Meningococcal Outer Membrane Protein NhhA Triggers Apoptosis in Macrophages Mikael Sjo¨ linder1, Georg Altenbacher1, Matthias Hagner1, Wei Sun2, Sophia Schedin-Weiss2¤, Hong Sjo¨ linder1* 1 Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden, 2 Department of Medical Biochemistry and Microbiology, Uppsala Biomedical Centre, Uppsala University, Uppsala, Sweden Abstract Phagocytotic cells play a fundamental role in the defense against bacterial pathogens. One mechanism whereby bacteria evade phagocytosis is to produce factors that trigger apoptosis. Here we identify for the first time a meningococcal protein capable of inducing macrophage apoptosis. The conserved meningococcal outer membrane protein NhhA (Neisseria hia/hsf homologue A, also known as Hsf) mediates bacterial adhesion and interacts with extracellular matrix components heparan sulphate and laminin. Meningococci lacking NhhA fail to colonise nasal mucosa in a mouse model of meningococcal disease. We found that exposure of macrophages to NhhA resulted in a highly increased rate of apoptosis that proceeded through caspase activation. Exposure of macrophages to NhhA also led to iNOS induction and nitric oxide production. However, neither nitric oxide production nor TNF-a signaling was found to be a prerequisite for NhhA-induced apoptosis. Macrophages exposed to wildtype NhhA-expressing meningococci were also found to undergo apoptosis whereas NhhA- deficient meningococci had a markedly decreased capacity to induce macrophage apoptosis. These data provide new insights on the role of NhhA in meningococcal disease. NhhA-induced macrophage apoptosis could be a mechanism whereby meningococci evade immunoregulatory and phagocytotic actions of macrophages. Citation: Sjo¨ linder M, Altenbacher G, Hagner M, Sun W, Schedin-Weiss S, et al. (2012) Meningococcal Outer Membrane Protein NhhA Triggers Apoptosis in Macrophages. PLoS ONE 7(1): e29586. doi:10.1371/journal.pone.0029586 Editor: Ray Borrow, Health Protection Agency, United Kingdom Received July 1, 2011; Accepted December 1, 2011; Published January 4, 2012 Copyright: 2012 Sjo¨ linder et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Swedish Research Council (Grant no. 2008-2572, 2008-3367, www.vr.se), the Swedish Society of Medicine (www.sls.se), the Magnus Bergvalls Foundation (http://www.magnbergvallsstiftelse.nu), the Harald Jeanssons Foundation (http://www.jeanssonsstiftelser.se), the Tore Nilsons Foundation (http://www.torenilsonsstiftelse.nu), and the A˚ke Wiberg Foundation (http://www.ake-wiberg.com). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hong.sjolinder@gmt.su.se ¤ Current address: Department of Neurobiology, Care Sciences and Society, Karolinska Institute, Novum, Huddinge, Sweden Introduction The pathogenic bacterium Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia. During asymptomatic carriage meningococci colonise the nasopharynx but under certain conditions bacteria spread to the bloodstream and meninges. Disease progression is rapid and associated with high mortality. Neisseria hia/hsf homologue (NhhA) is an outer membrane protein homologous to the Hia and Hsf adhesins of Haemophilus influenzae [1]. A strain survey showed that the NhhA gene was present in all (85) strains tested and immuno-electron microscopy demonstrated the presence of NhhA on the outer membrane [1]. In a screen for vaccine candidates against serogroup B meningococci, NhhA was identified as a surface-exposed antigen that could be used to produce an antibactericidal antibody response in mice [2–3]. The C-terminal domain of NhhA is able to trimerize and form a translocator domain that allows localization of the protein to the bacterial surface [4]. In agreement, a C-terminal point mutation was shown to block trimerisation of NhhA [5]. E. coli expressing recombinant Nhha can bind to epithelial cells and furthermore direct interaction between purified NhhA and laminin as well as heparan sulphate was observed [4]. Meningococci lacking NhhA are more sensitive to complement-mediated bacterial killing in human serum and can be more easily phagocytosed by macrophages. In addition, NhhA promotes the interaction between capsulated, wildtype meningococci and macrophages [6]. Using a mouse model of meningococcal disease, Nhha was found to be of importance for colonization of nasal mucosa. Bacterial survival upon intranasal administration could only be observed when using wildtype meningococci and not with meningococci lacking NhhA [6]. Programmed cell death of host cells can be triggered by the release of proapoptotic factors from pathogenic bacteria or exposure of proapoptotic factors on the bacterial surface. Several species of bacteria can induce apoptosis such as Pseudomonas aeruginosa [7] and enteropathogenic E. coli [8]. Apoptosis can play an important role during bacterial infections and be either induced or inhibited by bacterial factors under different pathological conditions. In N. meningitidis, the ability of highly pathogenic strains and isolates to cause invasive disease and tissue damage was found to be closely linked to pro-apoptotic activity [9]. In contrast, strains associated with asymptomatic carriage did not induce apoptosis [9]. The bacterial membrane component lipopolysaccharide (LPS) and related compounds such as lipooligosaccharide (LOS) of N. meningitidis are important apoptosis-inducing factors [10]. Howev- er, meningococci lacking LOS can also provoke proinflammatory responses [11] and induce apoptosis [9]. The N. meningitidis porin PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e29586