Pseudomonas Exotoxin A-Mediated Apoptosis Is Bak Dependent and Preceded by the Degradation of Mcl-1

Pseudomonas exotoxin A (PE) is a bacterial toxin that arrests protein synthesis and induces apoptosis. Here, we utilized mouse embryo fibroblasts (MEFs) deficient in Bak and Bax to determine the roles of these proteins in cell death induced by PE. PE induced a rapid and dose-dependent induction of apoptosis in wild-type (WT) and Bax knockout (Bax^−/−) MEFs but failed in Bak knockout (Bak^−/−) and Bax/Bak double-knockout (DKO) MEFs. Also a loss of mitochondrial membrane potential was observed in WT and Bax^−/− MEFs, but not in Bak^−/− or in DKO MEFs, indicating an effect of PE on mitochondrial permeability. PE-mediated inhibition of protein synthesis was identical in all 4 cell lines, indicating that differences in killing were due to steps after the ADP-ribosylation of EF2. Mcl-1, but not Bcl-x_L, was rapidly degraded after PE treatment, consistent with a role for Mcl-1 in the PE death pathway. Bak was associated with Mcl-1 and Bcl-x_L in MEFs and uncoupled from suppressed complexes after PE treatment. Overexpression of Mcl-1 and Bcl-x_L inhibited PE-induced MEF death. Our data suggest that Bak is the preferential mediator of PE-mediated apoptosis and that the rapid degradation of Mcl-1 unleashes Bak to activate apoptosis.Apoptosis is a mode of cell death utilized by multicellular organisms to remove unwanted cells. Also, many different cancer treatments, including chemotherapy and radiotherapy, induce apoptosis and result in the destruction of tumor cells. In some cases, apoptosis resistance can contribute to the failure of chemotherapy (14, 20, 24). Immunotoxins are a class of antitumor agents in which a powerful protein toxin is brought to the cancer cell by an antibody or an antibody fragment (for reviews, see references 28, 29, and 32). Several immunotoxins are currently in clinical trials, and one of these, BL22, targeting CD22, has shown excellent activity in drug-resistant hairy-cell leukemia (18, 19). Also, a fusion protein in which a fragment of diphtheria toxin is fused to the cytokine interleukin 2 (IL-2) (Ontak) is approved for the treatment of cutaneous T-cell lymphoma (26). Several studies carried out to determine how protein toxins and immunotoxins containing these toxins kill target cells have reported caspase activation (13, 16, 17, 30, 33). However, the steps leading up to caspase activation by these toxins that inhibit protein synthesis have not been elucidated.Bcl-2 family members are essential regulators of the mitochondrial (intrinsic) apoptosis pathway (1, 21). Proteins of this family have been divided into pro- and antiapoptotic proteins. Antiapoptotic proteins include the multi-Bcl-2 homology (BH) domain proteins Bcl-2, Bcl-x_L, Bcl-w, Mcl-1, Bcl-b, and Bcl2a1. Proapoptotic members can be further classified into two subfamilies, the multi-BH domain Bax homologues, including Bax, Bak, and Bok, and the BH3-only proteins, including Nbk/Bik, Noxa, Hrk, Bad, Bim, Puma, and Bmf. Bax and Bak are the most extensively studied central mediators in the mitochondrial apoptosis pathway (4, 6). Various stimuli, including pathogens, toxic drugs, irradiation, and starvation, induce a conformational change and activation of Bak/Bax, usually via BH3-only proapoptosis proteins. This results in the disruption of mitochondrial membranes and the release of apoptotic factors, such as cytochrome c, SMAC, and apoptosis-inducing factor, which lead to the activation of effector caspases (5, 37, 40, 42, 43).The roles of Bax and Bak can be redundant or nonredundant, depending on the apoptotic stimuli. Bak and Bax can compensate for each other in apoptosis induced by staurosporine, etoposide, UV irradiation, serum deprivation, tBid, Bim, Bad, or Noxa (37, 43). Bak plays an essential role for apoptosis induced by Semliki Forest virus, gliotoxin, Bcl-x_S, and vinblastine (22, 27, 34, 35), while Bax is favored for apoptosis induced by Nbk/Nik, a combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and ionizing irradiation, or TRAIL and 5-fluorouracil (5-FU) (9, 10, 36, 38). Silencing of either Bak or Bax resulted in resistance to apoptosis induced by Neisseria gonorrhoeae and cisplatin (15). Sometimes the same stimulus may result in different outcomes in different cell types. NBK/Bik mediated Bax-dependent cell death in one study (9), while in another study, NBK/Bik activated BAK-mediated apoptosis (31).In the current study, we utilized mutant mouse embryo fibroblasts (MEFs) deficient in Bak, Bax, or both proteins and provided evidence for an essential role of Bak in apoptosis induced by Pseudomonas exotoxin A (PE) and other protein synthesis inhibitors. We found that Bak^−/− cells are resistant to killing by PE and that Mcl-1, which binds to Bak, controls apoptosis induced by PE.     

An IcmF Family Protein,ImpLM, Is an Integral Inner Membrane Protein Interacting with ImpKL,and Its Walker A Motif Is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens

An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpL_M, an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpL_M and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of β-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpL_M is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impL_M mutants with substitutions or deletions in the Walker A motif failed to complement the impL_M deletion mutant for Hcp secretion, which provided evidence that ImpL_M may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpL_M and another essential T6SS component, ImpK_L. Topology and biochemical fractionation analyses suggested that ImpK_L is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpL_M-ImpK_L interaction domains suggested that ImpL_M interacts with ImpK_L via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpL_M interacts with ImpK_L, and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens.Many pathogenic gram-negative bacteria employ protein secretion systems formed by macromolecular complexes to deliver proteins or protein-DNA complexes across the bacterial membrane. In addition to the general secretory (Sec) pathway (18, 52) and twin-arginine translocation (Tat) pathway (7, 34), which transport proteins across the inner membrane into the periplasm, at least six distinct protein secretion systems occur in gram-negative bacteria (28, 46, 66). These systems are able to secrete proteins from the cytoplasm or periplasm to the external environment or the host cell and include the well-documented type I to type V secretion systems (T1SS to T5SS) (10, 15, 23, 26, 30) and a recently discovered type VI secretion system (T6SS) (4, 8, 22, 41, 48, 49). These systems use ATPase or a proton motive force to energize assembly of the protein secretion machinery and/or substrate translocation (2, 6, 41, 44, 60).Agrobacterium tumefaciens is a soilborne pathogenic gram-negative bacterium that causes crown gall disease in a wide range of plants. Using an archetypal T4SS (9), A. tumefaciens translocates oncogenic transferred DNA and effector proteins to the host and ultimately integrates transferred DNA into the host genome. Because of its unique interkingdom DNA transfer, this bacterium has been extensively studied and used to transform foreign DNA into plants and fungi (11, 24, 40, 67). In addition to the T4SS, A. tumefaciens encodes several other secretion systems, including the Sec pathway, the Tat pathway, T1SS, T5SS, and the recently identified T6SS (72). T6SS is highly conserved and widely distributed in animal- and plant-associated Proteobacteria and plays an important role in the virulence of several human and animal pathogens (14, 19, 41, 48, 56, 63, 74). However, T6SS seems to play only a minor role or even a negative role in infection or virulence of the plant-associated pathogens or symbionts studied to date (5, 37-39, 72).T6SS was initially designated IAHP (IcmF-associated homologous protein) clusters (13). Before T6SS was documented by Pukatzki et al. in Vibrio cholerae (48), mutations in this gene cluster in the plant symbiont Rhizobium leguminosarum (5) and the fish pathogen Edwardsiella tarda (51) caused defects in protein secretion. In V. cholerae, T6SS was responsible for the loss of cytotoxicity for amoebae and for secretion of two proteins lacking a signal peptide, hemolysin-coregulated protein (Hcp) and valine-glycine repeat protein (VgrG). Secretion of Hcp is the hallmark of T6SS. Interestingly, mutation of hcp blocks the secretion of VgrG proteins (VgrG-1, VgrG-2, and VgrG-3), and, conversely, vgrG-1 and vgrG-2 are both required for secretion of the Hcp and VgrG proteins from V. cholerae (47, 48). Similarly, a requirement of Hcp for VgrG secretion and a requirement of VgrG for Hcp secretion have also been shown for E. tarda (74). Because Hcp forms a hexameric ring (41) stacked in a tube-like structure in vitro (3, 35) and VgrG has a predicted trimeric phage tail spike-like structure similar to that of the T4 phage gp5-gp27 complex (47), Hcp and VgrG have been postulated to form an extracellular translocon. This model is further supported by two recent crystallography studies showing that Hcp, VgrG, and a T4 phage gp25-like protein resembled membrane penetration tails of bacteriophages (35, 45).Little is known about the topology and structure of T6SS machinery subunits and the distinction between genes encoding machinery subunits and genes encoding regulatory proteins. Posttranslational regulation via the phosphorylation of Fha1 by a serine-threonine kinase (PpkA) is required for Hcp secretion from Pseudomonas aeruginosa (42). Genetic evidence for P. aeruginosa suggested that the T6SS may utilize a ClpV-like AAA⁺ ATPase to provide the energy for machinery assembly or substrate translocation (41). A recent study of V. cholerae suggested that ClpV ATPase activity is responsible for remodeling the VipA/VipB tubules which are crucial for type VI substrate secretion (6). An outer membrane lipoprotein, SciN, is an essential T6SS component for mediating Hcp secretion from enteroaggregative Escherichia coli (1). A systematic study of the T6SS machinery in E. tarda revealed that 13 of 16 genes in the evp gene cluster are essential for secretion of T6S substrates (74), which suggests the core components of the T6SS. Interestingly, most of the core components conserved in T6SS are predicted soluble proteins without recognizable signal peptide and transmembrane (TM) domains.The intracellular multiplication F (IcmF) and H (IcmH) proteins are among the few core components with obvious TM domains (8). In Legionella pneumophila Dot/Icm T4SSb, IcmF and IcmH are both membrane localized and partially required for L. pneumophila replication in macrophages (58, 70, 75). IcmF and IcmH are thought to interact with each other in stabilizing the T4SS complex in L. pneumophila (58). In T6SS, IcmF is one of the essential components required for secretion of Hcp from several animal pathogens, including V. cholerae (48), Aeromonas hydrophila (63), E. tarda (74), and P. aeruginosa (41), as well as the plant pathogens A. tumefaciens (72) and Pectobacterium atrosepticum (39). In E. tarda, IcmF (EvpO) interacted with IcmH (EvpN), EvpL, and EvpA in a yeast two-hybrid assay, and its putative nucleotide-binding site (Walker A motif) was not essential for secretion of T6SS substrates (74).In this study, we characterized the topology and interactions of the IcmF and IcmH family proteins ImpL_M and ImpK_L, which are two essential components of the T6SS of A. tumefaciens. We adapted the nomenclature proposed by Cascales (8), using the annotated gene designation followed by the letter indicated by Shalom et al. (59). Our data indicate that ImpL_M and ImpK_L are both integral inner membrane proteins and interact with each other via their N-terminal domains residing in the cytoplasm. We also provide genetic evidence showing that ImpL_M may function as a nucleoside triphosphate (NTP)-binding protein or nucleoside triphosphatase to mediate T6S machinery assembly and/or substrate secretion.     

c-Jun NH2-Terminal Kinase Is Required for Lineage-Specific Differentiation but Not Stem Cell Self-Renewal

The c-Jun NH₂-terminal kinase (JNK) is implicated in proliferation. Mice with a deficiency of either the Jnk1 or the Jnk2 genes are viable, but a compound deficiency of both Jnk1 and Jnk2 causes early embryonic lethality. Studies using conditional gene ablation and chemical genetic approaches demonstrate that the combined loss of JNK1 and JNK2 protein kinase function results in rapid senescence. To test whether this role of JNK was required for stem cell proliferation, we isolated embryonic stem (ES) cells from wild-type and JNK-deficient mice. We found that Jnk1^−/− Jnk2^−/− ES cells underwent self-renewal, but these cells proliferated more rapidly than wild-type ES cells and exhibited major defects in lineage-specific differentiation. Together, these data demonstrate that JNK is not required for proliferation or self-renewal of ES cells, but JNK plays a key role in the differentiation of ES cells.The c-Jun NH₂-terminal kinase (JNK) is a member of the mitogen-activated protein (MAP) kinase group of signaling proteins. JNK is encoded by two ubiquitously expressed genes (Jnk1 and Jnk2) and by a third gene (Jnk3) that is selectively expressed in neurons (14). Gene disruption studies demonstrate that mice without Jnk1 or Jnk2 are viable, but compound deficiency of both Jnk1 and Jnk2 causes early embryonic lethality (14). Murine embryonic fibroblasts (MEFs) isolated from Jnk1^−/− Jnk2^−/− mice exhibit a severe growth retardation phenotype (54). The markedly reduced growth of Jnk1^−/− Jnk2^−/− MEFs is consistent with the finding that JNK is critically required for the regulation of AP1-dependent gene expression (56) that is implicated in cellular proliferation (26). Thus, Jnk1^−/− Jnk2^−/− MEFs express low levels of AP1 proteins (e.g., c-Jun and JunD) and exhibit marked defects in AP1 target gene expression (34, 56). This loss of AP1 function is mediated, in part, by reduced phosphorylation of the activation domain of Jun family proteins and ATF2 (56).More recent studies using a conditional gene ablation strategy have demonstrated that compound JNK deficiency causes rapid senescence (12). This conclusion was confirmed by using chemical genetic analysis with MEFs isolated from mice with a germ line mutation that sensitizes JNK to inhibition by a predesigned small-molecule drug (12, 25). This form of senescence was found to be p53 dependent (12) and resembles the p53-dependent senescence of c-Jun^−/− MEFs (49). These data indicate that JNK plays a critical role in cellular proliferation. Indeed, it is possible that the p53-dependent senescence observed in JNK-deficient cells may contribute to aging. This is because altered p53 function is established to be an important determinant of early aging (36, 55). Importantly, this role of p53 in aging appears to be distinct from p53-mediated tumor suppression and DNA damage responses (21, 39, 43).One aspect of the aging process is a reduction in the regenerative capacity of stem cells (50). Indeed, it has been established that altered p53 activity associated with aging causes decreased stem cell function (8, 18, 42) and that disruption of the p53 pathway can increase stem cell function (1). Since JNK can influence p53-dependent senescence (12), these data indicate that JNK may be important for stem cell proliferation and self-renewal potential.Embryonic stem (ES) cells proliferate and are capable of both self-renewal and differentiation to multiple cell types. Indeed, murine ES cells can differentiate to create all tissues within a mouse. The profound growth retardation and rapid p53-dependent senescence of Jnk1^−/− Jnk2^−/− MEFs (12) suggests that JNK may play a critical role in the normal function of ES cells, including self-renewal and differentiation potential. The purpose of the present study was to test this hypothesis. Our approach was to isolate ES cells from wild-type and JNK-deficient mice. We demonstrate that JNK is not required for self-renewal or the proliferation of ES cells. However, JNK is required for ES cell differentiation.     

Selective Expression of Human Immunodeficiency Virus Nef in Specific Immune Cell Populations of Transgenic Mice Is Associated with Distinct AIDS-Like Phenotypes

We previously reported that CD4C/human immunodeficiency virus (HIV)^Nef transgenic (Tg) mice, expressing Nef in CD4⁺ T cells and cells of the macrophage/dendritic cell (DC) lineage, develop a severe AIDS-like disease, characterized by depletion of CD4⁺ T cells, as well as lung, heart, and kidney diseases. In order to determine the contribution of distinct populations of hematopoietic cells to the development of this AIDS-like disease, five additional Tg strains expressing Nef through restricted cell-specific regulatory elements were generated. These Tg strains express Nef in CD4⁺ T cells, DCs, and macrophages (CD4E/HIV^Nef); in CD4⁺ T cells and DCs (mCD4/HIV^Nef and CD4F/HIV^Nef); in macrophages and DCs (CD68/HIV^Nef); or mainly in DCs (CD11c/HIV^Nef). None of these Tg strains developed significant lung and kidney diseases, suggesting the existence of as-yet-unidentified Nef-expressing cell subset(s) that are responsible for inducing organ disease in CD4C/HIV^Nef Tg mice. Mice from all five strains developed persistent oral carriage of Candida albicans, suggesting an impaired immune function. Only strains expressing Nef in CD4⁺ T cells showed CD4⁺ T-cell depletion, activation, and apoptosis. These results demonstrate that expression of Nef in CD4⁺ T cells is the primary determinant of their depletion. Therefore, the pattern of Nef expression in specific cell population(s) largely determines the nature of the resulting pathological changes.The major cell targets and reservoirs for human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) infection in vivo are CD4⁺ T lymphocytes and antigen-presenting cells (macrophages and dendritic cells [DC]) (21, 24, 51). The cell specificity of these viruses is largely dependent on the expression of CD4 and of its coreceptors, CCR5 and CXCR-4, at the cell surface (29, 66). Infection of these immune cells leads to the severe disease, AIDS, showing widespread manifestations, including progressive immunodeficiency, immune activation, CD4⁺ T-cell depletion, wasting, dementia, nephropathy, heart and lung diseases, and susceptibility to opportunistic pathogens, such as Candida albicans (1, 27, 31, 37, 41, 82, 93, 109). It is reasonable to assume that the various pathological changes in AIDS result from the expression of one or many HIV-1/SIV proteins in these immune target cells. However, assigning the contribution of each infected cell subset to each phenotype has been remarkably difficult, despite evidence that AIDS T-cell phenotypes can present very differently depending on the strains of infecting HIV-1 or SIV or on the cells targeted by the virus (4, 39, 49, 52, 72). For example, the T-cell-tropic X4 HIV strains have long been associated with late events and severe CD4⁺ T-cell depletion (22, 85, 96). However, there are a number of target cell subsets expressing CD4 and CXCR-4, and identifying which one is responsible for this enhanced virulence has not been achieved in vivo. Similarly, the replication of SIV in specific regions of the thymus (cortical versus medullary areas), has been associated with very different outcomes but, unfortunately, the critical target cells of the viruses were not identified either in these studies (60, 80). The task is even more complex, because HIV-1 or SIV can infect several cell subsets within a single cell population. In the thymus, double (CD4⁻ CD8⁻)-negative (DN) or triple (CD3⁻ CD4⁻ CD8⁻)-negative (TN) T cells, as well as double-positive (CD4⁺ CD8⁺) (DP) T cells, are infectible by HIV-1 in vitro (9, 28, 74, 84, 98, 99, 110) and in SCID-hu mice (2, 5, 91, 94). In peripheral organs, gut memory CCR5⁺ CD4⁺ T cells are primarily infected with R5 SIV, SHIV, or HIV, while circulating CD4⁺ T cells can be infected by X4 viruses (13, 42, 49, 69, 70, 100, 101, 104). Moreover, some detrimental effects on CD4⁺ T cells have been postulated to originate from HIV-1/SIV gene expression in bystander cells, such as macrophages or DC, suggesting that other infected target cells may contribute to the loss of CD4⁺ T cells (6, 7, 32, 36, 64, 90).Similarly, the infected cell population(s) required and sufficient to induce the organ diseases associated with HIV-1/SIV expression (brain, heart, and kidney) have not yet all been identified. For lung or kidney disease, HIV-specific cytotoxic CD8⁺ T cells (1, 75) or infected podocytes (50, 95), respectively, have been implicated. Activated macrophages have been postulated to play an important role in heart disease (108) and in AIDS dementia (35), although other target cells could be infected by macrophage-tropic viruses and may contribute significantly to the decrease of central nervous system functions (11, 86, 97), as previously pointed out (25).Therefore, because of the widespread nature of HIV-1 infection and the difficulty in extrapolating tropism of HIV-1/SIV in vitro to their cell targeting in vivo (8, 10, 71), alternative approaches are needed to establish the contribution of individual infected cell populations to the multiorgan phenotypes observed in AIDS. To this end, we developed a transgenic (Tg) mouse model of AIDS using a nonreplicating HIV-1 genome expressed through the regulatory sequences of the human CD4 gene (CD4C), in the same murine cells as those targeted by HIV-1 in humans, namely, in immature and mature CD4⁺ T cells, as well as in cells of the macrophage/DC lineages (47, 48, 77; unpublished data). These CD4C/HIV Tg mice develop a multitude of pathologies closely mimicking those of AIDS patients. These include a gradual destruction of the immune system, characterized among other things by thymic and lymphoid organ atrophy, depletion of mature and immature CD4⁺ T lymphocytes, activation of CD4⁺ and CD8⁺ T cells, susceptibility to mucosal candidiasis, HIV-associated nephropathy, and pulmonary and cardiac complications (26, 43, 44, 57, 76, 77, 79, 106). We demonstrated that Nef is the major determinant of the HIV-1 pathogenicity in CD4C/HIV Tg mice (44). The similarities of the AIDS-like phenotypes of these Tg mice to those in human AIDS strongly suggest that such a Tg mouse approach can be used to investigate the contribution of distinct HIV-1-expressing cell populations to their development.In the present study, we constructed and characterized five additional mouse Tg strains expressing Nef, through distinct regulatory elements, in cell populations more restricted than in CD4C/HIV Tg mice. The aim of this effort was to assess whether, and to what extent, the targeting of Nef in distinct immune cell populations affects disease development and progression.     

The Mitogen-Activated Protein Kinase Scaffold KSR1 Is Required for Recruitment of Extracellular Signal-Regulated Kinase to the Immunological Synapse

KSR1 is a mitogen-activated protein (MAP) kinase scaffold that enhances the activation of the MAP kinase extracellular signal-regulated kinase (ERK). The function of KSR1 in NK cell function is not known. Here we show that KSR1 is required for efficient NK-mediated cytolysis and polarization of cytolytic granules. Single-cell analysis showed that ERK is activated in an all-or-none fashion in both wild-type and KSR1-deficient cells. In the absence of KSR1, however, the efficiency of ERK activation is attenuated. Imaging studies showed that KSR1 is recruited to the immunological synapse during T-cell activation and that membrane recruitment of KSR1 is required for recruitment of active ERK to the synapse.Kinase suppressor of Ras was originally identified in Drosophila melanogaster (53) and Caenorhabditis elegans (19, 32, 52) as a positive regulator of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase signaling pathway. It is thought to function as a MAP kinase scaffold because it can bind to Raf, MEK, and ERK (18, 19, 27, 28, 44, 59). While the exact function of KSR is unknown, preassembling the three components of the ERK MAP kinase cascade could function to enhance the efficiency of ERK activation, potentially regulate the subcellular location of ERK activation, and promote access to specific subcellular substrates (16, 45, 46).While only one isoform of KSR is expressed in Drosophila (53), two KSR isoforms have been identified in C. elegans (19, 32, 52) and most higher organisms. They are referred to as KSR1 and KSR2 (32, 43). While KSR1 mRNA and protein are detectable in a wide variety of cells and tissues, including brain, thymus, and muscle (10, 11, 29), little is known about the expression pattern of KSR2.We previously reported the phenotype of KSR1-deficient mice (30). These mice are born at Mendelian ratios and develop without any obvious defects. Using gel filtration, we showed that KSR1 promotes the formation of large signaling complexes containing KSR1, Raf, MEK, and ERK (30). Using both primary T cells stimulated with antibodies to the T-cell receptor as well as fibroblasts stimulated with growth factors, we showed that KSR1-deficient cells exhibit an attenuation of ERK activation with defects in cell proliferation.Here we explored the role of KSR1 in NK cell-mediated cytolysis. The killing of a target cell by a cytolytic T cell or NK cell is a complicated process that involves cell polarization with microtubule-dependent movement of cytolytic granules to an area that is proximal to the contact surface or immunological synapse (7, 33, 34, 48-50, 54). A variety of different signaling molecules are also involved, including calcium (23), phosphatidylinositol-3,4,5-triphosphate (13, 17), and activation of the ERK MAP kinase (6, 42, 56). Recently, the recruitment of activated ERK to the immunological synapse (IS) has been shown to be a feature of successful killing of a target by cytotoxic T lymphocytes (58).How active ERK is recruited to the synapse is not known. Since KSR1 is known to be recruited to the plasma membrane by Ras activation (24), and since the immunological synapse is one of the major sites of Ras activation (26, 41), it seemed plausible to test the hypothesis that KSR1 recruitment to the plasma membrane functions to recruit ERK to the immunological synapse and facilitate its activation. We found that KSR1 was recruited to the immunological synapse and that KSR1 appeared to be required for the localization of active ERK at the contact site. As KSR1-deficient cells exhibit a defect in killing, this suggests that KSR1 recruitment to the synapse may be important in the cytolytic killing of target cells.     

Requirement of JIP1-Mediated c-Jun N-Terminal Kinase Activation for Obesity-Induced Insulin Resistance

The c-Jun NH₂-terminal kinase (JNK) interacting protein 1 (JIP1) has been proposed to act as a scaffold protein that mediates JNK activation. However, recent studies have implicated JIP1 in multiple biochemical processes. Physiological roles of JIP1 that are related to the JNK scaffold function of JIP1 are therefore unclear. To test the role of JIP1 in JNK activation, we created mice with a germ line point mutation in the Jip1 gene (Thr¹⁰³ replaced with Ala) that selectively blocks JIP1-mediated JNK activation. These mutant mice exhibit a severe defect in JNK activation caused by feeding of a high-fat diet. The loss of JIP1-mediated JNK activation protected the mutant mice against obesity-induced insulin resistance. We conclude that JIP1-mediated JNK activation plays a critical role in metabolic stress regulation of the JNK signaling pathway.Diet-induced obesity causes insulin resistance and metabolic syndrome, which can lead to β-cell dysfunction and type 2 diabetes (15). It is established that feeding mice a high-fat diet (HFD) causes activation of c-Jun NH₂-terminal kinase 1 (JNK1) (10). Moreover, Jnk1^−/− mice are protected against the effects of HFD-induced insulin resistance (10). Together, these observations indicate that JNK1 plays a critical role in the metabolic stress response. However, the mechanism that accounts for HFD-induced JNK1 activation is unclear. Recent studies have implicated the JIP1 scaffold protein in JNK1 activation caused by metabolic stress (23, 39).JIP1 can assemble a functional JNK activation module composed of a mitogen-activated protein kinase (MAPK) kinase kinase (a member of the mixed-lineage protein kinase [MLK] group), the MAPK kinase MKK7, and JNK (40, 42). This complex may be relevant to JNK activation caused by metabolic stress (23, 39). Indeed, MLK-deficient mice (14) and JIP1-deficient mice (13) exhibit defects in HFD-induced JNK activation and insulin resistance.The protection of Jip1^−/− mice against the effects of being fed an HFD may be mediated by loss of the JNK scaffold function of JIP1. However, JIP1 has also been reported to mediate other biochemical processes that would also be disrupted in Jip1^−/− mice. For example, JIP1 interacts with AKT and has been implicated in the mechanism of AKT activation (8, 17, 18, 34). Moreover, JIP1 interacts with members of the Src and Abl tyrosine kinase families (4, 16, 24), the lipid phosphatase SHIP2 (44), the MAPK phosphatase MKP7 (43), β-amyloid precursor protein (20, 31), the small GTPase regulatory proteins Ras-GRF1, p190-RhoGEF, RalGDS, and Tiam1 (2, 8, 21), ankyrin G (35), molecular chaperones (35), and the low-density-lipoprotein-related receptors LRP1, LRP2, and LRP8 (7, 37). JIP1 also interacts with other scaffold proteins, including the insulin receptor substrate proteins IRS1 and IRS2 (35). Finally, JIP1 may act as an adapter protein for kinesin-mediated (11, 12, 16, 38, 42) and dynein-mediated (35) trafficking on microtubules. The JNK scaffold properties of JIP1 therefore represent only one of the possible biochemical functions of JIP1 that are disrupted in Jip1^−/− mice.The purpose of this study was to test the role of JIP1 as a JNK scaffold protein in the response of mice to being fed an HFD. Our approach was to examine the effect of a point mutation that selectively prevents JIP1-induced JNK activation. It is established that phosphorylation of JIP1 on Thr¹⁰³ is required for JIP1-mediated JNK activation by the MLK pathway (25). Consequently, the phosphorylation-defective Thr103Ala JIP1 protein does not activate JNK (25). Here we describe the analysis of mice with a point mutation in the Jip1 gene that replaces the JIP1 phosphorylation site Thr¹⁰³ with Ala. We show that this mutation suppresses HFD-induced JNK activation and insulin resistance. These data demonstrate that JNK activation mediated by the JIP1 scaffold complex contributes to the response of mice to an HFD.     

A Novel Dehalobacter Species Is Involved in Extensive 4,5,6,7-Tetrachlorophthalide Dechlorination

The purpose of this study was the enrichment and phylogenetic identification of bacteria that dechlorinate 4,5,6,7-tetrachlorophthalide (commercially designated “fthalide”), an effective fungicide for rice blast disease. Sequential transfer culture of a paddy soil with lactate and fthalide produced a soil-free enrichment culture (designated the “KFL culture”) that dechlorinated fthalide by using hydrogen, which is produced from lactate. Phylogenetic analysis based on 16S rRNA genes revealed the dominance of two novel phylotypes of the genus Dehalobacter (FTH1 and FTH2) in the KFL culture. FTH1 and FTH2 disappeared during culture transfer in medium without fthalide and increased in abundance with the dechlorination of fthalide, indicating their growth dependence on the dechlorination of fthalide. Dehalobacter restrictus TEA is their closest relative, with 97.5% and 97.3% 16S rRNA gene similarities to FTH1 and FTH2, respectively.4,5,6,7-Tetrachlorophthalide (commercially designated “fthalide”) is an effective fungicide for rice blast disease, which inhibits melanin biosynthesis and the formation of the mature appressorial cells of the rice blast pathogen on the host plant (5, 16). Fthalide has been reported to be reductively dechlorinated in soil (16) and compost (28), although its fates in paddy soil and the fthalide-dechlorinating bacteria are unknown. Besides fthalide, polychlorinated aromatic compounds are known to be reductively dechlorinated by the bacteria of several phyla. Six strains of Desulfitobacterium spp. of the phylum Firmicutes (2, 3, 6, 10, 23, 29) and Desulfomonile tiedjei DCB-1 of the phylum Proteobacteria (21) can dechlorinate polychlorinated phenols. Three strains of the phylum Chloroflexi can dechlorinate a variety of compounds, including polychlorinated phenols, benzenes, biphenyls, or dibenzo-p-dioxins: Dehalococcoides ethenogenes 195 (9, 19), Dehalococcoides sp. strain CBDB1 (1, 4), and strain DF-1 of Chloroflexi, collectively called the “o-17/DF-1 group” (18). Dehalococcoides spp. utilize hydrogen as an electron donor and acetate as a carbon source for growth coupled to the reductive dechlorination of chlorinated compounds (1, 12, 13, 19, 26). In contrast, Desulfitobacterium spp. can dechlorinate chlorinated compounds not only with hydrogen, but also organic acids, such as formate, pyruvate, lactate, or butyrate (3, 10, 23). Strain DF-1 can utilize hydrogen and formate for the dechlorination of polychlorinated biphenyls (PCBs) (18).In this study, bacteria that dechlorinate fthalide were enriched from a paddy soil with sequentially transferred cultures using a soil-free medium supplemented with single organic acids. Acetate, formate, lactate, and butyrate were used in this study because they are frequently used in the enrichment of dechlorinators and release hydrogen at different concentrations (8, 11, 14). Fthalide-dechlorinating bacteria in the enriched culture were phylogenetically identified based on the 16S rRNA gene with PCR-denaturing gradient gel electrophoresis (DGGE), a 16S rRNA gene clone library, and quantitative real-time PCR (qPCR).     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Identification of a Novel System for Boron Transport: Atr1 Is a Main Boron Exporter in Yeast

Boron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1Δ mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1Δ cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1Δ cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.Boron has been proposed as an important micronutrient in plants and animals. Studies have shown the presence of several genes associated with boron transport and tolerance in plants (18, 25, 27); however, boron transport mechanisms in other organisms, including animals, remain unclear. In plants, boron functions as a cross-linker for rhammogalacturanon II in the cell membrane (9, 14, 21) and also as a structural component in cytoskeleton assembly (1). Arabidopsis thaliana BOR1 was the first gene shown to play a role in boron tolerance (28). Homologs of BOR1 were found in many organisms, including yeasts, plants, and mammals (22, 25, 29). A high level of boron leads to degradation of its own exporter, BOR1, in A. thaliana (27), and A. thaliana BOR1 cannot be used to produce genetically modified plants that grow in soil with high boron levels. However, transgenic plants expressing BOR4, one of six paralogs of BOR1, showed high tolerance to toxic levels of boron (18). Multicopy expression of BOT1, a BOR1 ortholog, provided boron tolerance to barley (25).The yeast Saccharomyces cerevisiae has been used as a model organism for characterization of plant boron tolerance genes (19, 20, 25, 26, 29). While 10 mM boric acid is lethal to Arabidopsis (18), yeast can grow in the presence of 80 mM boron and is considered a boron-tolerant organism (19, 20). Yeast Bor1 was characterized in detail (10). This protein is localized to the plasma membrane and functions as a boric acid exporter (26). The bor1Δ yeast strain overaccumulates boron (20, 28), and cells that overexpress BOR1 have less intracellular boron and show resistance to boron treatment (20). In addition to Bor1, two other proteins, Dur3 and Fps1, have been implicated in boron tolerance in yeast, but their functions are not clear (20). Dur3 is a plasma membrane transporter that plays a role in urea and polyamine transport (5, 31), and Fps1 is a member of the major intrinsic protein family and plays a role in glycerol, acetic acid, arsenite, and antimonite transport (16, 30, 33). Overexpression of FPS1 and DUR3 showed controversial effects on cellular boron levels. While FPS1 expression lowered the protoplasmic boron concentration, DUR3 expression led to a small increase in boron (20).The objective of this study was to identify proteins that are primarily responsible for boron transport in yeast. ATR1 was identified as a boron tolerance gene by screening a yeast DNA expression library. Yeast Atr1 is a member of the DHA2 family of drug-H⁺ antiporters with 14 predicted membrane-spanning segments (7). It was first characterized in a genetic screen as a high-copy-number suppressor of the 3-amino-1,2,4-triazole sensitivity of gcn4Δ mutants (11). It also conferred resistance to the DNA-damaging agent 4-nitroquinoline-N-oxide in a separate genetic screen (17). In this study, we demonstrated that high-copy-number expression of ATR1 conferred extreme resistance to boron and reduced intracellular levels of the element, whereas cells lacking the ATR1 gene were hypersensitive to boron and increased its intracellular levels. We analyzed changes in the global gene expression profile in response to boron and found that ATR1 is the most induced transporter gene. The Atr1-green fluorescent protein (GFP) fusion protein localized to the plasma membrane and vacuole. Taken together, our data show that Atr1 functions as a major boron efflux pump and provides tolerance of the element by pumping boron out of cells.