PorB has been reported to prevent apoptosis in B cells, Jurkat T cells and HeLa epithelial cells by translocating to and interacting with mitochondria [12]. Interestingly, contradictory reports claim that the closely related PorB of Neisseria gonorrhoeae causes calcium influx and apoptosis in HeLa cells and U937 monocytes [13–14]. In addition, norB and cycP, two N. meningitidis genes involved in nitric oxide metabolism, were found to play a role in preventing nitric oxide-dependent apoptosis during infection [15]. Here we demonstrate that the meningococcal outer membrane protein Nhha not only plays an important role in promoting bacterial adhesion. Macrophages are of major importance for the host defense during meningococcal infections. We found that macrophages undergo apoptosis in the presence of NhhA. Results NhhA binds to macrophages In order to perform functional studies of NhhA we expressed NhhA in soluble form by excluding the C-terminal membrane anchoring domain. A sequence encoding amino acids 51 to 510 of NhhA from FAM20, a N. meningitidis serogroup C strain, was cloned into the vector pET-21a and expressed in E. coli in fusion with a histidine tag. The N-terminal of NhhA contains a predicted leader peptide that was excluded from the NhhA construct. NhhA was purified by affinity chromatography followed by gel chromatography to avoid endotoxin contamination. The recom- binant NhhA was present as a monomer and did not undergo any detectable trimerisation (results not shown). Next, the ability of NhhA to bind to macrophages was investigated. RAW 264.7, a mouse macrophage cell line, was cultured under standard conditions and NhhA or an unrelated His-tagged control protein was added to the cell media. By immunofluorescence staining and immunoblotting we could detect binding of NhhA but not the control protein to macrophages (Fig. 1). NhhA induces apoptosis in macrophages We were interested to investigate if NhhA might have additional functions besides mediating bacterial adhesion to host cells. In initial experiments we noticed that exposure of macrophages to NhhA induced morphological changes associated with apoptosis such as blebbing and condensed and fragmented nuclei. To confirm an increased rate of apoptosis we stained NhhA-exposed cells with an AnnexinV antibody in combination with PI (Fig. 2A). Around 20% of the cells exposed to NhhA were found to stain positively for AnnexinV but not PI (Fig. 2A). Less than 2% of the NhhA-treated cells stained positively for PI but not AnnexinV (results not shown) indicating that NhhA-induced cell death proceeded mainly through apoptosis and not necrosis. To further study the capacity of NhhA to induce apoptosis we used a stain specifically labeling apoptotic cells exposing phosphatidyl serine on the cell surface (Fig. 2B). We found that after 20 h exposure to 400 nM NhhA, around 15% of the cells stained positively. The LPS antagonist Polymyxin B did not inhibit NhhA-induced apoptosis indicating that our preparations of purified recombinant NhhA were not contaminated with LPS. As expected, Polymyxin B inhibited LPS-induced apoptosis. In addition, the general caspase inhibitor ZVAD-FMK was found to inhibit NhhA- induced apoptosis indicating that NhhA triggers caspase-depen- dent apoptosis. The dose and time dependence of NhhA-induced apoptosis was investigated (Fig. 3). Already at a concentration of 10 nM NhhA the rate of apoptosis was significantly increased compared to an untreated control. An incubation time of 10 hours was required to induce statistically significant changes in the rate of apoptosis when using 400 nM NhhA. The reason that only a N. meningitidis NhhA Induces Macrophage Apoptosis Figure 1. NhhA binds to macrophages. A: The RAW 264.7 mouse macrophage cell line was grown on coverslips and incubated with 400 nM recombinant NhhA for 4 hours. Immunofluorescence analysis was performed using a monoclonal mouse anti His-tag antibody followed by an Alexa Fluor 488-labeled anti-mouse antibody. Control cells were incubated with an unrelated His-tagged protein (NMC0101). One representative experiment of three is shown. B: Macrophages were incubated with 400 nM recombinant NhhA (+ NhhA) or NMC0101, an unrelated His-tagged protein (2 NhhA) for 4 hours. Cells were washed in PBS, dissolved in SDS-PAGE sample buffer and cell-bound material or input material was analysed by immunoblot analysis using an anti His- tag antibody. Arrowhead indicates migration of NhhA. The sizes in kDa of a protein ladder is indicated. One representative experiment of three is shown. doi:10.1371/journal.pone.0029586.g001 minority of the cells were found to be apoptotic after NhhA exposure could be that macrophages are generally considered to be relatively resistant to apoptosis. In addition, only cells with ongoing apoptosis that are still viable are detected. To further confirm the specificity of NhhA-induced apoptosis we cloned, expressed and purified an unrelated His-tagged meningococcal protein (NMC0101). NMC0101 failed to induce apoptosis in RAW 264.7 cells (results not shown). In order to determine if endogenous meningococcal NhhA can play a role in regulating apoptosis we infected cells with the wildtype meningococcal strain FAM20 and with a FAM20 deletion mutant lacking NhhA [6]. We found that NhhA-deficient meningococci less efficiently induced apoptosis in macrophages, confirming that endogenous NhhA controls apoptosis in macro- phages (Fig. 3C). NhhA specifically targets the monocyte/macrophage lineage In order to investigate if NhhA specifically acted on macro- phages we also tested a human-derived pharyngeal epithelial cell line (FaDu) and human HL-60 promyelocytic leukemia cells. The HL-60 cells used in these experiments were undifferentiated or differentiated to a neutrophil-like phenotype using retinoic acid PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e29586