RESUMEN
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.
Asunto(s)
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.
Asunto(s)
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
[This corrects the article DOI: 10.3389/fmicb.2024.1347488.].
RESUMEN
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.
RESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
Infecciones por Klebsiella , Klebsiella pneumoniae , Animales , Ratones , Pulmón/microbiología , Fagocitosis , Macrófagos Alveolares , Neutrófilos , Infecciones por Klebsiella/microbiología , Antibacterianos/farmacologíaRESUMEN
Francisella tularensis is the causative agent of tularemia and is classified as a category A biodefense agent by the Centers for Disease Control and Prevention because of its highly infectious nature. F. tularensis infects leukocytes and exhibits an extracellular phase in the blood of the host. It is unknown, however, whether F. tularensis can infect erythrocytes; thus, we examined this possibility in vivo and in vitro. In the murine model of pulmonary type A tularemia, we showed the presence of intraerythrocytic bacteria by double-immunofluorescence microscopy and ex vivo gentamicin protection of the purified erythrocyte fraction. In vitro, F. tularensis invaded human erythrocytes, as shown in the gentamicin protection assays, double-immunofluorescence microscopy, flow cytometry, scanning electron microscopy, and transmission electron microscopy with immunogold labeling of the bacteria. Additional in vitro tests indicated that serum complement-dependent and complement-independent mechanisms contribute to erythrocyte invasion. Our results reveal a novel intraerythrocytic phase during F. tularensis infection.
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Eritrocitos/microbiología , Francisella tularensis/patogenicidad , Tularemia/microbiología , Tularemia/patología , Animales , Proteínas del Sistema Complemento/inmunología , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Francisella tularensis/crecimiento & desarrollo , Humanos , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Tularemia/epidemiologíaRESUMEN
Motile cilia project from the airway apical surface and directly interface with inhaled external environment. Owing to cilia's nanoscale dimension and high beating frequency, quantitative assessment of their motility remains a sophisticated task. Here we described a robust approach for reproducible engineering of apical-out airway organoid (AOAO) from a defined number of cells. Propelled by exterior-facing cilia beating, the mature AOAO exhibited stable rotational motion when surrounded by Matrigel. We developed a computational framework leveraging computer vision algorithms to quantify AOAO rotation and correlated it with the direct measurement of cilia motility. We further established the feasibility of using AOAO rotation to recapitulate and measure defective cilia motility caused by chemotherapy-induced toxicity and by CCDC39 mutations in cells from patients with primary ciliary dyskinesia. We expect our rotating AOAO model and the associated computational pipeline to offer a generalizable framework to expedite the modeling of and therapeutic development for genetic and environmental ciliopathies.
RESUMEN
Micavibrio aeruginosavorus is an obligate Gram-negative predatory bacterial species that feeds on other Gram-negative bacteria by attaching to the surface of its prey and feeding on the prey's cellular contents. In this study, Serratia marcescens with defined mutations in genes for extracellular cell structural components and secreted factors were used in predation experiments to identify structures that influence predation. No change was measured in the ability of the predator to prey on S. marcescens flagella, fimbria, surface layer, prodigiosin and phospholipase-A mutants. However, higher predation was measured on S. marcescens metalloprotease mutants. Complementation of the metalloprotease gene, prtS, into the protease mutant, as well as exogenous addition of purified serralysin metalloprotease, restored predation to wild type levels. Addition of purified serralysin also reduced the ability of M. aeruginosavorus to prey on Escherichia coli. Incubating M. aeruginosavorus with purified metalloprotease was found to not impact predator viability; however, pre-incubating prey, but not the predator, with purified metalloprotease was able to block predation. Finally, using flow cytometry and fluorescent microscopy, we were able to confirm that the ability of the predator to bind to the metalloprotease mutant was higher than that of the metalloprotease producing wild-type. The work presented in this study shows that metalloproteases from S. marcescens could offer elevated protection from predation.
Asunto(s)
Bacterias Gramnegativas/patogenicidad , Metaloproteasas/genética , Serratia marcescens/crecimiento & desarrollo , Proteínas Bacterianas/genética , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Metaloproteasas/metabolismo , Viabilidad Microbiana , Mutación , Serratia marcescens/enzimología , Serratia marcescens/genéticaRESUMEN
Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensis invades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU, or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks.
Asunto(s)
Eritrocitos/microbiología , Eritrocitos/fisiología , Francisella tularensis/patogenicidad , Tularemia/sangre , Tularemia/microbiología , Actinas , Animales , Proteínas Bacterianas/genética , Modelos Animales de Enfermedad , Endocitosis , Eritrocitos/patología , Femenino , Francisella tularensis/crecimiento & desarrollo , Genes Bacterianos/genética , Interacciones Huésped-Patógeno , Humanos , Concentración de Iones de Hidrógeno , Ixodes/microbiología , Ratones , Ratones Endogámicos C57BL , Mutación , Fagocitosis , Espectrina/farmacología , Enfermedades por Picaduras de Garrapatas/microbiología , Garrapatas/microbiología , Sistemas de Secreción Tipo VI/genéticaRESUMEN
Chronic obstructive pulmonary disease (COPD) involves aberrant airway inflammatory responses to cigarette smoke (CS) that are associated with epithelial cell dysfunction, cilia shortening, and mucociliary clearance disruption. Exposure to CS reduced cilia length and induced autophagy in vivo and in differentiated mouse tracheal epithelial cells (MTECs). Autophagy-impaired (Becn1+/- or Map1lc3B-/-) mice and MTECs resisted CS-induced cilia shortening. Furthermore, CS increased the autophagic turnover of ciliary proteins, indicating that autophagy may regulate cilia homeostasis. We identified cytosolic deacetylase HDAC6 as a critical regulator of autophagy-mediated cilia shortening during CS exposure. Mice bearing an X chromosome deletion of Hdac6 (Hdac6-/Y) and MTECs from these mice had reduced autophagy and were protected from CS-induced cilia shortening. Autophagy-impaired Becn1-/-, Map1lc3B-/-, and Hdac6-/Y mice or mice injected with an HDAC6 inhibitor were protected from CS-induced mucociliary clearance (MCC) disruption. MCC was preserved in mice given the chemical chaperone 4-phenylbutyric acid, but was disrupted in mice lacking the transcription factor NRF2, suggesting that oxidative stress and altered proteostasis contribute to the disruption of MCC. Analysis of human COPD specimens revealed epigenetic deregulation of HDAC6 by hypomethylation and increased protein expression in the airways. We conclude that an autophagy-dependent pathway regulates cilia length during CS exposure and has potential as a therapeutic target for COPD.
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Autofagia/fisiología , Cilios/fisiología , Histona Desacetilasas/fisiología , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Contaminación por Humo de Tabaco/efectos adversos , Animales , Proteínas Reguladoras de la Apoptosis/deficiencia , Beclina-1 , Células Cultivadas , Cilios/ultraestructura , Citosol/enzimología , Células Epiteliales/ultraestructura , Femenino , Histona Desacetilasa 6 , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Rastreo , Proteínas Asociadas a Microtúbulos/deficiencia , Moco , Factor 2 Relacionado con NF-E2/deficiencia , Factor 2 Relacionado con NF-E2/fisiología , Fenotipo , Fenilbutiratos/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/patología , Sirtuina 1/deficiencia , Sirtuina 1/fisiología , Productos de Tabaco , Tráquea/citología , UbiquitinaciónRESUMEN
AIMS: Veins are still the best conduits available for arterial bypass surgery. When these arterialized vein grafts fail, it is often due to the development of intimal hyperplasia (IH). We investigated the feasibility and efficacy of the ex vivo pre-treatment of vein grafts with soluble carbon monoxide (CO) in the inhibition of IH. METHODS AND RESULTS: The inferior vena cava was excised from donor rats and placed as an interposition graft into the abdominal aorta of syngeneic rats. Prior to implantation, vein grafts were stored in cold Lactated Ringer (LR) solution with or without CO saturation (bubbling of 100% CO) for 2 h. Three and 6 weeks following grafting, vein grafts treated with cold LR for 2 h developed IH, whereas grafts implanted immediately after harvest demonstrated significantly less IH. Treatment in CO-saturated LR significantly inhibited IH and reduced vascular endothelial cell (VEC) apoptosis. Electron microscopy revealed improved VEC integrity with less platelet/white blood cell aggregation in CO-treated grafts. The effects of CO in preventing IH were associated with activation of hypoxia inducible factor-1α (HIF-1α) and an increase in vascular endothelial growth factor (VEGF) expression at 3-6 h after grafting. Treatment with a HIF-1α inhibitor completely abrogated the induction of VEGF by CO and reversed the protective effects of CO on prevention of IH. CONCLUSION: Ex vivo treatment of vein grafts in CO-saturated LR preserved VEC integrity perioperatively and significantly reduced neointima formation. These effects appear to be mediated through the activation of the HIF1α/VEGF pathway.