Date Published: February 1, 2019
Publisher: Public Library of Science
Author(s): Charmaine N. Nganje, Scott A. Haynes, Christine M. Qabar, Rachel C. Lent, Elsa N. Bou Ghanem, Mara G. Shainheit, Samithamby Jeyaseelan.
Streptococcus pneumoniae (Spn) is an asymptomatic colonizer of the human nasopharynx but can also cause disease 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 CW-localized 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.
Streptococcus pneumoniae (Spn) is a Gram-positive bacterium that is a frequent, asymptomatic colonizer of the human upper respiratory tract. However, if it gains access to other anatomical sites in the human host, such as the lungs, inner ear, meninges or blood, it can cause a variety of diseases including pneumonia, otitis media, meningitis and sepsis, respectively [1–4]. Due to these invasive infections, about one million children die per year under the age of five, mostly in the developing world where access to healthcare is limited . Neutrophils are the most abundant white blood cell in the body and are often the first immune cell type to migrate to the site of infection [6, 7]. Neutrophils play a critical role in the effective clearance of Spn via the process of opsonophagocytic killing. This multi-step process involves the tagging of Spn cells with complement proteins and subsequent internalization and degradation through the action of various factors including reactive oxygen and nitrogen species, antimicrobial peptides and a family of enzymes contained within the azurophilic granules, neutrophil serine proteases (NSPs) . Of this repertoire of anti-microbial factors, previous work demonstrated that NSPs are the most important component for effectively killing Spn in vitro  and play a vital, protective role in murine models of pneumococcal pneumonia . Furthermore, in individuals with Chediak-Higashi syndrome, a rare genetic disorder that impairs the mobilization of NSP-containing granules , neutrophils exhibited a reduced ability to kill Spn .
In neutrophils, non-oxidative mechanisms of killing phagocytosed Spn were shown to be essential both in vitro and in vivo and primarily involve the activity of NSPs, such as NE and CG [9, 10]. Further supporting the importance of NSPs, individuals with impaired granule mobilization, which affects the fusion of NSP-containing granules with the phagolysosome, exhibit a defect in neutrophil-mediated killing of Spn . In addition to playing a key role in controlling Spn infections, other studies demonstrated that NSPs have the capacity to directly kill or degrade virulence factors produced by a variety of pathogens including P. aeruginosa, E. coli, S. flexneri, K. pneumoniae and S. aureus [20–25]. Although previous reports highlight the importance of NSPs in bacterial clearance, little is known about the identity of the Spn proteins that are degraded by NSPs and thus facilitate effective killing.