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When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.
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Proteínas Serina-Treonina Quinasas , Fosforilación , Tamaño de la CélulaRESUMEN
BACKGROUND: Klebsiella pneumoniae carbapenemase-producing K pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. METHODS: We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct enzyme-linked immunosorbent assay, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. RESULTS: We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In 5 genetically related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsonophagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. CONCLUSIONS: Loss of function in wcaJ led to increased complement resistance, complement binding, and opsonophagocytosis, which may promote KPC-Kp persistence by enabling coexistence of increased bloodstream fitness and reduced tissue virulence.
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Cápsulas Bacterianas , Proteínas del Sistema Complemento , Infecciones por Klebsiella , Klebsiella pneumoniae , Fagocitosis , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/inmunología , Humanos , Infecciones por Klebsiella/inmunología , Infecciones por Klebsiella/microbiología , Animales , Cápsulas Bacterianas/inmunología , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Ratones , Proteínas del Sistema Complemento/inmunología , Mutación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuenciación Completa del Genoma , Reinfección/microbiología , Reinfección/inmunología , Bacteriemia/microbiología , Bacteriemia/inmunología , FemeninoRESUMEN
Vascularization plays a critical role in organ maturation and cell-type development. Drug discovery, organ mimicry, and ultimately transplantation hinge on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcame this hurdle by combining a human induced pluripotent stem cell (iPSC) line containing an inducible ETS translocation variant 2 (ETV2) (a transcription factor playing a role in endothelial cell development) that directs endothelial differentiation in vitro, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive endothelialization with a cellular identity most closely related to human kidney endothelia. Endothelialized kidney organoids also show increased maturation of nephron structures, an associated fenestrated endothelium with de novo formation of glomerular and venous subtypes, and the emergence of drug-responsive renin expressing cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Thus, incorporation of an engineered endothelial niche into a previously published kidney organoid protocol allowed the orthogonal differentiation of endothelial and parenchymal cell types, demonstrating the potential for applicability to other basic and translational organoid studies.
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A polyphasic taxonomic study was carried out on strain ES2T, isolated from sediment of a wetland created to remediate acid drainage from a coal mine. The rod-shaped bacterium formed yellow/orange pigmented colonies and produced the pigment flexirubin. The 16S rRNA gene sequence results assigned the strain to Chryseobacterium, with 98.9 and 98.3â% similarity to Chryseobacterium vietnamense and Chryseobacterium cucumeris, respectively. Computation of the average nucleotide identity and digital DNA-DNA hybridization values with the closest phylogenetic neighbours of ES2T revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. The dominant fatty acids of strain ES2T were iso-C15â:â0, iso-C17â:â1 ω9c, iso C17â:â0 3-OH, and iso-C15â:â0 2-OH. The DNA G+C content was 35.5 mol%. The major polar lipid was phosphatidylethanolamine while menaquinone-6 was the only menaquinone found. This bacterium has been previously shown to possess metallophore activity towards rare earth elements, and based on genome sequencing, possesses all required genes for siderophore production/activity, possibly identifying the source of this unique ability. On the basis of the results obtained here, this bacterium is assigned to the genus Chryseobacterium as representing a new species with the name Chryseobacterium metallicongregator sp. nov., type strain ES2T (=NRRL B-65679T=KCTC 102120T).
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Chryseobacterium , Ácidos Grasos , Ácidos Grasos/química , Filogenia , ARN Ribosómico 16S/genética , Vitamina K 2 , ADN Bacteriano/genética , Composición de Base , Técnicas de Tipificación Bacteriana , Análisis de Secuencia de ADNRESUMEN
A polyphasic taxonomic study was carried out on strain TSed Te1T, isolated from sediment of a stream contaminated with acid drainage from a coal mine. The bacterium forms pink-pigmented colonies and has a rod-coccus growth cycle, which also includes some coryneform arrangements. This bacterium is capable of growing in the presence of up to 750 µg ml-1 tellurite and 5000 µg ml-1 selenite, reducing each to elemental form. Nearly complete 16S rRNA gene sequence analysis associated the strain with Gordonia, with 99.5 and 99.3â% similarity to Gordonia namibiensis and Gordonia rubripertincta, respectively. Computation of the average nucleotide identity and digital DNA-DNA hybridization comparisons with the closest phylogenetic neighbour of TSed Te1T revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. The dominant fatty acids were C16â:â0, C18â:â1, C16â:â1 and tuberculostearic acid. The DNA G+C content was 67.6 mol%. Major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside, while MK-9(H2) was the only menaquinone found. Mycolic acids of C56-C60 were present. Whole-cell hydrolysates contained meso-diaminopimelic acid along with arabinose and galactose as the major cell-wall sugars. On the basis of the results obtained in this study, the bacterium was assigned to the genus Gordonia and represents a new species with the name Gordonia metallireducens sp. nov. The type strain is TSed Te1T (=NRRL B-65678T=DSM 114093T).
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Ácidos Grasos , Bacteria Gordonia , Ácidos Grasos/química , Filogenia , ARN Ribosómico 16S/genética , Ríos , ADN Bacteriano/genética , Análisis de Secuencia de ADN , Composición de Base , Técnicas de Tipificación Bacteriana , Vitamina K 2RESUMEN
Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of ß-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of ß-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-ß signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of ß-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by ß-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.
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Colestasis Intrahepática/patología , Factor Nuclear 4 del Hepatocito/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , beta Catenina/metabolismo , gamma Catenina/metabolismo , Uniones Adherentes/metabolismo , Animales , Línea Celular Tumoral , Polaridad Celular , Factor Nuclear 4 del Hepatocito/genética , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Ratones , Ratones Noqueados , Factor de Crecimiento Transformador beta/genética , beta Catenina/genética , gamma Catenina/economía , gamma Catenina/genéticaRESUMEN
Viral cancers show oncogene addiction to viral oncoproteins, which are required for survival and proliferation of the dedifferentiated cancer cell. Human Merkel cell carcinomas (MCCs) that harbor a clonally integrated Merkel cell polyomavirus (MCV) genome have low mutation burden and require viral T antigen expression for tumor growth. Here, we showed that MCV+ MCC cells cocultured with keratinocytes undergo neuron-like differentiation with neurite outgrowth, secretory vesicle accumulation, and the generation of sodium-dependent action potentials, hallmarks of a neuronal cell lineage. Cocultured keratinocytes are essential for induction of the neuronal phenotype. Keratinocyte-conditioned medium was insufficient to induce this phenotype. Single-cell RNA sequencing revealed that T antigen knockdown inhibited cell cycle gene expression and reduced expression of key Merkel cell lineage/MCC marker genes, including HES6, SOX2, ATOH1, and KRT20 Of these, T antigen knockdown directly inhibited Sox2 and Atoh1 expression. MCV large T up-regulated Sox2 through its retinoblastoma protein-inhibition domain, which in turn activated Atoh1 expression. The knockdown of Sox2 in MCV+ MCCs mimicked T antigen knockdown by inducing MCC cell growth arrest and neuron-like differentiation. These results show Sox2-dependent conversion of an undifferentiated, aggressive cancer cell to a differentiated neuron-like phenotype and suggest that the ontology of MCC arises from a neuronal cell precursor.
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Antígenos Virales de Tumores/genética , Carcinoma de Células de Merkel/etiología , Carcinoma de Células de Merkel/metabolismo , Poliomavirus de Células de Merkel/genética , Fenotipo , Infecciones por Polyomavirus/complicaciones , Factores de Transcripción SOXB1/genética , Antígenos Virales de Tumores/inmunología , Antígenos Virales de Tumores/metabolismo , Carcinoma de Células de Merkel/patología , Ciclo Celular/genética , Línea Celular Tumoral , Linaje de la Célula/genética , Transformación Celular Viral , Técnicas de Silenciamiento del Gen , Humanos , Queratinocitos , Células de Merkel/metabolismo , Poliomavirus de Células de Merkel/inmunología , Neuritas/metabolismo , Neuronas/metabolismo , Infecciones por Polyomavirus/inmunología , Infecciones por Polyomavirus/virología , Factores de Transcripción SOXB1/metabolismo , Infecciones Tumorales por Virus/complicaciones , Infecciones Tumorales por Virus/inmunología , Infecciones Tumorales por Virus/virologíaRESUMEN
Osteoblasts in vivo form an epithelial-like layer with tight junctions between cells. Bone formation involves mineral transport into the matrix and acid transport to balance pH levels. To study the importance of the pH gradient in vitro, we used Transwell inserts composed of polyethylene terephthalate (PET) membranes with 0.4 µm pores at a density of (2 ± 0.4) x 106 pores per cm2. Mesenchymal stem cells (MSCs) prepared from murine bone marrow were used to investigate alternative conditions whereby osteoblast differentiation would better emulate in vivo bone development. MSCs were characterized by flow cytometry with more than 90% CD44 and 75% Sca-1 labeling. Mineralization was validated with paracellular alkaline phosphatase activity, collagen birefringence, and mineral deposition confirming MSCs identity. We demonstrate that MSCs cultured and differentiated on PET inserts form an epithelial-like layer while mineralizing. Measurement of the transepithelial resistance was â¼1400 Ωâ¢cm2 at three weeks of differentiation. The pH value of the media above and under the cells were measured while cells were in proliferation and differentiation. In mineralizing cells, a difference of 0.145 pH unit was observed between the medium above and under the cells indicating a transepithelial gradient. A significant difference in pH units was observed between the medium above and below the cells in proliferation compared to differentiation. Data on pH below membranes were confirmed by pH-dependent SNARF1 fluorescence. Control cells in proliferative medium did not form an epithelial-like layer, displayed low transepithelial resistance, and there was no significant pH gradient. By transmission electron microscopy, membrane attached osteoblasts in vitro had abundant mitochondria consistent with active transport that occurs in vivo by surface osteoblasts. In keeping with osteoblastic differentiation, scanning electron microscopy identified deposition of extracellular collagen surrounded by hydroxyapatite. This in vitro model is a major advancement in modeling bone in vivo for understanding of osteoblast bone matrix production.
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Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Animales , Calcificación Fisiológica , Proliferación Celular , Células Cultivadas , Células Epiteliales/citología , Concentración de Iones de Hidrógeno , Membranas Artificiales , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteogénesis , Tereftalatos Polietilenos/químicaRESUMEN
Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S. marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection.
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Toxinas Bacterianas/metabolismo , Células Epiteliales/metabolismo , Epitelio Corneal/metabolismo , Infecciones por Proteus/metabolismo , Proteus/metabolismo , Infecciones por Serratia/metabolismo , Serratia marcescens/metabolismo , Animales , Toxinas Bacterianas/genética , Muerte Celular , Células Epiteliales/microbiología , Células Epiteliales/patología , Epitelio Corneal/microbiología , Epitelio Corneal/patología , Humanos , Ratones , Perforina/genética , Perforina/metabolismo , Proteus/genética , Infecciones por Proteus/genética , Infecciones por Proteus/microbiología , Infecciones por Proteus/patología , Células RAW 264.7 , Infecciones por Serratia/genética , Infecciones por Serratia/microbiología , Infecciones por Serratia/patología , Serratia marcescens/genética , Porcinos , Sistemas de Secreción Tipo V/genética , Sistemas de Secreción Tipo V/metabolismoRESUMEN
Degeneration of retinal pigment epithelium (RPE) is one of the most critical phenotypic changes of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. While cultured polarized RPE cells with original properties are valuable in in vitro models to study RPE biology and the consequences of genetic and/or pharmacological manipulations, the procedure to establish mouse primary PRE cell culture or pluripotent stem cell-derived RPE cells is time-consuming and yields a limited number of cells. Thus, establishing a mouse in situ RPE culture system is highly desirable. Here we describe a novel and efficient method for RPE explant culture that allows for obtaining biologically relevant RPE cells in situ. These RPE explants (herein referred to as RPE flatmounts) are viable in culture for at least 7 days, can be efficiently transduced with adenoviral constructs, and/or treated with a variety of drugs/chemicals followed by downstream analysis of the signaling pathways/biological processes of interest, such as assessment of the autophagy flux, inflammatory response, and receptor tyrosine kinases stimulation. This method of RPE explant culture is highly beneficial for pharmacological and mechanistic studies in the field of RPE biology and AMD research.
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Adenoviridae/genética , Vectores Genéticos/administración & dosificación , Degeneración Macular/patología , Técnicas de Cultivo de Órganos/métodos , Epitelio Pigmentado de la Retina/citología , Transgenes , Animales , Células Cultivadas , Degeneración Macular/genética , Degeneración Macular/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Epitelio Pigmentado de la Retina/metabolismo , Transducción GenéticaRESUMEN
The keratin cytoskeleton and associated desmosomes contribute to the mechanical stability of epithelial tissues, but their organization in bladder umbrella cells and their responses to bladder filling are poorly understood. Using super-resolution confocal microscopy, along with 3D image reconstruction and platinum replica electron microscopy, we observed that the apical keratin network of umbrella cells was organized as a dense tile-like mesh comprised of tesserae bordered on their edges by cortical actin filaments, filled with woven keratin filaments, and crosslinked by plectin. A band of keratin was also observed at the cell periphery that was linked to the junction-associated actin ring by plectin. During bladder filling, the junction-localized desmosomal necklace expanded, and a subjacent girded layer was formed that linked the keratin network to desmosomes, including those at the umbrella cell-intermediate cell interface. Disruption of plectin led to focal keratin network dissolution, loss of the junction-associated band of keratin, perturbation of tight junction continuity, and loss of cell-cell cohesion. Our studies reveal a novel tile-like organization of the umbrella cell keratin cytoskeleton that is dependent on plectin, that reorganizes in response to bladder filling, and that likely serves to maintain umbrella cell continuity in the face of mechanical distension.
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[This corrects the article DOI: 10.3389/fmicb.2024.1347488.].
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Francisella tularensis is a gram-negative, intracellular pathogen which can cause serious, potentially fatal, illness in humans. Species of F. tularensis are found across the Northern Hemisphere and can infect a broad range of host species, including humans. Factors affecting the persistence of F. tularensis in the environment and its epidemiology are not well understood, however, the ability of F. tularensis to enter a viable but non-culturable state (VBNC) may be important. A broad range of bacteria, including many pathogens, have been observed to enter the VBNC state in response to stressful environmental conditions, such as nutrient limitation, osmotic or oxidative stress or low temperature. To investigate the transition into the VBNC state for F. tularensis, we analyzed the attenuated live vaccine strain, F. tularensis LVS grown under standard laboratory conditions. We found that F. tularensis LVS rapidly and spontaneously enters a VBNC state in broth culture at 37°C and that this transition coincides with morphological differentiation of the cells. The VBNC bacteria retained an ability to interact with both murine macrophages and human erythrocytes in in vitro assays and were insensitive to treatment with gentamicin. Finally, we present the first transcriptomic analysis of VBNC F. tularensis, which revealed clear differences in gene expression, and we identify sets of differentially regulated genes which are specific to the VBNC state. Identification of these VBNC specific genes will pave the way for future research aimed at dissecting the molecular mechanisms driving entry into the VBNC state.
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Non-neovascular or dry age-related macular degeneration (AMD) is a multi-factorial disease with degeneration of the aging retinal-pigmented epithelium (RPE). Lysosomes play a crucial role in RPE health via phagocytosis and autophagy, which are regulated by transcription factor EB/E3 (TFEB/E3). Here, we find that increased AKT2 inhibits PGC-1α to downregulate SIRT5, which we identify as an AKT2 binding partner. Crosstalk between SIRT5 and AKT2 facilitates TFEB-dependent lysosomal function in the RPE. AKT2/SIRT5/TFEB pathway inhibition in the RPE induced lysosome/autophagy signaling abnormalities, disrupted mitochondrial function and induced release of debris contributing to drusen. Accordingly, AKT2 overexpression in the RPE caused a dry AMD-like phenotype in aging Akt2 KI mice, as evident from decline in retinal function. Importantly, we show that induced pluripotent stem cell-derived RPE encoding the major risk variant associated with AMD (complement factor H; CFH Y402H) express increased AKT2, impairing TFEB/TFE3-dependent lysosomal function. Collectively, these findings suggest that targeting the AKT2/SIRT5/TFEB pathway may be an effective therapy to delay the progression of dry AMD.
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Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Lisosomas , Degeneración Macular , Proteínas Proto-Oncogénicas c-akt , Epitelio Pigmentado de la Retina , Transducción de Señal , Sirtuinas , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sirtuinas/metabolismo , Sirtuinas/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Degeneración Macular/genética , Humanos , Ratones , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Lisosomas/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Modelos Animales de Enfermedad , Células Madre Pluripotentes Inducidas/metabolismo , MasculinoRESUMEN
Purpose: Pseudomonas aeruginosa keratitis is a severe ocular infection that can lead to perforation of the cornea. In this study we evaluated the role of bacterial quorum sensing in generating corneal perforation and bacterial proliferation and tested whether co-injection of the predatory bacteria Bdellovibrio bacteriovorus could alter the clinical outcome. P. aeruginosa with lasR mutations were observed among keratitis isolates from a study collecting samples from India, so an isogenic lasR mutant strain of P. aeruginosa was included. Methods: Rabbit corneas were intracorneally infected with P. aeruginosa strain PA14 or an isogenic Δ lasR mutant and co-injected with PBS or B. bacteriovorus . After 24 h, eyes were evaluated for clinical signs of infection. Samples were analyzed by scanning electron microscopy, optical coherence tomography, sectioned for histology, and corneas were homogenized for CFU enumeration and for inflammatory cytokines. Results: We observed that 54% of corneas infected by wild-type PA14 presented with a corneal perforation (n=24), whereas only 4% of PA14 infected corneas that were co-infected with B. bacteriovorus perforate (n=25). Wild-type P. aeruginosa proliferation was reduced 7-fold in the predatory bacteria treated eyes. The Δ lasR mutant was less able to proliferate compared to the wild-type, but was largely unaffected by B. bacteriovorus . Conclusion: These studies indicate a role for bacterial quorum sensing in the ability of P. aeruginosa to proliferate and cause perforation of the rabbit cornea. Additionally, this study suggests that predatory bacteria can reduce the virulence of P. aeruginosa in an ocular prophylaxis model.
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PURPOSE: Pseudomonas aeruginosa keratitis is a severe ocular infection that can lead to perforation of the cornea. In this study we evaluated the role of bacterial quorum sensing in generating corneal perforation and bacterial proliferation and tested whether co-injection of the predatory bacteria Bdellovibrio bacteriovorus could alter the clinical outcome. P. aeruginosa with lasR mutations were observed among keratitis isolates from a study collecting samples from India, so an isogenic lasR mutant strain of P. aeruginosa was included. METHODS: Rabbit corneas were intracorneally infected with P. aeruginosa strain PA14 or an isogenic ΔlasR mutant and co-injected with PBS or B. bacteriovorus. After 24 h, eyes were evaluated for clinical signs of infection. Samples were analyzed by scanning electron microscopy, optical coherence tomography, sectioned for histology, and corneas were homogenized for CFU enumeration and for inflammatory cytokines. RESULTS: We observed that 54% of corneas infected by wild-type PA14 presented with a corneal perforation (n = 24), whereas only 4% of PA14 infected corneas that were co-infected with B. bacteriovorus perforate (n = 25). Wild-type P. aeruginosa proliferation was reduced 7-fold in the predatory bacteria treated eyes. The ΔlasR mutant was less able to proliferate compared to the wild-type, but was largely unaffected by B. bacteriovorus. CONCLUSION: These studies indicate a role for bacterial quorum sensing in the ability of P. aeruginosa to proliferate and cause perforation of the rabbit cornea. Additionally, this study suggests that predatory bacteria can reduce the virulence of P. aeruginosa in an ocular prophylaxis model.
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Perforación Corneal , Infecciones Bacterianas del Ojo , Queratitis , Infecciones por Pseudomonas , Animales , Conejos , Pseudomonas aeruginosa , Infecciones por Pseudomonas/microbiología , Queratitis/tratamiento farmacológico , Córnea/patología , Bacterias , Proliferación Celular , Infecciones Bacterianas del Ojo/microbiologíaRESUMEN
BACKGROUND AND AIMS: TM6SF2 rs58542926 (E167K) is associated with an increase in the prevalence of Metabolic Disfunction-Associated Steatotic Liver Disease (MASLD). Despite all the investigation related to the role of this variant in lipid metabolism, conflicting results in mouse studies underscore the importance of creating a human model for understanding the TM6SF2 mechanism. Therefore, the aim of this study is to generate a reliable human in vitro model that mimic the effects of the TM6SF2 E167K mutation and can be used for future mechanism studies. APPROACH AND RESULTS: We performed gene editing on human-induced pluripotent stem cells (iPSC) derived from a healthy individual to obtain the cells carrying the TM6SF2 E167K mutation. After hepatic differentiation, a decrease in TM6SF2 protein expression was observed in the mutated-induced hepatocyte. An increase in intracellular lipid droplets and a decrease in the efflux of cholesterol and ApoB100 were also observed. Transcriptomics analysis showed up-regulation of genes related to the transport, flux, and oxidation of lipids, fatty acids, and cholesterol in TM6SF2 E167K cells. Additionally, signs of cellular stress were observed in the ER and mitochondria. CONCLUSIONS: Our findings indicate that induced hepatocytes generated from iPSC carrying the TM6SF2 E167K recapitulate the effects observed in human hepatocytes from individuals with the TM6SF2 mutation. This study characterizes an in vitro model that can be used as a platform to help in the identification of potential clinical targets and therapies and to understand the mechanism by which the TM6SF2 E167K variant leads to vulnerability to MASLD.
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Introduction: Klebsiella pneumoniae (Kp) is a common cause of hospital-acquired pneumonia. Although previous studies have suggested that evasion of phagocytic uptake is a virulence determinant of Kp, few studies have examined phagocytosis sensitivity in clinical Kp isolates. Methods: We screened 19 clinical respiratory Kp isolates that were previously assessed for mucoviscosity for their sensitivity to macrophage phagocytic uptake, and evaluated phagocytosis as a functional correlate of in vivo Kp pathogenicity. Results: The respiratory Kp isolates displayed heterogeneity in the susceptibility to macrophage phagocytic uptake, with 14 out of 19 Kp isolates displaying relative phagocytosis-sensitivity compared to the reference Kp strain ATCC 43816, and 5 out of 19 Kp isolates displaying relative phagocytosis-resistance. Intratracheal infection with the non-mucoviscous phagocytosis-sensitive isolate S17 resulted in a significantly lower bacterial burden compared to infection with the mucoviscous phagocytosis-resistant isolate W42. In addition, infection with S17 was associated with a reduced inflammatory response, including reduced bronchoalveolar lavage fluid (BAL) polymorphonuclear (PMN) cell count, and reduced BAL TNF, IL-1ß, and IL-12p40 levels. Importantly, host control of infection with the phagocytosis-sensitive S17 isolate was impaired in alveolar macrophage (AM)-depleted mice, whereas AM-depletion had no significant impact on host defense against infection with the phagocytosis-resistant W42 isolate. Conclusion: Altogether, these findings show that phagocytosis is a primary determinant of pulmonary clearance of clinical Kp isolates.
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Infecciones por Klebsiella , Klebsiella pneumoniae , Animales , Ratones , Pulmón/microbiología , Fagocitosis , Macrófagos Alveolares , Neutrófilos , Infecciones por Klebsiella/microbiología , Antibacterianos/farmacologíaRESUMEN
Vascularization plays a critical role in organ maturation and cell type development. Drug discovery, organ mimicry, and ultimately transplantation in a clinical setting thereby hinges on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcome this hurdle by combining an inducible ETS translocation variant 2 (ETV2) human induced pluripotent stem cell (iPSC) line, which directs endothelial fate, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive vascularization by endothelial cells with an identity most closely related to endogenous kidney endothelia. Vascularized organoids also show increased maturation of nephron structures including more mature podocytes with improved marker expression, foot process interdigitation, an associated fenestrated endothelium, and the presence of renin+ cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Furthermore, this approach is orthogonal to native tissue differentiation paths, hence readily adaptable to other organoid systems and thus has the potential for a broad impact on basic and translational organoid studies.
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Age-related macular degeneration (AMD), the leading cause of geriatric blindness, is a multi-factorial disease with retinal-pigmented epithelial (RPE) cell dysfunction as a central pathogenic driver. With RPE degeneration, lysosomal function is a core process that is disrupted. Transcription factors EB/E3 (TFEB/E3) tightly control lysosomal function; their disruption can cause aging disorders, such as AMD. Here, we show that induced pluripotent stem cells (iPSC)-derived RPE cells with the complement factor H variant [ CFH (Y402H)] have increased AKT2, which impairs TFEB/TFE3 nuclear translocation and lysosomal function. Increased AKT2 can inhibit PGC1α, which downregulates SIRT5, an AKT2 binding partner. SIRT5 and AKT2 co-regulate each other, thereby modulating TFEB-dependent lysosomal function in the RPE. Failure of the AKT2/SIRT5/TFEB pathway in the RPE induced abnormalities in the autophagy-lysosome cellular axis by upregulating secretory autophagy, thereby releasing a plethora of factors that likely contribute to drusen formation, a hallmark of AMD. Finally, overexpressing AKT2 in RPE cells in mice led to an AMD-like phenotype. Thus, targeting the AKT2/SIRT5/TFEB pathway could be a potential therapy for atrophic AMD.