Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors Daphne A. C. Stapelsa, Kasra X. Ramyarb, Markus Bischoffc, Maren von Köckritz-Blickweded, Fin J. Mildera, Maartje Ruykena, Janina Eisenbeisc, William J. McWhortere, Mathias Herrmannc, Kok P. M. van Kessela, Brian V. Geisbrechtb,1, and Suzan H. M. Rooijakkersa,1,2 aMedical Microbiology, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands; bDepartment of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506; cInstitute of Medical Microbiology and Hygiene, Saarland University Faculty of Medicine and Medical Center, D-66421 Homburg/Saar, Germany; dDepartment of Physiological Chemistry, University of Veterinary Medicine, D-30559 Hannover, Germany; and eSchool of Biological Sciences, University of Missouri–Kansas City, Kansas City, MO 64110 Edited by Richard P. Novick, New York University School of Medicine, New York, NY, and approved July 31, 2014 (received for review April 28, 2014) Neutrophils are indispensable for clearing infections with the Results prominent human pathogen Staphylococcus aureus. Here, we re- Extracellular Adherence Proteins of S. aureus Inhibit NE. To in- port that S. aureus secretes a family of proteins that potently vestigate whether S. aureus secretes inhibitors of NSPs, we in- inhibits the activity of neutrophil serine proteases (NSPs): neutro- cubated NE with concentrated culture supernatants of different phil elastase (NE), proteinase 3, and cathepsin G. The NSPs, but not S. aureus strains and quantified residual NE activity toward a related serine proteases, are specifically blocked by the extracel- fluorescent peptide substrate. Indeed, we found that NE was lular adherence protein (Eap) and the functionally orphan Eap inhibited by supernatants of all tested S. aureus strains (Fig. 1A). homologs EapH1 and EapH2, with inhibitory-constant values in Fractionation of the supernatant of S. aureus Newman by ion- the low-nanomolar range. Eap proteins are together essential for NSP inhibition by S. aureus in vitro and promote staphylococcal exchange and size-exclusion chromatography yielded two protein infection in vivo. The crystal structure of the EapH1/NE complex bands that corresponded with the NE inhibitory activity. These showed that Eap molecules constitute a unique class of noncova- bands were identified by mass spectrometry as Eap and immu- B lent protease inhibitors that occlude the catalytic cleft of NSPs. nodominant surface antigen B (IsaB) (Fig. 1 ). Further analysis MICROBIOLOGY These findings increase our insights into the complex pathogene- revealed that Eap is the NE inhibitor, because NE activity was sis of S. aureus infections and create opportunities to design novel not affected by the presence of recombinant IsaB, but fully treatment strategies for inflammatory conditions related to exces- blocked by the presence of recombinant Eap (Fig. 1C). Eap is sive NSP activity. a 50- to 70-kDa protein that consists of multiple (most often four or five) repetitive EAP domains (11 kDa). The short linkers immune evasion | bacteria | phagocytes between EAP domains are susceptible to proteolysis (9), which likely explains why the band identified as Eap was only approx- nfections with the human pathogen Staphylococcus aureus imately 25 kDa (Fig. 1B). Iconstitute a major risk to human health. Although this bacte- rium harmlessly colonizes more than 30% of the population via Significance the nose or skin, it causes severe morbidity and mortality upon invasion of deeper tissues (1). To avert these serious infections, Neutrophils are among the first immune cells to migrate to the neutrophils play an indispensable role (2). Neutrophil serine site of infection and clear invading bacteria. They store large proteases (NSPs), including neutrophil elastase (NE), proteinase amounts of neutrophil serine proteases (NSPs) that play key 3 (PR3), and cathepsin G (CG), are important for various neu- roles in immune defense. Unfortunately, NSPs also contribute trophil functions. Active NSPs are stored within the azurophilic to tissue destruction in a variety of inflammatory disorders. In granules (3), but upon neutrophil activation, they either enter this study we discover that the pathogenic bacterium Staphy- the nucleus to regulate extracellular trap (NET) formation (4) or lococcus aureus secretes a family of highly potent and specific they are released into the extracellular milieu to kill certain NSP inhibitors that promote the pathogenicity of this bacte- bacteria (5), cleave bacterial virulence factors (5, 6), or regulate rium in vivo. From crystallography experiments, we conclude immune responses by cleaving chemokines and receptors (7). that these proteins constitute a unique class of NSP inhibitors, which can be used to design novel treatment strategies against Recently, a fourth neutrophil serine protease, denoted NSP4, excessive NSP activity. Furthermore, this study significantly in- was identified (8). creases our understanding of the complex nature of S. aureus Given the central role of NSPs in neutrophil function, we infections. wondered whether S. aureus had evolved mechanisms to cope S. aureus with NSPs. In this study, we discover that secretes Author contributions: D.A.C.S., M.B., M.v.K.-B., F.J.M., M.H., K.P.M.v.K., B.V.G., and S.H.M.R. a family of proteins that specifically and potently block NSPs: designed research; D.A.C.S., K.X.R., M.B., M.R., J.E., W.J.M., and B.V.G. performed research; extracellular adherence protein (Eap) and the hitherto func- D.A.C.S., M.B., and B.V.G. analyzed data; D.A.C.S., B.V.G., and S.H.M.R. wrote the paper; M.v.K.-B., F.J.M., M.H., and K.P.M.v.K. interpreted results; and B.V.G. and S.H.M.R. super- tional orphans Eap-homologue (EapH) 1 and 2. Structural vised the project. studies presented here show that Eap molecules represent a The authors declare no conflict of interest. unique class of noncovalent NSP inhibitors that is distinct from This article is a PNAS Direct Submission. the well-known chelonianin class of inhibitors. These mecha- Freely available online through the PNAS open access option. nistic insights can initiate development of novel, broad-range Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, NSP inhibitors to be used in various inflammatory conditions. www.pdb.org (PDB ID code 4NZL). Furthermore, these insights increase our understanding of the 1B.V.G. and S.H.M.R. contributed equally to this work. S. aureus pathogenicity of and underline the exceptional capa- 2To whom correspondence should be addressed. Email: [email protected]. bility of this pathogen to adapt to its host by modulating the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. immune response. 1073/pnas.1407616111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1407616111 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 isalsofoundinthetwoS. aureus proteins that are homologous to Eap but do not share the above described functions: EapH1 (12 kDa) and EapH2 (13 kDa) (11). Likewise, we found that EapH1 and EapH2 also inhibit NE (Fig. 1C). NE inhibition is specific for the Eap protein family, because all other tested pro- teins of S. aureus could not inhibit NE (Fig. 1C), including mol- ecules characterized by a similar beta-grasp–type fold as the Eap proteins (Fig. 1C, gray bars) (11, 12). Eap Proteins Specifically Inhibit Neutrophil Serine Proteases. NE belongs to the chymotrypsin family of serine proteases, and its amino acid sequence is most similar to that of PR3 and CG (55% and 37% amino acid identity, respectively). More distantly related chymotrypsin-like proteases include plasmin, plasma kallikrein, and thrombin. Of these six proteases, only the three NSPs appeared to be dose-dependently inhibited by the Eap proteins (Fig. 2 A–F). Next we determined the inhibitory constant (Ki)valuesofeach Eap protein versus the three NSPs and also measured the Ki values of the endogenous human NE/CG inhibitor SLPI (secreted leukocyte protease inhibitor) as a control. The Ki values observed for all Eap/NSP combinations were in the low-nanomolar range, which is consistent with a very potent inhibition (Fig. 2G). They are also within the same order of magnitude as the Ki of SLPI for both NE and CG. Because previous work has reported a lower Ki for SLPI/NE and SLPI/CG (0.3 nM and 10 nM, respectively) (13), our particular assay system may have even underestimated the inhibitory capacity of the Eap proteins. Importantly, the experi- mentally determined Ki values are all lower than the endogenous expression levels of the Eap proteins by S. aureus in culture (∼10 μg/mL or 200 nM) (14), indicating that Eap inhibition of NSPs is physiologically relevant. Eap Proteins Are Essential for NSP Inhibition and Promote Staphylococcal Infection. The genes for the Eap proteins lie interspersed throughout the genome, and at least two of three are present in all sequenced S. aureus strains. The eap gene is located upstream, and therefore outside, of the beta-hemolysin–converting prophage (phiNM3) that contains other immune-evasion proteins like staphylococcal com- plement inhibitor (SCIN) (scn) and chemotaxis inhibitory protein of S. aureus (CHIPS) (chp)(15,16)(Fig.3A). Neither eapH1 nor eapH2 lie in close proximity of phage-associated genes. Using se- quential gene deletions by homologous recombination, we con- Fig. 1. Extracellular adherence protein (Eap) family of S. aureus inhibits NE structed a panel of three isogenic eap mutants in S. aureus strain activity. (A) Residual activity of 60 nM NE upon incubation with culture Newman: Δeap, ΔeapΔH1,andΔeapΔH1ΔH2 (eap-triple mutant). supernatants of multiple S. aureus strains. (B) Silver staining analysis of the As a control, the eap-triple mutant was complemented with the fractions after gel filtration (Left). The boxes mark the protein bands ana- individual genes integrated into their original genomic location lyzed by mass spectrometry and the numbers indicate the sizes of reference ΔeapΔH1ΔH2 proteins (kilodaltons).