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• W2985716470 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Perforin-2 (MPEG1) is a pore-forming, antibacterial protein with broad-spectrum activity. Perforin-2 is expressed constitutively in phagocytes and inducibly in parenchymal, tissue-forming cells. In vitro, Perforin-2 prevents the intracellular replication and proliferation of bacterial pathogens in these cells. Perforin-2 knockout mice are unable to control the systemic dissemination of methicillin-resistant Staphylococcus aureus (MRSA) or Salmonella typhimurium and perish shortly after epicutaneous or orogastric infection respectively. In contrast, Perforin-2-sufficient littermates clear the infection. Perforin-2 is a transmembrane protein of cytosolic vesicles -derived from multiple organelles- that translocate to and fuse with bacterium containing vesicles. Subsequently, Perforin-2 polymerizes and forms large clusters of 100 Å pores in the bacterial surface with Perforin-2 cleavage products present in bacteria. Perforin-2 is also required for the bactericidal activity of reactive oxygen and nitrogen species and hydrolytic enzymes. Perforin-2 constitutes a novel and apparently essential bactericidal effector molecule of the innate immune system. https://doi.org/10.7554/eLife.06508.001 eLife digest An effective defense against foreign invaders is fundamental to an organism's survival. It is likely that immunity began to develop shortly after the emergence of Earth's first single-celled organisms and a remnant of that distant past still exists in our present day immune system in the form of Perforin-2. This ancient protein has been highly conserved throughout evolution from sea sponges to humans. Some studies have suggested that Perforin-2 may have an antimicrobial role in invertebrates (including clams, mussels, and snails) and fish. However, its mechanism of killing and its role in the mammalian immune systems has remained largely unknown. McCormack et al. now report that Perforin-2 is a crucial component of host defense against a wide spectrum of infectious bacteria in both mice and humans. This was shown when mice lacking Perforin-2 died from bacterial infections that are not normally lethal. Somewhat unexpectedly, other bactericidal molecules were also found to be less effective in the absence of Perforin-2. This indicates that Perforin-2 is required for the activity of multiple aspects of the mammalian immune system. McCormack et al. demonstrated that Perforin-2 kills by punching holes in bacteria. Unlike other pore-forming proteins that are only present in specific cells, all mammalian cells can express Perforin-2. McCormack et al. also showed that when Perforin-2 is produced at optimal levels, cells are able to combat otherwise lethal, drug-resistant bacteria, including methicillin resistant Staphylococcus aureus (MRSA). This means that Perforin-2 provides a rapid self-defense mechanism for cells against bacterial invaders. The protein's dual role as a pore-forming protein and a supporter of other antibacterial molecules is unprecedented. In the future, these findings could inform the development of treatments that activate and optimize Perforin-2 production to target and eradicate bacterial infections. https://doi.org/10.7554/eLife.06508.002 Introduction Multicellular eukaryotes deploy pore-forming proteins to disrupt the cellular integrity of bacterial pathogens and virally infected cells. The first immunologically relevant discovery of a pore-former was the spontaneous polymerization and refolding of the hydrophilic complement component C9 into a membrane-associated cylindrical complex (Podack and Tschopp, 1982; Tschopp et al., 1982). This finding resolved the question of the molecular nature of the membrane attack complex of complement (MAC) (Humphrey and Dourmashkin, 1969; Mayer, 1972; Muller-Eberhard, 1975; Bhakdi and Tranum-Jensen, 1978) where C5b-8 complexes, first assembled around membrane-bound C3b, trigger C9 to polymerize and form 100 Å pores in bacterial surfaces (Schreiber et al., 1979; Podack and Tschopp, 1982; Tschopp et al., 1982). The recognition that a single protein species, C9, was able to form pores by polymerization suggested the possibility that cytotoxic lymphocytes may be equipped with a similar pore-forming protein. Analysis of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) identified Perforin-1 as the pore-forming killer protein for virus-infected cells and tumor cells (Dennert and Podack, 1983; Podack and Dennert, 1983; Blumenthal et al., 1984). Sequence alignment of Perforin-1 and C9 identified a conserved domain, named the Membrane Attack Complex/Perforin (MACPF) domain in reference to its founding members (Lichtenheld et al., 1988). During polymerization, the MACPF-domains of individual protomers refold and expose an amphipathic helix that inserts into the targeted membranes (Rosado et al., 2007; Baran et al., 2009; Kondos et al., 2010; Law et al., 2010). The hydrophilic surface of the membrane-inserted portion of polymerizing MACPF forms the inner, hydrophilic lining of the nascent pore driving the displacement of hydrophobic membrane components. MACPF generated pores disrupt the innate barrier function of membranes and provide access for chemical or enzymatic effectors that finalize destruction of the target (Schreiber et al., 1979; Masson and Tschopp, 1987; Trapani et al., 1988; Shiver et al., 1992; Smyth et al., 1994). Macrophage Expressed Gene 1 (MPEG1) is the most recently identified protein with a MACPF-domain (Spilsbury et al., 1995). We renamed the new MACPF-containing protein Perforin-2 when we confirmed that it also was a pore forming protein. Evolutionary studies of Perforin-2, have demonstrated that Perforin-2 is one of the oldest eukaryotic MACPF members, present in early metazoan phyla including Porifera (sponges) (D'Angelo et al., 2012; Wiens et al., 2005; McCormack et al., 2013a; McCormack et al., 2013b). Orthologues of Perforin-2 are highly conserved throughout the animal kingdom (Mah et al., 2004; Wiens et al., 2005; Wang et al., 2008; He et al., 2011; Kemp and Coyne, 2011; Green et al., 2014). Recent studies in vertebrates (mammalia) demonstrate that expression of Perforin-2 is not limited to macrophages, as it was also detected in murine embryonic fibroblasts (MEF) and human epithelial cells after bacterial infection (Fields et al., 2013; McCormack et al., 2013a) suggesting that Perforin-2 expression is tied to antibacterial activity. Similarly, in Zebrafish one of its two isoforms, MPEG1.2, is induced following bacterial infection and limits bacterial burden (Benard et al., 2015). Here we show that Perforin-2 is a major antibacterial effector protein of the innate immune system in phagocytic and in tissue forming cells. Perforin-2 is an essential innate effector protein that kills gram-positive, gram-negative, and acid-fast bacteria. The absence of Perforin-2 enables survival of pathogenic bacteria in vitro and systemic dissemination in vivo indicating that expression of Perforin-2 in professional phagocytes and in parenchymal cells is required to eliminate pathogenic bacteria in vitro and in vivo. We demonstrate that Perforin-2 can polymerize to form pores visible by negative staining transmission electron microscopy in bacterial surfaces. The presence of Perforin-2 potentiates the antibacterial activity of other known effectors including reactive oxygen and nitrogen species. In our accompanying manuscript we report some of the molecular mechanisms of Perforin-2 activation and describe how a bacterial virulence factor blocks Perforin-2 function. Results Perforin-2-deficient neutrophils and macrophages are unable to kill pathogenic bacteria, including Mycobacterium tuberculosis Professional phagocytes avidly ingest and kill bacteria. To elucidate the contribution of Perforin-2 towards their bactericidal activity, we compared professional phagocytes from Perforin-2 deficient mice with Perforin-2 heterozygous and wild-type phagocytes. Perforin-2-deficient murine peritoneal exudate macrophages (PEM), neutrophils, and bone marrow-derived macrophages (BMDM) are unable to kill three different species of Mycobacteria (Mycobacterium smegmatis, Mycobacterium avium, M. tuberculosis), as indicated by significant intracellular bacterial replication in MPEG1 (Perforin-2) −/− compared to +/+ or +/− phagocytes (Figure 1A–C, Figure 1—figure supplement 1). Although BMDM express Perforin-2 constitutively, they must be activated with IFN and LPS in order to mediate Perforin-2-dependent growth inhibition of M. tuberculosis (Mtb) (Figure 1—figure supplement 2). This suggest that the destruction of Mtb requires both the expression and activation of Perforin-2. Figure 1 with 4 supplements see all Download asset Open asset Perforin-2 deficiency or siRNA knockdown abrogates intracellular killing of pathogenic bacteria. (A-C) Perforin-2 knockout, heterozygous, and wild-type macrophages and neutrophils were infected with Mycobacterium species (A) PEM infected with Mycobacterium smegmatis, (B) Neutrophils infected with Mycobacterium avium, and (C) BMDM infected with Mycobacterium tuberculosis. (D-H) Perforin-2 knockdown can be complemented in BV2 microglia cells infected with (D) M. avium, (E) M. smegmatis, (F) Salmonella typhimurium, and (G) MRSA. (H) Western blot demonstrating protein levels after complementation: BV2 transfected with (Lane 1) Perforin-2-RFP and Perforin-2 siRNA, (Lane 2) RFP and Perforin-2 siRNA, (Lane 3) RFP and Perforin-2 scramble siRNA, and (Lane 4) Perforin-2 siRNA alone. In western blots, Perforin-2-RFP is detected as a 105 kD band compared to the 72 kD band seen for endogenous Perforin-2 (lane 1 and 3 respectively). (I) Human MDM infection with MRSA. = MPEG1 (Perforin-2) wild-type cells (+/+), = MPEG1 (Perforin-2) heterozygous cells (+/−), • MPEG1 (Perforin-2) knockout cells (−/−). ■= RFP + Perforin-2 siRNA transfected cells, □= RFP + scramble siRNA transfected cells. ▼= Perforin-2-RFP + Perforin-2 siRNA transfected cells. One-way ANOVA with Tukey's multiple comparisons post-hoc test was used for A–G. (A–C) *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type cells; *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type and Perforin-2 knockout:Perforin-2 heterozygous cells. (D–G) *p < 0.05 between RFP + Perforin-2 siRNA:RFP + scramble siRNA and RFP + Perforin-2 siRNA:Perforin-2-RFP + Perforin-2 siRNA. (I) *p < 0.05 multiple t-tests with post-hoc correction for multiple comparisons using the Holm-Sidak method. https://doi.org/10.7554/eLife.06508.003 We also used Perforin-2 siRNA to ablate Perforin-2 in cells in vitro. To exclude the possibility of off-target protein effects by Perforin-2-siRNA, we performed complementation assays in BV2-microglia with C-terminally tagged Perforin-2-RFP (Figure 1D–G). Endogenous Perforin-2 was silenced (Figure 1H, lane 2) with siRNA specific for the 3′-untranslated sequence and the cells were complemented by transfection with siRNA-resistant Perforin-2-RFP (Figure 1H, lane 1). Only Perforin-2-RFP but not control RFP transfection restored bactericidal activity. The data indicated that RFP-tagged Perforin-2 was fully active and that siRNA ablation of Perforin-2 has negligible off-target effects on bactericidal activity. The results suggested that Perforin-2 is critical for intracellular bacterial killing. We also sought to determine the role of Perforin-2 in human professional phagocytes. Human macrophages and neutrophils express Perforin-2 protein constitutively (Figure 1—figure supplement 3). Human monocyte derived macrophages (MDM) efficiently killed intracellular MRSA. Perforin-2 knockdown by siRNA abrogated killing of MRSA and resulted in MRSA replication (Figure 1 I). In addition, we used the human promyelocytic cell line HL-60 that differentiates into Perforin-2-expressing neutrophils upon treatment with retinoic acid (RA). Perforin-2 siRNA silencing in RA-differentiated HL-60 cells abolished Perforin-2 protein expression (Figure 1—figure supplement 3) and was associated with intracellular replication of Salmonella typhimurium, MRSA, and M. smegmatis (Figure 1—figure supplement 4). In contrast, scramble-transfected controls continued to express endogenous Perforin-2 and the number of recovered bacteria was reduced over several hours. This result suggested that Perforin-2 was also required for the killing of bacteria in human neutrophils (Figure 1—figure supplement 4). In summary, the results indicate that professional murine and human phagocytes require Perforin-2 to kill phagocytosed bacteria. This finding raised the question of the function of the other known bactericidal mediators in relation to Perforin-2. Reactive oxygen and nitrogen species enhance the bactericidal activity of Perforin-2 but have little microbicidal activity when Perforin-2 is absent Reactive oxygen (ROS) and reactive nitrogen (RNS) species are recognized for their bactericidal activity in phagocytic cells. PEM activated by IFN-γ and LPS produce both families of effectors (Figure 2—figure supplement 1). We used the well-characterized chemical inhibitors N-acetyl-L-cysteine (NAC) and L-NG-nitroarginine methyl ester (L-NAME) to block ROS and nitric oxide (NO) respectively as Perforin-2 and ROS or Perforin-2 and RNS knockout animals are not currently available (Vazquez-Torres et al., 2000; Mantena et al., 2008; Sohn et al., 2011). First, we established that the addition of the inhibitors reduced levels of ROS and NO produced by activated PEMs (Figure 2—figure supplement 1). The role of endogenous ROS and NO on cellular bactericidal activity in PEM in the presence and absence endogenous of Perforin-2 was assessed in two complementary ways. First, we assessed the effect of chemical inhibitors of ROS and NO on killing of intracellular wild-type S. typhimurium (Figure 2A–D). ROS is known to be active and produced during the first 4 hr after S. typhimurium infection in PEM (Mastroeni et al., 2000). In Perforin-2 deficient PEM, S. typhimurium replicated equally well regardless of ROS inhibition (Figure 2B). This suggests ROS had minimal influence on intracellular replication of S. typhimurium in the absence of Perforin-2. In contrast, with PEM that express Perforin-2, the inhibition of endogenous ROS by NAC enables S. typhimurium to replicate significantly more than mock treatment during the first 4 hr after infection, suggesting that ROS in combination with Perforin-2 helps to restrain S. typhimurium replication during this period (Figure 2A). Figure 2 with 2 supplements see all Download asset Open asset Antimicrobial compounds (ROS and NO) enhance Perforin-2 mediated killing of S. typhimurium by PEM but have limited activity in the absence of Perforin-2. (A–D) Wild-type S. typhimurium infection of PEMs isolated from either MPEG1 (Perforin-2) +/+ (A, C), or MPEG1 (Perforin-2) −/− mice (B, D). Non-filled symbols indicated MPEG1 (Perforin-2) +/+ PEMs; whereas filled symbols are MPEG1 (Perforin-2) −/− PEMs. Cells were incubated with NAC (blue line), NAME (green line), or mock (black line). To assess bacterial resistance mechanisms against these effectors, (E) SodC1 or (F) HmpA knockout S. typhimurium were used to infect MPEG1 (Perforin-2) −/− or +/+ PEMs. The above experiments were conducted with six biologic replicates and are representative of four independent experiments. Statistical analysis was performed utilizing multiple T-tests with correction for multiple comparisons using the Holm-Sidak method. *p < 0.05. https://doi.org/10.7554/eLife.06508.008 Inhibition of endogenous NO production with L-NAME in the presence of Perforin-2 also allowed increased intracellular S. typhimurium replication beginning several hours post-infection (Figure 2C) coinciding with the known time period required for onset of NO production (see Figure 2—figure supplement 1) (Mastroeni et al., 2000). As with ROS, endogenous NO had little effect on S. typhimurium replication in the absence of Perforin-2 (Figure 2D). The results indicate that although ROS and NO contribute towards the intracellular killing of bacterial pathogens, their bactericidal activity is largely dependent upon the presence of Perforin-2. To validate that the cooperation between Perforin-2 and ROS and NO was not specific to S. typhimurium, we repeated the above with M. smegmatis. The cooperative activity of Perforin-2 with ROS and NO was also evident in killing of M. smegmatis in PEM (Figure 2—figure supplement 2). Pharmacologic inhibitors significantly increased bacterial survival only when Perforin-2 was present, with little additional effect when Perforin-2 was absent. This data suggested that the critical importance of Perforin-2 in facilitating ROS and NO activity is not specific to only S. typhimurium. To further investigate the dependence of ROS and NO bactericidal activity upon Perforin-2 we utilized S. typhimurium mutants lacking the periplasmic superoxide dismutase (sodC1) or flavohemoglobin (hmpA) genes, which neutralize ROS or NO, respectively, in intracellular killing assays. (Stevanin et al., 2002; Uzzau et al., 2002; Krishnakumar et al., 2004; Prior et al., 2009). If ROS and NO activity is dependent upon Perforin-2 we hypothesized that bacterial ROS and NO defense mechanisms would be unnecessary in Perforin-2 knockout cells. Unlike other superoxide dismutases that protect bacteria from oxygen radicals produced intracellularly as a byproduct of cellular respiration, SodC1 is a periplasmic superoxide dismutase. In vivo, sodC1 mutants are significantly attenuated relative to wild-type S. typhimurium (De Groote et al., 1997; Fang et al., 1999; Krishnakumar et al., 2004). Flavohemoglobin acts by either catalyzing an O2-dependent denitrosylase reaction converting NO to a nitrate ion or N2O, or an anoxic reductive reaction forming NO−. As with SodC1, in vivo and in vitro studies substantiate the role of HmpA with significant attenuation observed with HmpA deficient bacteria (Stevanin et al., 2002; Bang et al., 2006). In the presence of Perforin-2, SodC1-deficient S. typhimurium were killed more efficiently than wild-type S. typhimurium. However, SodC1-deficient S. typhimurium replicate similar to wild-type S. typhimurium when Perforin-2 was absent (Figure 2E). Similarly, hmpA mutants are more susceptible to killing by NO than wild-type bacteria, but only when Perforin-2 is present. In the absence of Perforin-2, Flavohemoglobin does not enhance the survival and replication of wild-type S. typhimurium relative to the hmpA mutant. Thus, both chemical and genetic analyses indicate that Perforin-2 is required for the bactericidal activity of ROS and NO in macrophages. Perforin-2 is required for bactericidal activity of parenchymal, tissue forming cells The induction of Perforin-2 in certain parenchymal cells was reported previously (Fields et al., 2013; McCormack et al., 2013). We expanded this analysis for many human and murine primary cells and established cell lines, ranging from epithelial to endothelial cells, from astrocytes to myoblasts, and from neural cells to secretory cells (Table 1, Table 2). Every cell type derived from ectodermal, neuroectodermal, endodermal, or mesodermal lineage tested to date is able to express Perforin-2 message either constitutively or after type I or II IFN induction. Table 1 (murine cells) and Table 2 (human cells) summarize these results while their respective supplements (Supplementary files 1, 2) show the inducibility of Perforin-2′s mRNA and protein (qPCR of ΔCT of Perforin-2 normalized to GAPDH and western blot analysis). Moreover, all cell types analyzed (54 out of 54) are able to kill bacteria in an in vitro bactericidal assay when Perforin-2 is expressed. When infection occurs prior to Perforin-2 induction or when Perforin-2 is siRNA-ablated or genetically deficient using the above assay, bacteria were not killed by cells and consequently replicate. In contrast, cells that express Perforin-2 were bactericidal. These results suggest that Perforin-2 can be expressed ubiquitously to defend cells against bacterial invasion. Table 1 Murine perforin-2 expression https://doi.org/10.7554/eLife.06508.011 Cell type:Perforin-2 expression:Peritoneal macrophageConstitutiveBone marrow derived macrophage (BMDM)ConstitutiveBone marrow derived dendritic cell (BMDC)ConstitutiveBV-2 microglia cell lineConstitutiveRaw264.7 macrophage cell lineConstitutiveJ774A.1 macrophage cell lineConstitutiveMicrogliaConstitutiveNeutrophil (peritoneum stimulation)ConstitutiveNeutrophil (bone marrow)ConstitutiveGamma delta (γδ) T cell (from Skin)ConstitutiveGamma delta (γδ) T cell (from Gut)ConstitutiveGamma delta (γδ) T cell (from Vagina)ConstitutiveMarginal zone B cellConstitutiveKeratinocyte (Back)ConstitutiveIntestinal epithelial cellsConstitutiveSplenocytesConstitutiveOT1 CD8 T cell induced with TGFβ, RA, and IL2ConstitutiveOT1 CD8 T cellInducibleCD4 T cellInducibleB cellInducibleAstrocyteInducibleNeuronInducibleCath.a neuroblastoma cell lineInducibleNeuro-2A neuroblastoma cell lineInducibleAdult CNS fibroblastInducibleEmbryonic fibroblastInducibleNIH 3T3 fibroblast cell lineInducibleBalb/c 3T3 fibroblast cell lineInducibleC2C12 myoblast cell lineInducibleNeonatal ventricular myocytesInducibleCMT-93 rectal carcinoma cell lineInducibleCT26 colon carcinoma cell lineInducibleB16-F10 melanoma cell lineInducibleB16-F0 melanoma cell lineInducibleMOVCAR 5009 ovarian cancer cell lineInducibleMOVCAR 5447 ovarian cancer cell lineInducibleLL/2 Lewis lung carcinoma cell lineInducibleED-1 lung adenocarcinoma cell lineInducible Italics: Ex vivo primary cells utilized for analysis. Table 2 Human peforin-2 expression https://doi.org/10.7554/eLife.06508.012 Cell type:Perforin-2 expression:Monocyte derived macrophage (MDM)ConstitutiveMonocyte derived dendritic cell (MDC)ConstitutivePBMC isolated NK cellConstitutivePolymorphonuclear granulocyte (neutrophil)ConstitutiveHL-60 promyelocyte cell line RA differentiated to PMNConstitutiveHL-60 cell line PMA differentiated to MacrophageConstitutiveFetal keratinocyteConstitutiveAdult keratinocyteConstitutivePMA differentiated Thp-1 monocyte cell lineConstitutiveNK-92 cell lineConstitutiveNormal colon biopsyConstitutiveNormal skin biopsyConstitutiveUmbilical endothelial cell (HUVEC)InducibleHeLa cervical carcinoma cell lineInducibleA2EN endocervical epithelial cell lineInducibleUM-UC-3 bladder cancer cell lineInducibleUM-UC-9 bladder cancer cell lineInducibleCaCo-2 colorectal carcinoma cell lineInducibleHEK293 embryonal kidney cell lineInducibleMIA-PaCa-2 pancreatic cancer cell lineInducibleSkin fibroblastInducibleThp-1 monocyte cell lineInducibleHL-60 promyelocyte cell lineInducibleOVCAR3 ovarian carcinoma cell lineInducibleA549 alveolar adenocarcinoma cell lineInducibleU-1752 bronchiolar epithelial cell lineInducibleJeg-3 placental choriocarcinoma cell lineInducible Italics: Ex vivo primary cells utilized for analysis. Perforin-2 siRNA knockdown was used to determine its contribution towards intracellular killing of bacteria by IFN-induced murine and human parenchymal cells. Although IFN induces hundreds of antimicrobial genes in addition to Perforin-2, silencing of Perforin-2 alone was sufficient to cause bacterial replication. Without exception, Perforin-2 expression and function were essential for killing a diverse array of intracellular pathogenic bacteria by parenchymal or phagocytic cells. Examples of bactericidal activity include human vascular endothelial cells (HUVEC); human pancreatic cells (MIA-PaCa-2); human uroepithelial cells (UM-UC-9); murine ovarian epithelial cells (MOVCAR 5009); murine colon epithelial cells (CT26); and murine cardiac myoblasts (C2C12), respectively (Figure 3 and Supplementary file 3). Examples of human and murine siRNA-mediated Perforin-2 protein knockdown include HUVECs and myoblasts (Figure 3, Supplementary file 4). To certify that Perforin-2 siRNA targeting was specific, siRNA resistant Perforin-2-RFP was utilized to complement Perforin-2 siRNA in parenchymal tissue forming cells. Figure 3—figure supplement 1 highlights representative examples of Perforin-2 complementation in myoblasts, intestinal epithelial cells, and PEM. Figure 3 with 2 supplements see all Download asset Open asset Perforin-2 significantly contributes to intracellular killing in non-hematopoietically derived cells. One day prior to the infection, cells were transfected with either a pool of scramble (□) or Perforin-2 specific (■) siRNA and 14 hr prior to the infection induced with IFN-γ. (A) HUVEC cells infected with M. smegmatis, (B) MIA-PaCa-2 cells infected with S. typhimurium, (C) UM-UC-9 infected with MRSA, (D) Perforin-2 MEF infected with MRSA, (E) Human Kc infected with MRSA induced with IFN-γ, (F) Human Kc infected with MRSA with no IFN-γ induction. = MPEG1 (Perforin-2) +/+, = MPEG1 (Perforin-2) +/−, •= MPEG1 (Perforin-2) −/−. (A–C, E, F) The above graphs contain 5–9 biologic replicates, and are representative of 3–7 independent experiments. Statistical analysis was performed utilizing multiple T-tests with correction for multiple comparisons using the Holm-Sidak method. *p < 0.05. (D) One-way ANOVA with Tukey post-hoc multiple comparisons. *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type mice *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type and Perforin-2 knockout:Perforin-2 heterozygous mice. *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type, Perforin-2 knockout:Perforin-2 heterozygous, and Perforin-2 heterozygous:Perforin-2 wild-type. https://doi.org/10.7554/eLife.06508.013 To further validate the requirement of Perforin-2 for bactericidal activity in non-phagocytic cells, we used genetically deficient MEFs obtained from MPEG1 (Perforin-2) +/+, +/−, and −/− littermates. After overnight induction with IFN-γ, MPEG1 (Perforin-2) +/+ MEFs eliminate MRSA. In contrast, IFN-γ treated Perforin-2 −/− MEFs enable MRSA to replicate. Heterozygous MEFs had intermediate bactericidal activity (Figure 3D) suggesting a gene dose effect of Perforin-2. Keratinocytes (Kc) represent the first cellular barrier to infection in the skin (Song et al., 2002; Mempel et al., 2003; Bernard and Gallo, 2011). Unlike other parenchymal cells that need to be induced, primary human Kc express Perforin-2 constitutively. The identity of human Perforin-2 in Kc was confirmed by western blotting (Figure 3—figure supplement 2). Densitometry analysis suggests similar Perforin-2 protein levels in MDM and Kc (Figure 3—figure supplement 2). Expression could also be silenced with human Perforin-2 siRNA (Figure 3—figure supplement 2, lane 2), but not with scramble siRNA (Figure 3—figure supplement 2, lane 3). The inhibition of Perforin-2 expression abrogated the bactericidal activity of Kc (Figure 3E) that were able to kill MRSA, irrespective of prior IFN activation (Figure 3F). Cumulatively, our results suggest that Perforin-2 is an effector for cellular defense against pathogenic bacteria in professional phagocytes and in other cells. The ubiquity of Perforin-2 expression suggests a critical importance in cellular defenses of many, if not all, tissue against pathogenic bacteria. The findings raise the question of the molecular mechanisms by which Perforin-2 exerts its potent bactericidal function. Perforin-2 accumulates in membranes enclosing bacteria and is associated with bacterial lysis The MACPF domain of Perforin-2 suggests that it is a pore-forming protein similar to the pore-formers of complement (C9) and cytotoxic lymphocytes (Perforin-1) (Podack and Tschopp, 1982; Dennert and Podack, 1983; Law et al., 2010). In analogy to C9 and perforin-1, pore-formation by the MACPF domain of Perforin-2 on the bacterial surface may constitute the lethal hit. However, Perforin-2, unlike C9 and Perforin-1, is anchored in membrane vesicles with its MACPF domain predicted to reside inside the vesicle or outside on the plasma membrane (Figure 4A). Therefore we decided to study the cell biology of Perforin-2 in resting cells and following bacterial infection. Figure 4 with 3 supplements see all Download asset Open asset Endogenous Perforin-2 is located in intracellular sites allowing for rapid translocation to bacteria. (A) Schematic demonstrating proposed orientation of Perforin-2 in vesicles. (B) Fractionation results of endogenous Perforin-2 from human macrophages. (Lane L) is a post-nuclear lysate control, (Lane 1–8) are individual fractions corresponding with specific indicated organelles. (C) Overexpression of murine Perforin-2-GFP in murine BV2 microglial cells. (D–F) Confocal images taken 5 min after S. typhimurium infection in Perforin-2-GFP + Perforin-2 siRNA transfected BV2 cells. White arrows denote extracellular S. typhimurium, red arrows highlight a DNA cloud corresponding with S. typhimurium (D) DAPI only, (E) Perforin-2-GFP only, (F) Merge of DAPI and Perforin-2-GFP. (G–I) Confocal images taken 5 min after Escherichia coli-GFP infection in Perforin-2-RFP + Perforin-2 siRNA transfected BV2 cells. Arrows point to extracellular E. coli-GFP that has made contact but is still extracellular with normal bacilli morphology maintained. (G) E. coli-GFP only, (H) Perforin-2-RFP only, and (I) merge E. coli-GFP and Perforin-2-RFP. Fractions in B were probed as follows: Cytoplasm—MEK1/2; E" @default.
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• W2985716470 title "Author response: Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria" @default.
• W2985716470 doi "https://doi.org/10.7554/elife.06508.037" @default.
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