Sigma 1- and mu 1-Adaptin homologues of Leishmania mexicana are required for parasite survival in the infected host

The sorting of membrane-bound proteins from the trans-Golgi network to lysosomal/endosomal compartments is achieved by preferential inclusion into clathrin-coated vesicles. Contained within the cytoplasmic domains of such proteins, specific sequence motifs have been identified (tyrosine-based and/or di-leucine-based) that are essential for targeting and are recognized by a family of heterotetrameric adaptor complexes, which then recruit clathrin. These cytosolic protein complexes, which have been found in a wide variety of higher eukaryotic organisms, are essential for the development of multicellular organisms. In trypanosomatids, the adaptin-mediated sorting of proteins is largely uncharacterized. In order to identify components of the adaptor-complex machinery, this study reports the cloning and characterization of sigma 1- and mu 1-adaptin gene homologues from the eukaryotic protozoan parasite, Leishmania mexicana. Generation of sigma 1- and mu 1-adaptin gene deletion mutants shows that these promastigote parasites are viable in culture, but are unable to establish infection of macrophages or mice, indicating that adaptin function is crucial for pathogenesis in these unicellular organisms.     

A mitogen-activated protein (MAP) kinase homologue of Leishmania mexicana is essential for parasite survival in the infected host.

The parasitic protozoon Leishmania mexicana undergoes two major developmental stages in its life cycle exhibiting profound physiological and morphological differences, the promastigotes in the insect vector and the amastigotes in mammalian macrophages. A deletion mutant, Deltalmsap1/2, for the secreted acid phosphatase (SAP) gene locus, comprising the two SAP genes separated by an intergenic region of approximately 11.5 kb, lost its ability to cause a progressive disease in Balb/c mice. While in vitro growth of promastigotes, invasion of host cells and differentiation from promastigotes to amastigotes was indistinguishable from the wild-type, the mutant parasites ceased to proliferate when transformed to amastigotes in infected macrophages or in a macrophage-free in vitro differentiation system, suggesting a stage-specific growth arrest. This phenotype could be reverted by complementation with 6 kb of the intergenic region of the SAP gene locus. Sequence analysis identified two open reading frames, both encoding single copy genes; one gene product shows high homology to mitogen-activated protein (MAP) kinases. Complementation experiments revealed that the MAP kinase homologue, designated LMPK, is required and is sufficient to restore the infectivity of the Deltalmsap1/2 mutant. Therefore, LMPK is a kinase that is essential for the survival of L.mexicana in the infected host by affecting the cell division of the amastigotes.     

Regulation of translation is required for dendritic cell function and survival during activation

In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation (maturation) that exhibits specific mechanisms to control antigen processing and presentation. Here, we show that in response to lipopolysaccharides, protein synthesis is rapidly enhanced in DCs. This enhancement occurs via a PI3K-dependent signaling pathway and is key for DC activation. In addition, we show that later on, in a manner similar to viral or apoptotic stress, DC activation leads to the phosphorylation and proteolysis of important translation initiation factors, thus inhibiting cap-dependent translation. This inhibition correlates with major changes in the origin of the peptides presented by MHC class I and the ability of mature DCs to prevent cell death. Our observations have important implications in linking translation regulation with DC function and survival during the immune response.     

mTOR is required for asymmetric division through small GTPases in mouse oocytes

Mammalian target of rapamycin (mTOR) is central to the control of cell proliferation, growth, and survival in mammalian cells. Prolonged treatment with rapamycin inhibits mTOR complex 2 (mTORC2) activity, and both the mTORC1-mediated S6K1 and 4E-BP1/eIF4E pathways are essential for TORC2-mediated RhoA, Cdc42, and Rac1 expression during cell motility and F-actin reorganization. The functions of mTOR in the mouse oocyte remain unclear, however. The present study shows that rapamycin affects mTOR expression and cytoskeleton reorganization during meiotic maturation of mouse oocytes. mTOR mRNA was expressed in germinal vesicles (GV) until metaphase I (MI), and increased during metaphase II (MII). Immunostaining showed that mTOR localized around the spindle and in the cytoplasm of oocytes. Treatment of oocytes with rapamycin decreased mTOR at the RNA and protein level, and altered asymmetric division. Formation of the actin cap and the cortical granule-free domain were also disrupted after rapamycin treatment, indicating the failure of spindle migration. Injection of an anti-mTOR antibody yielded results consistent with those obtained for rapamycin treatment, further confirming the involvement of mTOR in oocyte polarity. Furthermore, rapamycin treatment reduced the mRNA expression of small GTPases (RhoA, Cdc42, and Rac1), which are crucial regulatory factors for cytoskeleton reorganization. Taken together, these results suggest that rapamycin inhibits spindle migration and asymmetric division during mouse oocyte maturation via mTOR-mediated small GTPase signaling pathways.     

JNK1 is required for T cell-mediated immunity against Leishmania major infection

c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase that plays important regulatory roles in helper T cell differentiation. In the current study, we used Jnk1-deficient mice to examine the function of JNK during an in vivo pathogenic infection, leishmaniasis, which is strongly influenced by Th1/Th2 effector mechanisms. The data show that Jnk1-deficient mice, despite their usually genetically resistant background, were unable to resolve Leishmania infections. Jnk1-/- mice displayed reduced delayed-type hypersensitivity in response to the pathogen, which was associated with a T cell defect. We found that, although these mice can direct an apparent Th1-response, there is also simultaneous generation of Leishmania-specific Th2 responses, which possibly down-modulate protective Th1-mediated immune function. These findings demonstrate that the negative regulation of Th2 cytokine production by the JNK1 signaling pathway is essential for generating Th1-polarized immunity against intracellular pathogens, such as Leishmania major.     

Calcineurin is required for Leishmania major stress response pathways and for virulence in the mammalian host

Leishmania parasites must adapt to elevated temperatures and other environmental stresses during infection of their mammalian hosts. How these environmental cues are sensed is poorly understood. In this study we show that calcium uptake is required for parasite thermotolerance at 34-37°C. To identify potential downstream targets of calcium influx, a Leishmania major mutant lacking the essential regulatory subunit (CnB) of the Ca(2+) /calmodulin-dependent serine/threonine-specific phosphatase, calcineurin, was generated. The Δcnb mutant grew as well as wild-type parasites at 27°C and differentiated normally to infective metacyclic promastigotes. However, Δcnb parasites lost viability when exposed to increased temperature (34°C) and were hypersensitive to endoplasmic reticulum and membrane stress, induced by tunicamycin and inhibitors of sterol and sphingolipid biosynthesis respectively. Δcnb promastigotes were internalized by macrophages, but their differentiation to the heat adapted amastigote stage was delayed and the resulting parasites failed to proliferate. Strikingly, the Δcnb parasites were completely cleared by susceptible BALB/c mice. Complementation of Δcnb parasites with CnB restored thermotolerance and infectivity in both macrophages and animal models. Our results suggest that Ca(2+) influx and calcineurin signalling are required for both early and long-term adaptive parasite responses to environmental stresses encountered in the mammalian host.     

Interleukin-6 is required for parasite specific response and host resistance to Trypanosoma cruzi.

Infection with Trypanosoma cruzi, the agent of Chagas' disease, results in elevated levels of interleukin-6 (IL-6) in serum and infected tissues. However, it remains unknown whether IL-6 plays a role in host defence against T. cruzi. To determine whether IL-6 underlies disease progression, we followed the time course of T. cruzi-infected mice bearing IL-6 +/+ and minus sign/minus sign genotypes, respectively. We found that IL-6 minus sign/minus sign mice were more susceptible to T. cruzi infection as they exhibited about 3-fold higher parasitaemia and died earlier than wild-type animals. Unlike what might be expected, T. cruzi-infected IL-6 minus sign/minus sign mice did not show at peak infection a decrease in the secretion of IFN-gamma, a Th1 cytokine crucial for controlling the parasite. Instead, they exhibited a much reduced splenocyte recall response to T. cruzi antigens. Our results suggest that IL-6 mediates anti-parasite protective responses against T. cruzi.     

The Bin3 RNA methyltransferase is required for repression of caudal translation in the Drosophila embryo

Bin3 was first identified as a Bicoid-interacting protein in a yeast two-hybrid screen. In human cells, a Bin3 ortholog (BCDIN3) methylates the 5′ end of 7SK RNA, but its role in vivo is unknown. Here, we show that in Drosophila, Bin3 is important for dorso-ventral patterning in oogenesis and for anterior–posterior pattern formation during embryogenesis. Embryos that lack Bin3 fail to repress the translation of caudal mRNA and exhibit head involution defects. bin3 mutants also show (1) a severe reduction in the level of 7SK RNA, (2) reduced binding of Bicoid to the caudal 3′ UTR, and (3) genetic interactions with bicoid, and with genes encoding eIF4E, Larp1, polyA binding protein (PABP), and Ago2. 7SK RNA coimmunoprecipitated with Bin3 and is present in Bicoid complexes. These data suggest a model in which Bicoid recruits Bin3 to the caudal 3′ UTR. Bin3's role is to bind and stabilize 7SK RNA, thereby promoting formation of a repressive RNA–protein complex that includes the RNA-binding proteins Larp1, PABP, and Ago2. This complex would prevent translation by blocking eIF4E interactions required for initiation. Our results, together with prior network analysis in human cells, suggest that Bin3 interacts with multiple partner proteins, methylates small non-coding RNAs, and plays diverse roles in development.     

Lipophosphoglycan is not required for infection of macrophages or mice by Leishmania mexicana

Cell surface lipophosphoglycan (LPG) is commonly regarded as a multifunctional Leishmania virulence factor required for survival and development of these parasites in mammals. In this study, the LPG biosynthesis gene lpg1 was deleted in Leishmania mexicana by targeted gene replacement. The resulting mutants are deficient in LPG synthesis but still display on their surface and secrete phosphoglycan-modified molecules, most likely in the form of proteophosphoglycans, whose expression appears to be up-regulated. LPG-deficient L.mexicana promastigotes show no significant differences to LPG-expressing parasites with respect to attachment to, uptake into and multiplication inside macrophages. Moreover, in Balb/c and C57/BL6 mice, LPG-deficient L.mexicana clones are at least as virulent as the parental wild-type strain and lead to lethal disseminated disease. The results demonstrate that at least L. mexicana does not require LPG for experimental infections of macrophages or mice. Leishmania mexicana LPG is therefore not a virulence factor in the mammalian host.     

Leishmania major parasite stage-dependent host cell invasion and immune evasion

Leishmania pathogenesis is primarily studied using the disease-inducing promastigote stage of Leishmania major. Despite many efforts, all attempts so far have failed to culture the disease-relevant multiplying amastigote stage of L. major. Here, we established a stably growing axenic L. major amastigote culture system that was characterized genetically, morphologically, and by stage-specific DsRed protein expression. We found parasite stage-specific disease development in resistant C57BL/6 mice. Human neutrophils, as first host cells for promastigotes, do not take up amastigotes. In human macrophages, we observed an amastigote-specific complement receptor 3-mediated, endocytotic entry mechanism, whereas promastigotes are taken up by complement receptor 1-mediated phagocytosis. Promastigote infection of macrophages induced the inflammatory mediators TNF, CCL3, and CCL4, whereas amastigote infection was silent and resulted in significantly increased parasite numbers: from 7.1 ± 1.4 (after 3 h) to 20.1 ± 7.9 parasites/cell (after 96 h). Our study identifies Leishmania stage-specific disease development, host cell preference, entry mechanism, and immune evasion. Since the amastigote stage is the disease-propagating form found in the infected mammalian host, the newly developed L. major axenic cultures will serve as an important tool in better understanding the amastigote-driven immune response in leishmaniasis.     

Leishmania major Hsp100 is required chiefly in the mammalian stage of the parasite.

In Leishmania major a 100-kDa heat shock protein, Hsp100, is abundant in the intracellular amastigote stage which persists in the mammalian host. A replacement of both clpB alleles which encode Hsp100 does not affect promastigote viability under standard culture conditions but impairs thermotolerance in vitro. In experimental infections of BALB/c inbred mice, the lack of Hsp100 in the gene replacement mutants results in a markedly delayed lesion development compared with that in infections with wild-type L. major. Overexpression of exogenous clpB gene copies can partly restore virulence to the gene replacement mutants. Genetic-selection experiments also reveal a strong pressure for Hsp100 expression in the mammalian stage. This requirement for Hsp100 was also observed in in vitro infection experiments with mouse peritoneal macrophages. These experiments indicated a role for Hsp100 during the development from the promastigote to the amastigote stage. Our results suggest an important role for this parasite heat shock protein during the initial stages of a mammalian infection.     

Groucho oligomerization is required for repression in vivo

Drosophila Groucho (Gro) is a member of a family of metazoan corepressors with widespread roles in development. Previous studies indicated that a conserved domain in Gro, termed the Q domain, was required for repression in cultured cells and mediated homotetramerization. Evidence presented here suggests that the Q domain contains two coiled-coil motifs required for oligomerization and repression in vivo. Mutagenesis of the putative hydrophobic faces of these motifs, but not of the hydrophilic faces, prevents the formation of both tetramers and higher order oligomers. Mutagenesis of the hydrophobic faces of both coiled-coil motifs in the context of a Gal4-Gro fusion protein prevents repression of a Gal4-responsive reporter in S2 cells, while mutagenesis of a single motif weakens repression. The finding that the repression directed by the single mutants depends on endogenous wild-type Gro further supports the idea that oligomerization plays a role in repression. Overexpression in the fly of forms of Gro able to oligomerize, but not of a form of Gro unable to oligomerize, results in developmental defects and ectopic repression of Gro target genes in the wing disk. Although the function of several corepressors is suspected to involve oligomerization, these studies represent one of the first direct links between corepressor oligomerization and repression in vivo.     

Partial cleavage of RasGAP by caspases is required for cell survival in mild stress conditions

Tight control of apoptosis is required for proper development and maintenance of homeostasis in multicellular organisms. Cells can protect themselves from potentially lethal stimuli by expressing antiapoptotic factors, such as inhibitors of apoptosis, FLICE (caspase 8)-inhibitory proteins, and members of the Bcl2 family. Here, we describe a mechanism that allows cells to survive once executioner caspases have been activated. This mechanism relies on the partial cleavage of RasGAP by caspase 3 into an amino-terminal fragment called fragment N. Generation of this fragment leads to the activation of the antiapoptotic Akt kinase, preventing further amplification of caspase activity. Partial cleavage of RasGAP is required for cell survival under stress conditions because cells expressing an uncleavable RasGAP mutant cannot activate Akt, cannot prevent amplification of caspase 3 activity, and eventually undergo apoptosis. Executioner caspases therefore control the extent of their own activation by a feedback regulatory mechanism initiated by the partial cleavage of RasGAP that is crucial for cell survival under adverse conditions.     

Ribosomal protein L33 is required for ribosome biogenesis, subunit joining, and repression of GCN4 translation

We identified a mutation in the 60S ribosomal protein L33A (rpl33a-G76R) that elicits derepression of GCN4 translation (Gcd- phenotype) by allowing scanning preinitiation complexes to bypass inhibitory upstream open reading frame 4 (uORF4) independently of prior uORF1 translation and reinitiation. At 37 degrees C, rpl33a-G76R confers defects in 60S biogenesis comparable to those produced by the deletion of RPL33A (DeltaA). At 28 degrees C, however, the 60S biogenesis defect is less severe in rpl33a-G76R than in DeltaA cells, yet rpl33a-G76R confers greater derepression of GCN4 and a larger reduction in general translation. Hence, it appears that rpl33a-G76R has a stronger effect on ribosomal-subunit joining than does a comparable reduction of wild-type 60S levels conferred by DeltaA. We suggest that rpl33a-G76R alters the 60S subunit in a way that impedes ribosomal-subunit joining and thereby allows 48S rRNA complexes to abort initiation at uORF4, resume scanning, and initiate downstream at GCN4. Because overexpressing tRNAiMet suppresses the Gcd- phenotype of rpl33a-G76R cells, dissociation of tRNAiMet from the 40S subunit may be responsible for abortive initiation at uORF4 in this mutant. We further demonstrate that rpl33a-G76R impairs the efficient processing of 35S and 27S pre-rRNAs and reduces the accumulation of all four mature rRNAs, indicating an important role for L33 in the biogenesis of both ribosomal subunits.     

Pyk2 is required for neutrophil degranulation and host defense responses to bacterial infection

The appropriate regulation of neutrophil activation is critical for maintaining host defense and limiting inflammation. Polymorphonuclear neutrophils (PMNs) express a number of cytoplasmic tyrosine kinases that regulate signaling pathways leading to activation. One of the most highly expressed, but least studied, kinases in PMNs is proline rich kinase 2 (Pyk2). By analogy to the related focal adhesion kinase, Pyk2 has been implicated in regulating PMN adhesion and migration; however, its physiologic function has yet to be described. Using pyk2(-/-) mice, we found that this kinase was required for integrin-mediated degranulation responses, but was not involved in adhesion-induced cell spreading or activation of superoxide production. Pyk2-deficient PMNs also manifested reduced migration on fibrinogen-coated surfaces. The absence of Pyk2 resulted in a severe reduction in paxillin and Vav phosphorylation following integrin ligation, which likely accounts for the poor degranulation and cell migration. Pyk2(-/-) mice were unable to efficiently clear infection with Staphylococcus aureus in a skin abscess model, owing in part to the poor release of granule contents at the site of infection. However, Pyk2-deficient PMNs responded normally to soluble agonists, demonstrating that this kinase functions mainly in the integrin pathway. These data demonstrate the unrealized physiologic role of this kinase in regulating the adhesion-mediated release of PMN granule contents.