RESUMEN
The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and in vivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.
Asunto(s)
Movimiento Celular , Pulmón , Mecanotransducción Celular , Neutrófilos , Animales , Ratones , Membrana Celular , Canales Iónicos/genética , Neutrófilos/metabolismo , Neutrófilos/microbiología , Actividad Bactericida de la Sangre/genética , Mecanotransducción Celular/genéticaRESUMEN
Lipids stored in lipid-bodies (LBs) in host cells are potential sources of fatty acids for pathogens. However, the mechanism of recruitment of LBs from the host cells by pathogens to acquire fatty acids is not known. Here, we have found that Leishmania specifically upregulates the expression of host Rab18 and its GEF, TRAPPC9 by downregulating the expression of miR-1914-3p by reducing the level of Dicer in macrophages via their metalloprotease gp63. Our results also show that miR-1914-3p negatively regulates the expression of Rab18 and its GEF in cells. Subsequently, Leishmania containing parasitophorous vacuoles (Ld-PVs) recruit and retain host Rab18 and TRAPPC9. Leishmania infection also induces LB biogenesis in host cells and recruits LBs on Ld-PVs and acquires FLC12-labeled fatty acids from LBs. Moreover, overexpression of miR-1914-3p in macrophages significantly inhibits the recruitment of LBs and thereby suppresses the multiplication of parasites in macrophages as parasites are unable to acquire fatty acids. These results demonstrate a novel mechanism how Leishmania acquire fatty acids from LBs for their growth in macrophages.
Asunto(s)
Leishmania , MicroARNs , Gotas Lipídicas/metabolismo , Macrófagos/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Ácidos Grasos/metabolismo , Proliferación CelularRESUMEN
Metalloprotease-gp63 is a virulence factor secreted by Leishmania. However, secretory pathway in Leishmania is not well defined. Here, we cloned and expressed the GRASP homolog from Leishmania. We found that Leishmania expresses one GRASP homolog of 58 kDa protein (LdGRASP) which localizes in LdRab1- and LPG2-positive Golgi compartment in Leishmania. LdGRASP was found to bind with COPII complex, LdARF1, LdRab1 and LdRab11 indicating its role in ER and Golgi transport in Leishmania. To determine the function of LdGRASP, we generated LdGRASP knockout parasites using CRISPR-Cas9. We found fragmentation of Golgi in Ld:GRASPKO parasites. Our results showed enhanced transport of non-GPI-anchored gp63 to the cell surface leading to higher secretion of this form of gp63 in Ld:GRASPKO parasites in comparison to Ld:WT cells. In contrast, we found that transport of GPI-anchored gp63 to the cell surface is blocked in Ld:GRASPKO parasites and thereby inhibits its secretion. The overexpression of dominant-negative mutant of LdRab1 or LdSar1 in Ld:GRASPKO parasites significantly blocked the secretion of non-GPI-anchored gp63. Interestingly, we found that survival of transgenic parasites overexpressing Ld:GRASP-GFP is significantly compromised in macrophages in comparison to Ld:WT and Ld:GRASPKO parasites. These results demonstrated that LdGRASP differentially regulates Ldgp63 secretory pathway in Leishmania.
Asunto(s)
Leishmania , Metaloendopeptidasas , Proteínas Protozoarias , Factores de Virulencia , Animales , Sistemas CRISPR-Cas , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Matriz de Golgi/metabolismo , Proteínas de la Matriz de Golgi/genética , Leishmania/metabolismo , Leishmania/genética , Macrófagos/parasitología , Macrófagos/metabolismo , Metaloendopeptidasas/metabolismo , Metaloendopeptidasas/genética , Transporte de Proteínas , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Factores de Virulencia/metabolismo , Factores de Virulencia/genéticaRESUMEN
The pathogenesis of lung fibrosis involves hyperactivation of innate and adaptive immune pathways that release inflammatory cytokines and growth factors such as tumor growth factor (TGF)ß1 and induce aberrant extracellular matrix protein production. During the genesis of pulmonary fibrosis, resident alveolar macrophages are replaced by a population of newly arrived monocyte-derived interstitial macrophages that subsequently transition into alveolar macrophages (Mo-AMs). These transitioning cells initiate fibrosis by releasing profibrotic cytokines and remodeling the matrix. Here, we describe a strategy for leveraging the up-regulation of the mannose receptor CD206 in interstitial macrophages and Mo-AM to treat lung fibrosis. We engineered mannosylated albumin nanoparticles, which were found to be internalized by fibrogenic CD206+ monocyte derived macrophages (Mo-Macs). Mannosylated albumin nanoparticles incorporating TGFß1 small-interfering RNA (siRNA) targeted the profibrotic subpopulation of CD206+ macrophages and prevented lung fibrosis. The findings point to the potential utility of mannosylated albumin nanoparticles in delivering TGFß-siRNA into CD206+ profibrotic macrophages as an antilung fibrosis strategy.
Asunto(s)
Linfotoxina-alfa , Macrófagos Alveolares , Nanopartículas , Fibrosis Pulmonar , ARN Interferente Pequeño , Animales , Bleomicina/farmacología , Modelos Animales de Enfermedad , Linfotoxina-alfa/genética , Macrófagos Alveolares/inmunología , Receptor de Manosa , Ratones , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/terapia , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genéticaRESUMEN
TLR4 signaling via endotoxemia in macrophages promotes macrophage transition to the inflammatory phenotype through NLRP3 inflammasome activation. This transition event has the potential to trigger acute lung injury (ALI). However, relatively little is known about the regulation of NLRP3 and its role in the pathogenesis of ALI. Here we interrogated the signaling pathway activated by CD38, an ectoenzyme expressed in macrophages, in preventing ALI through suppressing NLRP3 activation. Wild-type and Cd38-knockout (Cd38-/-) mice were used to assess inflammatory lung injury, and isolated macrophages were used to delineate underlying TLR4 signaling pathway. We showed that CD38 suppressed TLR4 signaling in macrophages by inhibiting Bruton's tyrosine kinase (Btk) through the recruitment of Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) and resulting in the dephosphorylation of activated Btk. Cd38-/- mice show enhanced lung polymorphonuclear leukocyte extravasation and severe lung injury. LPS- or polymicrobial sepsis-induced mortality in Cd38-/- mice were markedly augmented compared with wild types. CD38 in macrophages functioned by inhibiting Btk activation through activation of SHP2 and resulting dephosphorylation of Btk, and thereby preventing activation of downstream targets NF-κB and NLRP3. Cd38-/- macrophages displayed markedly increased activation of Btk, NF-κB, and NLRP3, whereas in vivo administration of the Btk inhibitor ibrutinib (a Food and Drug Administration-approved drug) prevented augmented TLR4-induced inflammatory lung injury seen in Cd38-/- mice. Our findings together show upregulation of CD38 activity and inhibition of Btk activation downstream of TLR4 activation as potential strategies to prevent endotoxemic ALI.
Asunto(s)
ADP-Ribosil Ciclasa 1/fisiología , Lesión Pulmonar Aguda/prevención & control , Adenina/análogos & derivados , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Endotoxemia/prevención & control , Inflamasomas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Glicoproteínas de Membrana/fisiología , Piperidinas/farmacología , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Adenina/farmacología , Agammaglobulinemia Tirosina Quinasa/genética , Agammaglobulinemia Tirosina Quinasa/metabolismo , Animales , Endotoxemia/etiología , Endotoxemia/metabolismo , Endotoxemia/patología , Femenino , Inflamasomas/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Transducción de SeñalRESUMEN
Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the Leishmania containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus Leishmania is postulated to be residing in the phagolysosomes in macrophages. Here, we report that Leishmania donovani specifically upregulates the expression of Rab5a by degrading c-Jun via their metalloprotease gp63 to downregulate the expression of miR-494 in THP-1 differentiated human macrophages. Our results also show that miR-494 negatively regulates the expression of Rab5a in cells. Subsequently, L. donovani recruits and retains Rab5a and EEA1 on PV to reside in early endosomes and inhibits transport to lysosomes in human macrophages. Similarly, we have also observed that Leishmania PV also recruits Rab5a by upregulating its expression in human PBMC differentiated macrophages. However, the parasite modulates the endosome by recruiting Lamp1 and inactive pro-CathepsinD on PV via the overexpression of Rab5a in infected cells. Furthermore, siRNA knockdown of Rab5a or overexpression of miR-494 in human macrophages significantly inhibits the survival of the parasites. These results provide the first mechanistic insights of parasite-mediated remodeling of endo-lysosomal trafficking to reside in a specialized early endocytic compartment.
Asunto(s)
Endosomas/parasitología , Leishmania donovani/fisiología , Leucocitos Mononucleares/parasitología , Macrófagos/parasitología , MicroARNs/genética , Proteínas de Unión al GTP rab5/metabolismo , Animales , Regulación hacia Abajo , Endosomas/microbiología , Humanos , Lisosomas/metabolismo , Fagosomas/microbiología , Activación Transcripcional/genética , Regulación hacia Arriba , Vacuolas/parasitologíaRESUMEN
During the 1991 Gulf War, oil wells in the oil fields of Kuwait were set aflame and destroyed. This resulted in severe crude oil pollution of the countries only fresh water aquifers. Here, for the first time the natural attenuation and biodegradation of the persisting groundwater contamination was investigated to assess potential processes in the aquifer. Biodegradation experiments were conducted under aerobic and multiple anaerobic conditions using microcosms of the contaminated groundwater from Kuwait. Under the conditions tested, a portion of the total petroleum hydrocarbon (TPH) component was degraded, however there was only a slight change in the bulk concentration of the contaminant measured as dissolved organic carbon (DOC), suggesting the presence of a recalcitrant pollutant. Changes in the associated microbial community composition under different reduction-oxidation conditions were observed and known hydrocarbon degraders identified. The results of this study indicate that lingering contaminant still persists in the groundwater and is recalcitrant to further biodegradation, which presents challenges for future remediation plans.
Asunto(s)
Agua Subterránea/microbiología , Guerra del Golfo , Contaminación por Petróleo/análisis , Petróleo/análisis , Bacterias/metabolismo , Biodegradación Ambiental , Biodiversidad , Agua Subterránea/química , Kuwait , Contaminantes Químicos del Agua/análisisRESUMEN
Leishmania donovani possess two isoforms of Rab5 (Rab5a and Rab5b), which are involved in fluid phase and receptor-mediated endocytosis, respectively. We have characterized the solution structure and dynamics of a stabilized truncated LdRab5a mutant. For the purpose of NMR structure determination, protein stability was enhanced by systematically introducing various deletions and mutations. Deletion of hypervariable C-terminal and the 20 residues LdRab5a specific insert slightly enhanced the stability, which was further improved by C107S mutation. The final construct, truncated LdRab5a with C107S mutation, was found to be stable for longer durations at higher concentration, with an increase in melting temperature by 10°C. Solution structure of truncated LdRab5a shows the characteristic GTPase fold having nucleotide and effector binding sites. Orientation of switch I and switch II regions match well with that of guanosine 5'-(ß, γ-imido)triphosphate (GppNHp)-bound human Rab5a, indicating that the truncated LdRab5a attains the canonical GTP bound state. However, the backbone dynamics of the P-loop, switch I, and switch II regions were slower than that observed for guanosine 5'-(ß, γ-imido)triphosphate (GMPPNP)-bound H-Ras. This dynamic profile may further complement the residue-specific complementarity in determining the specificity of interaction with the effectors. In parallel, biophysical investigations revealed the urea induced unfolding of truncated LdRab5a to be a four-state process that involved two intermediates, I1 and I2. The maximal 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (Bis-ANS) binding was observed for I2 state, which was inferred to have molten globule like characteristics. Overall, the strategy presented would have significant impact for studying other Rab and small GTPase proteins by NMR spectroscopy.
Asunto(s)
Leishmania donovani , Proteínas de Unión al GTP rab5/química , Proteínas de Unión al GTP rab5/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Estabilidad Proteica , Desplegamiento Proteico , Alineación de Secuencia , Eliminación de Secuencia , Temperatura , Proteínas de Unión al GTP rab5/genéticaRESUMEN
Metalloprotease gp63 (Leishmania donovani gp63 (Ldgp63)) is a critical virulence factor secreted by Leishmania However, how newly synthesized Ldgp63 exits the endoplasmic reticulum (ER) and is secreted by this parasite is unknown. Here, we cloned, expressed, and characterized the GTPase LdSar1 and other COPII components like LdSec23, LdSec24, LdSec13, and LdSec31 from Leishmania to understand their role in ER exit of Ldgp63. Using dominant-positive (LdSar1:H74L) and dominant-negative (LdSar1:T34N) mutants of LdSar1, we found that GTP-bound LdSar1 specifically binds to LdSec23, which binds, in turn, with LdSec24(1-702) to form a prebudding complex. Moreover, LdSec13 specifically interacted with His6-LdSec31(1-603), and LdSec31 bound the prebudding complex via LdSec23. Interestingly, dileucine 594/595 and valine 597 residues present in the Ldgp63 C-terminal domain were critical for binding with LdSec24(703-966), and GFP-Ldgp63L594A/L595A or GFP-Ldgp63V597S mutants failed to exit from the ER. Moreover, Ldgp63-containing COPII vesicle budding from the ER was inhibited by LdSar1:T34N in an in vitro budding assay, indicating that GTP-bound LdSar1 is required for budding of Ldgp63-containing COPII vesicles. To directly demonstrate the function of LdSar1 in Ldgp63 trafficking, we coexpressed RFP-Ldgp63 along with LdSar1:WT-GFP or LdSar1:T34N-GFP and found that LdSar1:T34N overexpression blocks Ldgp63 trafficking and secretion in Leishmania Finally, we noted significantly compromised survival of LdSar1:T34N-GFP-overexpressing transgenic parasites in macrophages. Taken together, these results indicated that Ldgp63 interacts with the COPII complex via LdSec24 for Ldgp63 ER exit and subsequent secretion.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/enzimología , GTP Fosfohidrolasas/metabolismo , Leishmania donovani/metabolismo , Macrófagos/parasitología , Metaloendopeptidasas/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Virulencia/metabolismo , Sustitución de Aminoácidos , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Línea Celular Tumoral , Citosol/enzimología , Citosol/metabolismo , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Humanos , Membranas Intracelulares/enzimología , Membranas Intracelulares/metabolismo , Leishmania donovani/citología , Leishmania donovani/genética , Leishmania donovani/crecimiento & desarrollo , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/genética , Mutagénesis Sitio-Dirigida , Mutación , Organismos Modificados Genéticamente , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
Differential functions of Rab5 isoforms in endocytosis are not well characterized. Here, we cloned, expressed, and characterized Rab5a and Rab5b from Leishmania and found that both of them are localized in the early endosome. To understand the role of LdRab5 isoforms in different modes of endocytosis in Leishmania, we generated transgenic parasites overexpressing LdRab5a, LdRab5b, or their dominant-positive (LdRab5a:Q93L and LdRab5b:Q80L) or dominant-negative mutants (LdRab5a:N146I and LdRab5b:N133I). Using LdRab5a or its mutants overexpressing parasites, we found that LdRab5a specifically regulates the fluid-phase endocytosis of horseradish peroxidase and also specifically induced the transport of dextran-Texas Red to the lysosomes. In contrast, cells overexpressing LdRab5b or its mutants showed that LdRab5b explicitly controls receptor-mediated endocytosis of hemoglobin, and overexpression of LdRab5b:WT enhanced the transport of internalized Hb to the lysosomes in comparison with control cells. To unequivocally demonstrate the role of Rab5 isoforms in endocytosis in Leishmania, we tried to generate null-mutants of LdRab5a and LdRab5b parasites, but both were lethal indicating their essential functions in parasites. Therefore, we used heterozygous LdRab5a(+/-) and LdRab5b(+/-) cells. LdRab5a(+/-) Leishmania showed 50% inhibition of HRP uptake, but hemoglobin endocytosis was uninterrupted. In contrast, about 50% inhibition of Hb endocytosis was observed in LdRab5b(+/-) cells without any significant effect on HRP uptake. Finally, we tried to identify putative LdRab5a and LdRab5b effectors. We found that LdRab5b interacts with clathrin heavy chain and hemoglobin receptor. However, LdRab5a failed to interact with the clathrin heavy chain, and interaction with hemoglobin receptor was significantly less. Thus, our results showed that LdRab5a and LdRab5b differentially regulate fluid phase and receptor-mediated endocytosis in Leishmania.
Asunto(s)
Endocitosis/fisiología , Leishmania donovani/metabolismo , Isoformas de Proteínas/fisiología , Proteínas de Unión al GTP rab5/fisiología , Secuencia de Aminoácidos , Animales , Mutación , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rab5/química , Proteínas de Unión al GTP rab5/genéticaRESUMEN
Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania.
Asunto(s)
Leishmania/enzimología , Proteínas de Unión al GTP rab1/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Datos de Secuencia Molecular , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rab1/químicaRESUMEN
The regulation of cell-cell adhesion is important for the processes of tissue formation and morphogenesis. Here, we report that loss of 14-3-3γ leads to a decrease in cell-cell adhesion and a defect in the transport of plakoglobin and other desmosomal proteins to the cell border in HCT116 cells and cells of the mouse testis. 14-3-3γ binds to plakoglobin in a PKCµ-dependent fashion, resulting in microtubule-dependent transport of plakoglobin to cell borders. Transport of plakoglobin to the border is dependent on the KIF5B-KLC1 complex. Knockdown of KIF5B in HCT116 cells, or in the mouse testis, results in a phenotype similar to that observed upon 14-3-3γ knockdown. Our results suggest that loss of 14-3-3γ leads to decreased desmosome formation and a decrease in cell-cell adhesion in vitro, and in the mouse testis in vivo, leading to defects in testis organization and spermatogenesis.
Asunto(s)
Proteínas 14-3-3/metabolismo , Desmosomas/metabolismo , gamma Catenina/metabolismo , Animales , Transporte Biológico , Adhesión Celular/fisiología , Células HCT116 , Humanos , Técnicas In Vitro , Infertilidad Masculina/metabolismo , Cinesinas , Masculino , RatonesRESUMEN
Leishmania is auxotroph for heme. Previously, we have shown that Leishmania acquire heme from the degradation of endocytosed hemoglobin via a specific receptor located in the flagellar pocket. Here, we report the cloning and expression of clathrin heavy chain from Leishmania (Ld-CHC) and provide evidences that Ld-CHC is localized in flagellar pocket and regulates Hb-endocytosis in Leishmania. Kinetic analysis of Hb trafficking in GFP-Ld-CHC overexpressed Leishmania reveals that Hb is internalized through Ld-CHC coated region and remains associated with Ld-CHC containing vesicles at early time points of internalization and subsequently starts dissociating from Hb-containing vesicles at later time points indicating that clathrin-coating and uncoating regulate Hb trafficking in Leishmania. Interestingly, overexpression of dominant negative mutant of clathrin heavy chain of Leishmania (GFP-Ld-CHC-Hub) blocks the Hb internalization and causes severe growth defect in parasite. Moreover, we have shown that chlorpromazine, a pharmacological agent, blocks Hb internalization in Leishmania by depolymerizing Ld-CHC and thereby inhibits the growth of the parasites. Taken together, our results have shown that Hb endocytosis in Leishmania is a clathrin dependent process and is essential for the survival of the parasites.
Asunto(s)
Cadenas Pesadas de Clatrina , Endocitosis , Leishmania , Secuencia de Aminoácidos , Clorpromazina/farmacología , Cadenas Pesadas de Clatrina/genética , Cadenas Pesadas de Clatrina/metabolismo , Clonación Molecular , Endocitosis/efectos de los fármacos , Endocitosis/genética , Expresión Génica/efectos de los fármacos , Hemo/metabolismo , Hemoglobinas/metabolismo , Cinética , Leishmania/genética , Leishmania/metabolismo , Leishmania/parasitología , Datos de Secuencia MolecularRESUMEN
Numerical modeling of the recovery of moisture by injecting warm air in the unsaturated zone in a 100 m × 100 m plot of agricultural land in Kuwait, a country located in an arid environment, was conducted to provide "proof of concept" of the technique. If technically and economically feasible, it will be a potential additional source of water that could be exploited for farming activities and other uses. The COMSOL software was used to develop the model and, based on the results of the scenario runs, the effects of different hydraulic and operational parameters, including that of well spacing, on moisture recovery were assessed. In general, the results suggested that the recovery should increase with the increase in the hydraulic conductivity of the unsaturated zone, the amount of heat input, and the pressure differential between the unsaturated zone and the well head. Within the period examined (0 to 11 days), the recovery decreases with the increase in the soil moisture content, possibly due to the fall in relative permeability to moisture-rich air with the increased water contents in the pore spaces, although the effects may change over a longer period as water contents decrease with moisture recovery. The moisture recovery from the unsaturated zone through the injection of warm air appears to be a feasible proposition from this study that should be demonstrated through a pilot scale experiment in the field.
RESUMEN
Leishmania donovani is an auxotroph for heme. Parasite acquires heme by clathrin-mediated endocytosis of hemoglobin by specific receptor. However, the regulation of receptor recycling pathway is not known in Leishmania. Here, we have cloned, expressed and characterized the Rab4 homologue from L. donovani. We have found that LdRab4 localizes in both early endosomes and Golgi in L. donovani. To understand the role of LdRab4 in L. donovani, we have generated transgenic parasites overexpressing GFP-LdRab4:WT, GFP-LdRab4:Q67L, and GFP-LdRab4:S22N. Our results have shown that overexpression of GFP-LdRab4:Q67L or GFP-LdRab4:S22N does not alter the cell surface localization of hemoglobin receptor in L. donovani. Surprisingly, we have found that overexpression of GFP-LdRab4:S22N significantly blocks the transport of Ldgp63 to the cell surface whereas the trafficking of Ldgp63 is induced to the cell surface in GFP-LdRab4:WT and GFP-LdRab4:Q67L overexpressing parasites. Consequently, we have found significant inhibition of gp63 secretion by GFP-LdRab4:S22N overexpressing parasites whereas secretion of Ldgp63 is enhanced in GFP-LdRab4:WT and GFP-LdRab4:Q67L overexpressing parasites in comparison to untransfected control parasites. Moreover, we have found that survival of transgenic parasites overexpressing GFP-LdRab4:S22N is severely compromised in macrophages in comparison to GFP-LdRab4:WT and GFP-LdRab4:Q67L expressing parasites. These results demonstrated that LdRab4 unconventionally regulates the secretory pathway in L. donovani.
Asunto(s)
Leishmania donovani , Vías Secretoras , Animales , Leishmania donovani/genética , Animales Modificados Genéticamente/metabolismo , Proteínas Portadoras/metabolismo , Hemoglobinas/metabolismo , Hemo/metabolismoRESUMEN
Several intracellular pathogens have developed diverse strategies to avoid targeting to lysosomes. However, they universally recruit lysosome-associated membrane protein 1 (LAMP1); the mechanism of LAMP1 recruitment remains unclear. Here, we report that a Salmonella effector protein, SipC, specifically binds with host Syntaxin6 through its C terminus and thereby recruits Syntaxin6 and other accessory molecules like VAMP2, Rab6, and Rab8 on Salmonella-containing phagosomes (SCP) and acquires LAMP1 by fusing with LAMP1-containing Golgi-derived vesicles. In contrast, sipC knock-out:SCP (sipC(-):SCP) or sipC(M398K):SCP fails to obtain significant amounts of Syntaxin6 and is unable to acquire LAMP1. Moreover, phagosomes containing respective knock-out Salmonella like sipA(-), sipB(-), sipD(-), sopB(-), or sopE(-) recruit LAMP1, demonstrating the specificity of SipC in this process. In addition, depletion of Syntaxin6 by shRNA in macrophages significantly inhibits LAMP1 recruitment on SCP. Additionally, survival of sipC(-):Salmonella in mice is found to be significantly inhibited in comparison with WT:Salmonella. Our results reveal a novel mechanism showing how Salmonella acquires LAMP1 through a SipC-Syntaxin6-mediated interaction probably to stabilize their niche in macrophages and also suggest that similar modalities might be used by other intracellular pathogens to recruit LAMP1.
Asunto(s)
Proteínas Bacterianas/metabolismo , Aparato de Golgi/metabolismo , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Fagosomas/metabolismo , Fagosomas/microbiología , Proteínas Qa-SNARE/metabolismo , Salmonella typhimurium/metabolismo , Animales , Proteínas Bacterianas/genética , Línea Celular , Aparato de Golgi/microbiología , Espacio Intracelular/metabolismo , Espacio Intracelular/microbiología , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Mutación , Transporte de Proteínas , Salmonella typhimurium/genética , Salmonella typhimurium/fisiología , Especificidad por SustratoRESUMEN
Vascular endothelial cadherin (VE-cadherin) expressed at endothelial adherens junctions (AJs) is vital for vascular integrity and endothelial homeostasis. Here we identify the requirement of the ubiquitin E3-ligase CHFR as a key mechanism of ubiquitylation-dependent degradation of VE-cadherin. CHFR was essential for disrupting the endothelium through control of the VE-cadherin protein expression at AJs. We observe augmented expression of VE-cadherin in endothelial cell (EC)-restricted Chfr knockout (ChfrΔEC) mice. We also observe abrogation of LPS-induced degradation of VE-cadherin in ChfrΔEC mice, suggesting the pathophysiological relevance of CHFR in regulating the endothelial junctional barrier in inflammation. Lung endothelial barrier breakdown, inflammatory neutrophil extravasation, and mortality induced by LPS were all suppressed in ChfrΔEC mice. We find that the transcription factor FoxO1 is a key upstream regulator of CHFR expression. These findings demonstrate the requisite role of the endothelial cell-expressed E3-ligase CHFR in regulating the expression of VE-cadherin, and thereby endothelial junctional barrier integrity.
Asunto(s)
Uniones Adherentes , Ubiquitina , Animales , Ratones , Uniones Adherentes/metabolismo , Ubiquitina/metabolismo , Ligasas/metabolismo , Lipopolisacáridos/farmacología , Cadherinas/genética , Cadherinas/metabolismo , Endotelio/metabolismo , Ubiquitinación , Endotelio Vascular/metabolismo , Células CultivadasRESUMEN
Recent studies suggest that training of innate immune cells such as tissue-resident macrophages by repeated noxious stimuli can heighten host defense responses. However, it remains unclear whether trained immunity of tissue-resident macrophages also enhances injury resolution to counterbalance the heightened inflammatory responses. Here, we studied lung-resident alveolar macrophages (AMs) prechallenged with either the bacterial endotoxin or with Pseudomonas aeruginosa and observed that these trained AMs showed greater resilience to pathogen-induced cell death. Transcriptomic analysis and functional assays showed greater capacity of trained AMs for efferocytosis of cellular debris and injury resolution. Single-cell high-dimensional mass cytometry analysis and lineage tracing demonstrated that training induces an expansion of a MERTKhiMarcohiCD163+F4/80low lung-resident AM subset with a proresolving phenotype. Reprogrammed AMs upregulated expression of the efferocytosis receptor MERTK mediated by the transcription factor KLF4. Adoptive transfer of these trained AMs restricted inflammatory lung injury in recipient mice exposed to lethal P. aeruginosa. Thus, our study has identified a subset of tissue-resident trained macrophages that prevent hyperinflammation and restore tissue homeostasis following repeated pathogen challenges.
Asunto(s)
Macrófagos Alveolares , Inmunidad Entrenada , Animales , Ratones , Traslado Adoptivo , Tirosina Quinasa c-Mer/genética , FagocitosisRESUMEN
Kuwait, an arid country, wants to have a reserve of water for emergency use and has planned to create an underground reserve of water through multi-well artificial recharge at Kabd area. Numerical modeling of different recharge-recovery scenarios was carried out to chalk out an optimum strategy for implementation of the project. These scenario runs suggested that apart from the aquifer parameters and the quality of the groundwater and the recharge water, the well spacing and well construction, orientation of the field with respect to the prevailing hydraulic gradient, and the injection and the pumping schedule would determine the overall recovery efficiency of the setup. For the selected site, following strategies were found to have positive impact on meeting the goals of the creation of a reserve of water for use in an emergency: (1) during the reserve creation stage, simultaneous recharge and pumping through alternate wells; (2) the compensation of the water lost from the reserve created due to the flow down the hydraulic gradient during the waiting period; and (3) the orientation of the long axis of the field perpendicular to the regional hydraulic gradient. The adoption of cyclic injection and recovery option for the creation of the reserve eliminates the need of the first two steps but calls for several cycles of injection followed by recovery and adequate and consistent supply of water for injection over that period. An optimum field design and two alternative recharge options have been suggested based on the above observations.