The NK cell receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells.

Natural killer (NK) cell kill infected, transformed and stressed cells when an activating NK cell receptor is triggered1. Most NK cells and some innate lymphoid cells express the activating receptor NKp46, encoded by NCR1, the most evolutionarily ancient NK cell receptor2,3. Blockage of NKp46 inhibits NK killing of many cancer targets4. Although a few infectious NKp46 ligands have been identified, the endogenous NKp46 cell surface ligand is unknown. Here we show that NKp46 recognizes externalized calreticulin (ecto-CRT), which translocates from the endoplasmic reticulum (ER) to the cell membrane during ER stress. ER stress and ecto-CRT are hallmarks of chemotherapy-induced immunogenic cell death5,6, flavivirus infection and senescence. NKp46 recognition of the P domain of ecto-CRT triggers NK cell signalling and NKp46 caps with ecto-CRT in NK immune synapses. NKp46-mediated killing is inhibited by knockout or knockdown of CALR, the gene encoding CRT, or CRT antibodies, and is enhanced by ectopic expression of glycosylphosphatidylinositol-anchored CRT. NCR1)-deficient human (and Nrc1-deficient mouse) NK cells are impaired in the killing of ZIKV-infected, ER-stressed and senescent cells and ecto-CRT-expressing cancer cells. Importantly, NKp46 recognition of ecto-CRT controls mouse B16 melanoma and RAS-driven lung cancers and enhances tumour-infiltrating NK cell degranulation and cytokine secretion. Thus, NKp46 recognition of ecto-CRT as a danger-associated molecular pattern eliminates ER-stressed cells.

Costimulatory CD226 Signaling Regulates Proliferation of Memory-like NK Cells in Healthy Individuals with Latent Mycobacterium tuberculosis Infection.

It is now widely accepted that NK cells can acquire memory, and this makes them more effective to protect against some pathogens. Prior reports indicate memory-like NK cells (mlNKs) in murine model of Mycobacterium tuberculosis (Mtb) as well as in healthy individuals with latent TB infection (LTBI). The increased expression of CD226 was evident in mlNKs from LTBI+ people after stimulation with γ-irradiated Mtb (γ-Mtb). We thus evaluated the contribution of costimulatory CD226 signaling in the functionality of mlNKs in LTBI+ people. We found that blockade of CD226 signaling using the antibody- or CRISPR/Cas9-mediated deletion of the CD226 gene in NK cells diminished the proliferation of mlNKs from LTBI+ people. Blocking CD226 signaling also reduced the phosphorylation of FOXO1 and cMyc expression. Additionally, cMyc inhibition using a chemical inhibitor reduced proliferation by mlNKs from LTBI+ people. Moreover, blocking CD226 signaling reduced glycolysis in NK cells, and the inhibition of glycolysis led to reduced effector function of mlNKs from LTBI+ people. Overall, our results provide a role for CD226 signaling in mlNK responses to Mtb.

Decidual NK cells kill Zika virus-infected trophoblasts.

Zika virus (ZIKV) during pregnancy infects fetal trophoblasts and causes placental damage and birth defects including microcephaly. Little is known about the anti-ZIKV cellular immune response at the maternal-fetal interface. Decidual natural killer cells (dNK), which directly contact fetal trophoblasts, are the dominant maternal immune cells in the first-trimester placenta, when ZIKV infection is most hazardous. Although dNK express all the cytolytic molecules needed to kill, they usually do not kill infected fetal cells but promote placentation. Here, we show that dNK degranulate and kill ZIKV-infected placental trophoblasts. ZIKV infection of trophoblasts causes endoplasmic reticulum (ER) stress, which makes them dNK targets by down-regulating HLA-C/G, natural killer (NK) inhibitory receptor ligands that help maintain tolerance of the semiallogeneic fetus. ER stress also activates the NK activating receptor NKp46. ZIKV infection of Ifnar1 -/- pregnant mice results in high viral titers and severe intrauterine growth restriction, which are exacerbated by depletion of NK or CD8 T cells, indicating that killer lymphocytes, on balance, protect the fetus from ZIKV by eliminating infected cells and reducing the spread of infection.

γδ T cells suppress Plasmodium falciparum blood-stage infection by direct killing and phagocytosis.

Activated Vγ9Vδ2 (γδ2) T lymphocytes that sense parasite-produced phosphoantigens are expanded in Plasmodium falciparum-infected patients. Although previous studies suggested that γδ2 T cells help control erythrocytic malaria, whether γδ2 T cells recognize infected red blood cells (iRBCs) was uncertain. Here we show that iRBCs stained for the phosphoantigen sensor butyrophilin 3A1 (BTN3A1). γδ2 T cells formed immune synapses and lysed iRBCs in a contact, phosphoantigen, BTN3A1 and degranulation-dependent manner, killing intracellular parasites. Granulysin released into the synapse lysed iRBCs and delivered death-inducing granzymes to the parasite. All intra-erythrocytic parasites were susceptible, but schizonts were most sensitive. A second protective γδ2 T cell mechanism was identified. In the presence of patient serum, γδ2 T cells phagocytosed and degraded opsonized iRBCs in a CD16-dependent manner, decreasing parasite multiplication. Thus, γδ2 T cells have two ways to control blood-stage malaria-γδ T cell antigen receptor (TCR)-mediated degranulation and phagocytosis of antibody-coated iRBCs.

Decidual NK Cells Transfer Granulysin to Selectively Kill Bacteria in Trophoblasts.

Maternal decidual NK (dNK) cells promote placentation, but how they protect against placental infection while maintaining fetal tolerance is unclear. Here we show that human dNK cells highly express the antimicrobial peptide granulysin (GNLY) and selectively transfer it via nanotubes to extravillous trophoblasts to kill intracellular Listeria monocytogenes (Lm) without killing the trophoblast. Transfer of GNLY, but not other cell death-inducing cytotoxic granule proteins, strongly inhibits Lm in human placental cultures and in mouse and human trophoblast cell lines. Placental and fetal Lm loads are lower and pregnancy success is greatly improved in pregnant Lm-infected GNLY-transgenic mice than in wild-type mice that lack GNLY. This immune defense is not restricted to pregnancy; peripheral NK (pNK) cells also transfer GNLY to kill bacteria in macrophages and dendritic cells without killing the host cell. Nanotube transfer of GNLY allows dNK to protect against infection while leaving the maternal-fetal barrier intact.

Cytotoxic CD8+ T cells recognize and kill Plasmodium vivax-infected reticulocytes.

Plasmodium vivax causes approximately 100 million clinical malaria cases yearly1,2. The basis of protective immunity is poorly understood and thought to be mediated by antibodies3,4. Cytotoxic CD8+ T cells protect against other intracellular parasites by detecting parasite peptides presented by human leukocyte antigen class I on host cells. Cytotoxic CD8+ T cells kill parasite-infected mammalian cells and intracellular parasites by releasing their cytotoxic granules5,6. Perforin delivers the antimicrobial peptide granulysin and death-inducing granzymes into the host cell, and granulysin then delivers granzymes into the parasite. Cytotoxic CD8+ T cells were thought to have no role against Plasmodium spp. blood stages because red blood cells generally do not express human leukocyte antigen class I7. However, P. vivax infects reticulocytes that retain the protein translation machinery. Here we show that P. vivax-infected reticulocytes express human leukocyte antigen class I. Infected patient circulating CD8+ T cells highly express cytotoxic proteins and recognize and form immunological synapses with P. vivax-infected reticulocytes in a human leukocyte antigen-dependent manner, releasing their cytotoxic granules to kill both host cell and intracellular parasite, preventing reinvasion. P. vivax-infected reticulocytes and parasite killing is perforin independent, but depends on granulysin, which generally efficiently forms pores only in microbial membranes8. We find that P. vivax depletes cholesterol from the P. vivax-infected reticulocyte cell membrane, rendering it granulysin-susceptible. This unexpected T cell defense might be mobilized to improve P. vivax vaccine efficacy.

Granzyme B Disrupts Central Metabolism and Protein Synthesis in Bacteria to Promote an Immune Cell Death Program.

Human cytotoxic lymphocytes kill intracellular microbes. The cytotoxic granule granzyme proteases released by cytotoxic lymphocytes trigger oxidative bacterial death by disrupting electron transport, generating superoxide anion and inactivating bacterial oxidative defenses. However, they also cause non-oxidative cell death because anaerobic bacteria are also killed. Here, we use differential proteomics to identify granzyme B substrates in three unrelated bacteria: Escherichia coli, Listeria monocytogenes, and Mycobacteria tuberculosis. Granzyme B cleaves a highly conserved set of proteins in all three bacteria, which function in vital biosynthetic and metabolic pathways that are critical for bacterial survival under diverse environmental conditions. Key proteins required for protein synthesis, folding, and degradation are also substrates, including multiple aminoacyl tRNA synthetases, ribosomal proteins, protein chaperones, and the Clp system. Because killer cells use a multipronged strategy to target vital pathways, bacteria may not easily become resistant to killer cell attack.

Control of catalysis in globin coupled adenylate cyclase by a globin-B domain.

The globin coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) has two globin domains (globin-A and globin-B). Globin-B domain (210-360 amino acids) may guide the interaction between globin-A and adenylate cyclase domains for the regulation of catalysis. We investigated the role of globin-B domain in HemAC-Lm by constructing a series of mutants namely Δ209 (209 amino acids deleted), Δ360 (360 amino acids deleted), H161A, H311A and H311A-Δ209. Spectroscopic data suggest that the Δ209 and H311A-Δ209 proteins to be Fe(2+)-O2 form and apo form, respectively, indicating that His311 residue in the globin-B domain is crucial for heme binding in Δ209 protein. However, the H311A mutant is still of the Fe(2+)-O2 form whereas H161A mutant shows the apo form, indicating that only His161 residue in the globin-A domain is responsible for heme binding in full length enzyme. cAMP measurements suggest that the activities of Δ360 and Δ209 proteins were ∼10 and ∼1000 times lesser than full length enzyme, respectively, leading to the fact that globin-B domain inhibited catalysis rather than activation in absence of globin-A domain. These data suggest that the O2 bound globin-A domain in HemAC-Lm allows the best cooperation of the catalytic domain interactions to generate optimum cAMP.

The ferrous-dioxy complex of Leishmania major globin coupled heme containing adenylate cyclase: the role of proximal histidine on its stability.

Recently we have described the globin-coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) that shows an O2 dependent cAMP signaling (Sen Santara, et. al. Proc. Natl. Acad. Sci. U.S.A. 110, 16790-16795 (2013)). The heme iron of HemAC-Lm is expected to participate in oxygen binding and activates adenylate cyclase activity during catalysis, but its interactions with O2 are uncharacterized. We have utilized the HemAC-Lm and stopped-flow methods to study the formation and decay of the HemAC-Lm oxygenated complex at 25°C. Mixing of the ferrous HemAC-Lm with air-saturated buffer generates a very stable oxygenated complex with absorption maxima at 414, 540 and 576nm. The distal axial ligand in the deoxygenated ferrous HemAC-Lm is displaced by O2 at a rate of ~10s(-1). To prepare apoprotein of heme iron in HemAC-Lm, we have mutated the proximal His161 to Ala and characterized the mutant protein. The apo as well as heme reconstituted ferric state of the mutant protein shows a ~30 fold lower catalytic activity compared to oxygenated form of wild type protein. The oxygenated form of heme reconstituted mutant protein is highly unstable (decay rate=6.1s(-1)). Decomposition of the oxygenated intermediate is independent of O2 concentration and is monophasic. Thus, the stabilization of ferrous-oxy species is an essential requirement in the wild type HemAC-Lm for a conformational alteration in the sensor domain that, sequentially, activates the adenylate cyclase domain, resulting in the synthesis of cAMP.

Identification of proximal and distal axial ligands in Leishmania major pseudoperoxidase.

Previous optical and electron paramagnetic resonance (EPR) spectroscopic studies of the newly discovered peroxynitrite scavenging pseudoperoxidase from Leishmania major (LmPP) suggested that ferric LmPP contained a six-coordinate low-spin (6cLS) heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. To identify the axial ligands of LmPP, we exploit a systematic mutational analysis of potential heme ligands. On the basis of UV-visible and EPR spectroscopy, we report that the substitution of the proximal His206 with alanine in LmPP alters the 6cLS to a five-coordinate high spin (5cHS) form at pH 4.0 that has a spectrum characteristic of a Cys-ligated 5cHS derivative. The electronic absorption and EPR analysis of all alanine-substituted Cys and Met single mutants establish that when Cys107 is replaced with alanine, a new species appears that has a spectrum characteristic of a histidine-ligated 5cHS derivative at pH 4.0. Together, these results suggest that His206 and Cys107 act as the proximal and distal axial ligands in ferric LmPP, respectively. However, the electronic properties of reduced wild-type LmPP are similar to those of known 5cHS His-ligated heme proteins at pH 8.8, indicating that the thiolate bond was broken upon reduction. Furthermore, the wild-type protein was only partially reduced at pH 4.0, but the E105L mutant was completely reduced to form a 5cHS ferrous heme. These results imply that the presence of an acidic residue near the distal site may prevent reduction of the heme iron at acidic pH.

Globin-coupled heme containing oxygen sensor soluble adenylate cyclase in Leishmania prevents cell death during hypoxia.

Globin and adenylate cyclase play individually numerous crucial roles in eukaryotic organisms. Comparison of the amino acid sequences of globins and adenylate cyclase from prokaryotic to eukaryotic organisms suggests that they share an early common ancestor, even though these proteins execute different functions in these two kingdoms. The latest studies of biological signaling molecules in both prokaryotic and eukaryotic organisms have discovered a new class of heme-containing proteins that act as sensors. The protein of the globin family is still unknown in the trypanosomatid parasites, Trypanosome and Leishmania. In addition, globin-coupled heme containing adenylate cyclase is undescribed in the literature. Here we report a globin-coupled heme containing adenylate cyclase (HemAC-Lm) in the unicellular eukaryotic organism Leishmania. The protein exhibits spectral properties similar to neuroglobin and cytoglobin. Localization studies and activity measurements demonstrate that the protein is present in cytosol and oxygen directly stimulates adenylate cyclase activity in vivo and in vitro. Gene knockdown and overexpression studies suggest that O2-dependent cAMP signaling via protein kinase A plays a fundamental role in cell survival through suppression of oxidative stress under hypoxia. In addition, the enzyme-dependent cAMP generation shows a stimulatory as well as inhibitory role in cell proliferation of Leishmania promastigotes during normoxia. Our work begins to clarify how O2-dependent cAMP generation by adenylate cyclase is likely to function in cellular adaptability under various O2 tensions.

Protection against peroxynitrite by pseudoperoxidase from Leishmania major.

Heme proteins share the ability to detoxify reactive nitrogen intermediates (NO and peroxynitrite). But, to date, no heme-containing enzymatic defense against toxic reactive nitrogen intermediates has been discovered in Leishmania species. We have cloned, expressed, and characterized a pseudoperoxidase from Leishmania major (LmPP) that is capable of detoxifying peroxynitrite (ONOO(-)). Optical, EPR, and resonance Raman spectral studies demonstrate that ONOO(-) can rapidly convert the six-coordinate ferric low-spin to a ferric high-spin form at neutral pH. Western blotting and immunofluorescence studies with anti-LmPP antibody show that the mature enzyme is located at the plasma membrane of amastigotes and is expressed eightfold higher in amastigotes compared to promastigotes. Moreover, to further investigate its exact physiological role in Leishmania, we have created LmPP-knockout mutants by gene replacement in L. major strains. IC(50) values for exogenously added H(2)O(2) or 3-morpholinosydnonimine (SIN1) show that deletion of LmPP in L. major renders the cell more susceptible to SIN1. The null mutant cells exhibit a marked decrease in virulence on infection with activated macrophages as well as inoculation into BALB/c mice. Collectively, these data provide strong evidence that LmPP plays an important role in the enzymatic defense against ONOO(-) within macrophages.

NAD(P)H cytochrome b5 oxidoreductase deficiency in Leishmania major results in impaired linoleate synthesis followed by increased oxidative stress and cell death.

NAD(P)H cytochrome b(5) oxidoreductase (Ncb5or), comprising cytochrome b(5) and cytochrome b(5) reductase domains, is widely distributed in eukaryotic organisms. Although Ncb5or plays a crucial role in lipid metabolism of mice, so far no Ncb5or gene has been reported in the unicellular parasitic protozoa Leishmania species. We have cloned, expressed, and characterized Ncb5or gene from Leishmania major. Steady state catalysis and spectral studies show that NADH can quickly reduce the ferric state of the enzyme to the ferrous state and is able to donate an electron(s) to external acceptors. To elucidate its exact physiological role in Leishmania, we attempted to create NAD(P)H cytochrome b(5) oxidoreductase from L. major (LmNcb5or) knock-out mutants by targeted gene replacement technique. A free fatty acid profile in knock-out (KO) cells reveals marked deficiency in linoleate and linolenate when compared with wild type (WT) or overexpressing cells. KO culture has a higher percentage of dead cells compared with both WT and overexpressing cells. Increased O(2) uptake, uncoupling and ATP synthesis, and loss of mitochondrial membrane potential are evident in KO cells. Flow cytometric analysis reveals the presence of a higher concentration of intracellular H(2)O(2), indicative of increased oxidative stress in parasites lacking LmNcb5or. Cell death is significantly reduced when the KO cells are pretreated with BSA bound linoleate. Real time PCR studies demonstrate a higher Δ12 desaturase, superoxide dismutase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA with a concomitant fall in Δ9 desaturase mRNA expression in LmNcb5or null cell line. Together these findings suggest that decreased linoleate synthesis, and increased oxidative stress and apoptosis are the major consequences of LmNcb5or deficiency in Leishmania.