aminopeptidase N (PepN) that is degraded by the NSP, neutrophil elastase (NE). We demonstrate that PepN is a bona fide cell 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 effective Spn killing. By better understanding how neutrophils kill Spn, we aim to inform the development of improved therapeutic interventions.