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.