NLRC5 senses NAD+ depletion, forming a PANoptosome and driving PANoptosis and inflammation.

NLRs constitute a large, highly conserved family of cytosolic pattern recognition receptors that are central to health and disease, making them key therapeutic targets. NLRC5 is an enigmatic NLR with mutations associated with inflammatory and infectious diseases, but little is known about its function as an innate immune sensor and cell death regulator. Therefore, we screened for NLRC5's role in response to infections, PAMPs, DAMPs, and cytokines. We identified that NLRC5 acts as an innate immune sensor to drive inflammatory cell death, PANoptosis, in response to specific ligands, including PAMP/heme and heme/cytokine combinations. NLRC5 interacted with NLRP12 and PANoptosome components to form a cell death complex, suggesting an NLR network forms similar to those in plants. Mechanistically, TLR signaling and NAD+ levels regulated NLRC5 expression and ROS production to control cell death. Furthermore, NLRC5-deficient mice were protected in hemolytic and inflammatory models, suggesting that NLRC5 could be a potential therapeutic target.

The NLR family of innate immune and cell death sensors.

Nucleotide-binding oligomerization domain (NOD)-like receptors, also known as nucleotide-binding leucine-rich repeat receptors (NLRs), are a family of cytosolic pattern recognition receptors that detect a wide variety of pathogenic and sterile triggers. Activation of specific NLRs initiates pro- or anti-inflammatory signaling cascades and the formation of inflammasomes-multi-protein complexes that induce caspase-1 activation to drive inflammatory cytokine maturation and lytic cell death, pyroptosis. Certain NLRs and inflammasomes act as integral components of larger cell death complexes-PANoptosomes-driving another form of lytic cell death, PANoptosis. Here, we review the current understanding of the evolution, structure, and function of NLRs in health and disease. We discuss the concept of NLR networks and their roles in driving cell death and immunity. An improved mechanistic understanding of NLRs may provide therapeutic strategies applicable across infectious and inflammatory diseases and in cancer.

NLRP12-PANoptosome activates PANoptosis and pathology in response to heme and PAMPs.

Cytosolic innate immune sensors are critical for host defense and form complexes, such as inflammasomes and PANoptosomes, that induce inflammatory cell death. The sensor NLRP12 is associated with infectious and inflammatory diseases, but its activating triggers and roles in cell death and inflammation remain unclear. Here, we discovered that NLRP12 drives inflammasome and PANoptosome activation, cell death, and inflammation in response to heme plus PAMPs or TNF. TLR2/4-mediated signaling through IRF1 induced Nlrp12 expression, which led to inflammasome formation to induce maturation of IL-1ß and IL-18. The inflammasome also served as an integral component of a larger NLRP12-PANoptosome that drove inflammatory cell death through caspase-8/RIPK3. Deletion of Nlrp12 protected mice from acute kidney injury and lethality in a hemolytic model. Overall, we identified NLRP12 as an essential cytosolic sensor for heme plus PAMPs-mediated PANoptosis, inflammation, and pathology, suggesting that NLRP12 and molecules in this pathway are potential drug targets for hemolytic and inflammatory diseases.

Single cell analysis of PANoptosome cell death complexes through an expansion microscopy method.

In response to infection or sterile insults, inflammatory programmed cell death is an essential component of the innate immune response to remove infected or damaged cells. PANoptosis is a unique innate immune inflammatory cell death pathway regulated by multifaceted macromolecular complexes called PANoptosomes, which integrate components from other cell death pathways. Growing evidence shows that PANoptosis can be triggered in many physiological conditions, including viral and bacterial infections, cytokine storms, and cancers. However, PANoptosomes at the single cell level have not yet been fully characterized. Initial investigations have suggested that key pyroptotic, apoptotic, and necroptotic molecules including the inflammasome adaptor protein ASC, apoptotic caspase-8 (CASP8), and necroptotic RIPK3 are conserved components of PANoptosomes. Here, we optimized an immunofluorescence procedure to probe the highly dynamic multiprotein PANoptosome complexes across various innate immune cell death-inducing conditions. We first identified and validated antibodies to stain endogenous mouse ASC, CASP8, and RIPK3, without residual staining in the respective knockout cells. We then assessed the formation of PANoptosomes across innate immune cell death-inducing conditions by monitoring the colocalization of ASC with CASP8 and/or RIPK3. Finally, we established an expansion microscopy procedure using these validated antibodies to image the organization of ASC, CASP8, and RIPK3 within the PANoptosome. This optimized protocol, which can be easily adapted to study other multiprotein complexes and other cell death triggers, provides confirmation of PANoptosome assembly in individual cells and forms the foundation for a deeper molecular understanding of the PANoptosome complex and PANoptosis to facilitate therapeutic targeting.

NLRC4 Deficiency Leads to Enhanced Phosphorylation of MLKL and Necroptosis.

Hosts rely on the innate immune system to clear pathogens in response to infection. Pathogen-associated molecular patterns bind to innate immune receptors and engage activation of downstream signaling to initiate a host immune response to fight infection. A key component of this innate response is programmed cell death. Recent work has highlighted significant cross-talk and functional redundancy between cell death pathways, leading to the discovery of PANoptosis, an inflammatory programmed cell death pathway dependent on PANoptosomes, which are innate immune danger-sensing complexes that activate inflammatory cell death and contain caspases with or without inflammasome components and receptor interacting protein homotypic interaction motif-containing proteins. Although PANoptosis has been characterized in response to a growing number of pathogens, inflammatory diseases, and cancer, its role and the functional consequences of PANoptotic component modulation during NLR family CARD domain-containing protein 4 (NLRC4) activation by Pseudomonas aeruginosa infection remain unknown. In this study, we show that P. aeruginosa can induce PANoptosis in mouse bone marrow-derived macrophages (BMDMs). Only the combined deletion of caspase-1, -11, -8, and RIPK3 protected mouse BMDMs from cell death. Moreover, we showed that PANoptotic components act in a compensatory manner; in the absence of NAIP5 and NLRC4 during P. aeruginosa challenge, activation of caspase-1, -3, -7, and -8 was reduced, whereas alternative cell death molecules such as RIPK1 and MLKL were activated in mouse BMDMs. Taken together, these data highlight the extensive cross-talk between cell death signaling molecules and showcase the plasticity of the system.

The Transcription Factor IRF9 Promotes Colorectal Cancer via Modulating the IL-6/STAT3 Signaling Axis.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, and innate immune responses and inflammation are known to affect the course of disease. Interferon (IFN) signaling in particular is critical for modulating inflammation-associated diseases including CRC. While the effects of IFN signaling in CRC have been studied, results have been conflicting. Furthermore, individual molecules in the IFN pathway that could be therapeutically targeted have distinct functions, with many of their diverse roles in CRC remaining unclear. Here, we found that IRF9 had an oncogenic effect in CRC; loss of IRF9 reduced tumorigenesis in both azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced and spontaneous CRC models. IRF9 also reduced DSS-induced colitis and inflammation in the colon, but it had no effect on the NF-κB and MAPK signaling activation. Instead, IRF9 enhanced the transcription and production of the inflammatory cytokine IL-6. By promoting IL-6 release, IRF9 drove the activation of pro-oncogenic STAT3 signaling in the colon. Overall, our study found that IRF9 promoted the development of CRC via modulation of the IL-6/STAT3 signaling axis, identifying multiple potential targets and suggesting new therapeutic strategies for the treatment of CRC.

BAFF Attenuates Immunosuppressive Monocytes in the Melanoma Tumor Microenvironment.

Emerging evidence indicates B-cell activating factor (BAFF, Tnfsf13b) to be an important cytokine for antitumor immunity. In this study, we generated a BAFF-overexpressing B16.F10 melanoma cell model and found that BAFF-expressing tumors grow more slowly in vivo than control tumors. The tumor microenvironment (TME) of BAFF-overexpressing tumors had decreased myeloid infiltrates with lower PD-L1 expression. Monocyte depletion and anti-PD-L1 antibody treatment confirmed the functional importance of monocytes for the phenotype of BAFF-mediated tumor growth delay. RNA sequencing analysis confirmed that monocytes isolated from BAFF-overexpressing tumors were characterized by a less exhaustive phenotype and were enriched for in genes involved in activating adaptive immune responses and NF-κB signaling. Evaluation of patients with late-stage metastatic melanoma treated with inhibitors of the PD-1/PD-L1 axis demonstrated a stratification of patients with high and low BAFF plasma levels. Patients with high BAFF levels experienced lower responses to anti-PD-1 immunotherapies. In summary, these results show that BAFF, through its effect on tumor-infiltrating monocytes, not only impacts primary tumor growth but can serve as a biomarker to predict response to anti-PD-1 immunotherapy in advanced disease. SIGNIFICANCE: The BAFF cytokine regulates monocytes in the melanoma microenvironment to suppress tumor growth, highlighting the importance of BAFF in antitumor immunity.

ADAR1 restricts ZBP1-mediated immune response and PANoptosis to promote tumorigenesis.

Cell death provides host defense and maintains homeostasis. Zα-containing molecules are essential for these processes. Z-DNA binding protein 1 (ZBP1) activates inflammatory cell death, PANoptosis, whereas adenosine deaminase acting on RNA 1 (ADAR1) serves as an RNA editor to maintain homeostasis. Here, we identify and characterize ADAR1's interaction with ZBP1, defining its role in cell death regulation and tumorigenesis. Combining interferons (IFNs) and nuclear export inhibitors (NEIs) activates ZBP1-dependent PANoptosis. ADAR1 suppresses this PANoptosis by interacting with the Zα2 domain of ZBP1 to limit ZBP1 and RIPK3 interactions. Adar1fl/flLysMcre mice are resistant to development of colorectal cancer and melanoma, but deletion of the ZBP1 Zα2 domain restores tumorigenesis in these mice. In addition, treating wild-type mice with IFN-γ and the NEI KPT-330 regresses melanoma in a ZBP1-dependent manner. Our findings suggest that ADAR1 suppresses ZBP1-mediated PANoptosis, promoting tumorigenesis. Defining the functions of ADAR1 and ZBP1 in cell death is fundamental to informing therapeutic strategies for cancer and other diseases.

Inflammatory Cell Death, PANoptosis, Mediated by Cytokines in Diverse Cancer Lineages Inhibits Tumor Growth.

Resistance to cell death is a hallmark of cancer. Immunotherapy, particularly immune checkpoint blockade therapy, drives immune-mediated cell death and has greatly improved treatment outcomes for some patients with cancer, but it often fails clinically. Its success relies on the cytokines and cytotoxic functions of effector immune cells to bypass the resistance to cell death and eliminate cancer cells. However, the specific cytokines capable of inducing cell death in tumors and the mechanisms that connect cytokines to cell death across cancer cell types remain unknown. In this study, we analyzed expression of several cytokines that are modulated in tumors and found correlations between cytokine expression and mortality. Of several cytokines tested for their ability to kill cancer cells, only TNF-α and IFN-γ together were able to induce cell death in 13 distinct human cancer cell lines derived from colon and lung cancer, melanoma, and leukemia. Further evaluation of the specific programmed cell death pathways activated by TNF-α and IFN-γ in these cancer lines identified PANoptosis, a form of inflammatory cell death that was previously shown to be activated by contemporaneous engagement of components from pyroptosis, apoptosis, and/or necroptosis. Specifically, TNF-α and IFN-γ triggered activation of gasdermin D, gasdermin E, caspase-8, caspase-3, caspase-7, and MLKL. Furthermore, the intratumoral administration of TNF-α and IFN-γ suppressed the growth of transplanted xenograft tumors in an NSG mouse model. Overall, this study shows that PANoptosis, induced by synergism of TNF-α and IFN-γ, is an important mechanism to kill cancer cells and suppress tumor growth that could be therapeutically targeted.

Advances in Understanding Activation and Function of the NLRC4 Inflammasome.

Innate immune receptors initiate a host immune response, or inflammatory response, upon detecting pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Among the innate immune receptors, nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) play a pivotal role in detecting cytosolic PAMPs and DAMPs. Some NLRs can form a multiprotein cytosolic complex known as the inflammasome. Inflammasome activation triggers caspase-1-mediated cleavage of the pore-forming protein gasdermin D (GSDMD), which drives a form of inflammatory cell death called pyroptosis. Parallelly, activated caspase-1 cleaves immature cytokines pro-IL-1ß and pro-IL-18 into their active forms, which can be released via GSDMD membrane pores. The NLR family apoptosis inhibitory proteins (NAIP)-NLR family caspase-associated recruitment domain-containing protein 4 (NLRC4) inflammasome is important for mounting an immune response against Gram-negative bacteria. NLRC4 is activated through NAIPs sensing type 3 secretion system (T3SS) proteins from Gram-negative bacteria, such as Salmonella Typhimurium. Mutations in NAIPs and NLRC4 are linked to autoinflammatory disorders in humans. In this review, we highlight the role of the NAIP/NLRC4 inflammasome in host defense, autoinflammatory diseases, cancer, and cell death. We also discuss evidence pointing to a role of NLRC4 in PANoptosis, which was recently identified as a unique inflammatory programmed cell death pathway with important physiological relevance in a range of diseases. Improved understanding of the NLRC4 inflammasome and its potential roles in PANoptosis paves the way for identifying new therapeutic strategies to target disease.

Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes.

COVID-19 is characterized by excessive production of pro-inflammatory cytokines and acute lung damage associated with patient mortality. While multiple inflammatory cytokines are produced by innate immune cells during SARS-CoV-2 infection, we found that only the combination of TNF-α and IFN-γ induced inflammatory cell death characterized by inflammatory cell death, PANoptosis. Mechanistically, TNF-α and IFN-γ co-treatment activated the JAK/STAT1/IRF1 axis, inducing nitric oxide production and driving caspase-8/FADD-mediated PANoptosis. TNF-α and IFN-γ caused a lethal cytokine shock in mice that mirrors the tissue damage and inflammation of COVID-19, and inhibiting PANoptosis protected mice from this pathology and death. Furthermore, treating with neutralizing antibodies against TNF-α and IFN-γ protected mice from mortality during SARS-CoV-2 infection, sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other infectious and autoinflammatory diseases by limiting tissue damage/inflammation.

Synergism of TNF-α and IFN-γ triggers inflammatory cell death, tissue damage, and mortality in SARS-CoV-2 infection and cytokine shock syndromes.

The COVID-19 pandemic has caused significant morbidity and mortality. Currently, there is a critical shortage of proven treatment options and an urgent need to understand the pathogenesis of multi-organ failure and lung damage. Cytokine storm is associated with severe inflammation and organ damage during COVID-19. However, a detailed molecular pathway defining this cytokine storm is lacking, and gaining mechanistic understanding of how SARS-CoV-2 elicits a hyperactive inflammatory response is critical to develop effective therapeutics. Of the multiple inflammatory cytokines produced by innate immune cells during SARS-CoV-2 infection, we found that the combined production of TNF-α and IFN-γ specifically induced inflammatory cell death, PANoptosis, characterized by gasdermin-mediated pyroptosis, caspase-8-mediated apoptosis, and MLKL-mediated necroptosis. Deletion of pyroptosis, apoptosis, or necroptosis mediators individually was not sufficient to protect against cell death. However, cells deficient in both RIPK3 and caspase-8 or RIPK3 and FADD were resistant to this cell death. Mechanistically, the JAK/STAT1/IRF1 axis activated by TNF-α and IFN-γ co-treatment induced iNOS for the production of nitric oxide. Pharmacological and genetic deletion of this pathway inhibited pyroptosis, apoptosis, and necroptosis in macrophages. Moreover, inhibition of PANoptosis protected mice from TNF-α and IFN-γ-induced lethal cytokine shock that mirrors the pathological symptoms of COVID-19. In vivo neutralization of both TNF-α and IFN-γ in multiple disease models associated with cytokine storm showed that this treatment provided substantial protection against not only SARS-CoV-2 infection, but also sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock models, demonstrating the broad physiological relevance of this mechanism. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other cytokine storm-driven syndromes by limiting inflammation and tissue damage. The findings also provide a molecular and mechanistic description for the term cytokine storm. Additionally, these results open new avenues for the treatment of other infectious and autoinflammatory diseases and cancers where TNF-α and IFN-γ synergism play key pathological roles.

Fragile X mental retardation protein protects against tumour necrosis factor-mediated cell death and liver injury.

OBJECTIVE: The Fragile X mental retardation (FMR) syndrome is a frequently inherited intellectual disability caused by decreased or absent expression of the FMR protein (FMRP). Lack of FMRP is associated with neuronal degradation and cognitive dysfunction but its role outside the central nervous system is insufficiently studied. Here, we identify a role of FMRP in liver disease. DESIGN: Mice lacking Fmr1 gene expression were used to study the role of FMRP during tumour necrosis factor (TNF)-induced liver damage in disease model systems. Liver damage and mechanistic studies were performed using real-time PCR, Western Blot, staining of tissue sections and clinical chemistry. RESULTS: Fmr1null mice exhibited increased liver damage during virus-mediated hepatitis following infection with the lymphocytic choriomeningitis virus. Exposure to TNF resulted in severe liver damage due to increased hepatocyte cell death. Consistently, we found increased caspase-8 and caspase-3 activation following TNF stimulation. Furthermore, we demonstrate FMRP to be critically important for regulating key molecules in TNF receptor 1 (TNFR1)-dependent apoptosis and necroptosis including CYLD, c-FLIPS and JNK, which contribute to prolonged RIPK1 expression. Accordingly, the RIPK1 inhibitor Necrostatin-1s could reduce liver cell death and alleviate liver damage in Fmr1null mice following TNF exposure. Consistently, FMRP-deficient mice developed increased pathology during acute cholestasis following bile duct ligation, which coincided with increased hepatic expression of RIPK1, RIPK3 and phosphorylation of MLKL. CONCLUSIONS: We show that FMRP plays a central role in the inhibition of TNF-mediated cell death during infection and liver disease.

iRhom2 inhibits bile duct obstruction-induced liver fibrosis.

Chronic liver disease can induce prolonged activation of hepatic stellate cells, which may result in liver fibrosis. Inactive rhomboid protein 2 (iRhom2) is required for the maturation of A disintegrin and metalloprotease 17 (ADAM17, also called TACE), which is responsible for the cleavage of membrane-bound tumor necrosis factor-α (TNF-α) and its receptors (TNFRs). Here, using the murine bile duct ligation (BDL) model, we showed that the abundance of iRhom2 and activation of ADAM17 increased during liver fibrosis. Consistent with this, concentrations of ADAM17 substrates were increased in plasma samples from mice after BDL and in patients suffering from liver cirrhosis. We observed increased liver fibrosis, accelerated disease progression, and an increase in activated stellate cells after BDL in mice lacking iRhom2 (Rhbdf2-/- ) compared to that in controls. In vitro primary mouse hepatic stellate cells exhibited iRhom2-dependent shedding of the ADAM17 substrates TNFR1 and TNFR2. In vivo TNFR shedding after BDL also depended on iRhom2. Treatment of Rhbdf2-/- mice with the TNF-α inhibitor etanercept reduced the presence of activated stellate cells and alleviated liver fibrosis after BDL. Together, these data suggest that iRhom2-mediated inhibition of TNFR signaling protects against liver fibrosis.

Renal control of disease tolerance to malaria.

Malaria, the disease caused by Plasmodium spp. infection, remains a major global cause of morbidity and mortality. Host protection from malaria relies on immune-driven resistance mechanisms that kill Plasmodium However, these mechanisms are not sufficient per se to avoid the development of severe forms of disease. This is accomplished instead via the establishment of disease tolerance to malaria, a defense strategy that does not target Plasmodium directly. Here we demonstrate that the establishment of disease tolerance to malaria relies on a tissue damage-control mechanism that operates specifically in renal proximal tubule epithelial cells (RPTEC). This protective response relies on the induction of heme oxygenase-1 (HMOX1; HO-1) and ferritin H chain (FTH) via a mechanism that involves the transcription-factor nuclear-factor E2-related factor-2 (NRF2). As it accumulates in plasma and urine during the blood stage of Plasmodium infection, labile heme is detoxified in RPTEC by HO-1 and FTH, preventing the development of acute kidney injury, a clinical hallmark of severe malaria.

Cholestasis induced liver pathology results in dysfunctional immune responses after arenavirus infection.

Immune responses are critical for defense against pathogens. However, prolonged viral infection can result in defective T cell immunity, leading to chronic viral infection. We studied immune activation in response to arenavirus infection during cholestasis using bile duct ligation (BDL). We monitored T cell responses, virus load and liver pathology markers after infection with lymphocytic choriomeningitis virus (LCMV). BDL mice failed to induce protective anti-viral immunity against LCMV and consequently exhibited chronic viral infection. BDL mice exhibited reduced anti-viral T cell immunity as well as reduced type 1 interferon production early after LCMV infection. Consistently, the presence of serum from BDL mice reduced the responsiveness of dendritic cell (DC) and T cell cultures when compared to Sham controls. Following fractionation and mass spectrometry analyses of sera, we identified several serum factors to be upregulated following BDL including bilirubin, bile acids, 78 kDa Glucose regulated protein (GRP78) and liver enzymes. Bilirubin and GRP78 were capable of inhibiting DC and T cell activation. In this work, we demonstrate that liver damage mediated by cholestasis results in defective immune induction following arenavirus infection.

B Cell-Mediated Maintenance of Cluster of Differentiation 169-Positive Cells Is Critical for Liver Regeneration.

The liver has an extraordinary capacity to regenerate through activation of key molecular pathways. However, central regulators controlling liver regeneration remain insufficiently studied. Here, we show that B cell-deficient animals failed to induce sufficient liver regeneration after partial hepatectomy (PHx). Consistently, adoptive transfer of B cells could rescue defective liver regeneration. B cell-mediated lymphotoxin beta production promoted recovery from PHx. Absence of B cells coincided with loss of splenic cluster of differentiation 169-positive (CD169+ ) macrophages. Moreover, depletion of CD169+ cells resulted in defective liver regeneration and decreased survival, which was associated with reduced hepatocyte proliferation. Mechanistically, CD169+ cells contributed to liver regeneration by inducing hepatic interleukin-6 (IL-6) production and signal transducer and activator of transcription 3 activation. Accordingly, treatment of CD169+ cell-depleted animals with IL-6/IL-6 receptor rescued liver regeneration and severe pathology following PHx. Conclusion: We identified CD169+ cells to be a central trigger for liver regeneration, by inducing key signaling pathways important for liver regeneration.

Deletions in the cytoplasmic domain of iRhom1 and iRhom2 promote shedding of the TNF receptor by the protease ADAM17.

The protease ADAM17 (a disintegrin and metalloproteinase 17) catalyzes the shedding of various transmembrane proteins from the surface of cells, including tumor necrosis factor (TNF) and its receptors. Liberation of TNF receptors (TNFRs) from cell surfaces can dampen the cellular response to TNF, a cytokine that is critical in the innate immune response and promotes programmed cell death but can also promote sepsis. Catalytically inactive members of the rhomboid family of proteases, iRhom1 and iRhom2, mediate the intracellular transport and maturation of ADAM17. Using a genetic screen, we found that the presence of either iRhom1 or iRhom2 lacking part of their extended amino-terminal cytoplasmic domain (herein referred to as ΔN) increases ADAM17 activity, TNFR shedding, and resistance to TNF-induced cell death in fibrosarcoma cells. Inhibitors of ADAM17, but not of other ADAM family members, prevented the effects of iRhom-ΔN expression. iRhom1 and iRhom2 were functionally redundant, suggesting a conserved role for the iRhom amino termini. Cells from patients with a dominantly inherited cancer susceptibility syndrome called tylosis with esophageal cancer (TOC) have amino-terminal mutations in iRhom2. Keratinocytes from TOC patients exhibited increased TNFR1 shedding compared with cells from healthy donors. Our results explain how loss of the amino terminus in iRhom1 and iRhom2 impairs TNF signaling, despite enhancing ADAM17 activity, and may explain how mutations in the amino-terminal region contribute to the cancer predisposition syndrome TOC.

Plasmodium berghei glycine cleavage system T-protein is non-essential for parasite survival in vertebrate and invertebrate hosts.

T-protein, an aminomethyltransferase, represents one of the four components of glycine cleavage system (GCS) and catalyzes the transfer of methylene group from H-protein intermediate to tetrahydrofolate (THF) forming N(5), N(10)-methylene THF (CH2-THF) with the release of ammonia. The malaria parasite genome encodes T-, H- and L-proteins, but not P-protein which is a glycine decarboxylase generating the aminomethylene group. A putative GCS has been considered to be functional in the parasite mitochondrion despite the absence of a detectable P-protein homologue. In the present study, the mitochondrial localization of T-protein in the malaria parasite was confirmed by immunofluorescence and its essentiality in the entire parasite life cycle was studied by targeting the T-protein locus in Plasmodium berghei (Pb). PbT knock out parasites did not show any growth defect in asexual, sexual and liver stages indicating that the T-protein is dispensable for parasite survival in vertebrate and invertebrate hosts. The absence of P-protein homologue and the non-essentiality of T protein suggest the possible redundancy of GCS activity in the malaria parasite. Nevertheless, the H- and L-proteins of GCS could be essential for malaria parasite because of their involvement in α-ketoacid dehydrogenase reactions.