scholarly journals Causes and Biophysical Consequences of Cellulose Production byPseudomonas fluorescensSBW25 at the Air-Liquid Interface

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Maxime Ardré ◽  
Djinthana Dufour ◽  
Paul B. Rainey
Keyword(s):  
Liquid Interface ◽  
Water Soluble ◽  
Diguanylate Cyclase ◽  
Content Type ◽  
Environmental Signals ◽  
Fluid Instabilities ◽  
Rayleigh Taylor Instability ◽  
Air Liquid Interface ◽  
Surface Production

ABSTRACTCellulose-overproducing wrinkly spreader mutants ofPseudomonas fluorescensSBW25 have been the focus of much investigation, but conditions promoting the production of cellulose in ancestral strain SBW25 and its effects and consequences have escaped in-depth investigation through lack of anin vitrophenotype. Here, using a custom-built device, we reveal that in static broth microcosms, ancestral SBW25 encounters environmental signals at the air-liquid interface that activate, via three diguanylate cyclase-encoding pathways (Wsp, Aws, and Mws), production of cellulose. Secretion of the polymer at the meniscus leads to modification of the environment and growth of numerous microcolonies that extend from the surface. Accumulation of cellulose and associated microbial growth leads to Rayleigh-Taylor instability resulting in bioconvection and rapid transport of water-soluble products over tens of millimeters. Drawing upon data, we built a mathematical model that recapitulates experimental results and captures the interactions between biological, chemical and physical processes.IMPORTANCEThis work reveals a hitherto unrecognized behavior that manifests at the air-liquid interface that depends on production of cellulose and hints at undiscovered dimensions to bacterial life at surfaces. Additionally, the study links activation of known diguanylate cyclase-encoding pathways to cellulose expression and to signals encountered at the meniscus. Further significance stems from recognition of the consequences of fluid instabilities arising from surface production of cellulose for transport of water-soluble products over large distances.

scholarly journals Causes and consequences of cellulose production byPseudomonas fluorescensSBW25 at the air-liquid interface

2019 ◽  
Author(s):  
Maxime Ardré ◽  
Paul B Rainey
Keyword(s):  
Liquid Interface ◽  
Water Soluble ◽  
Diguanylate Cyclase ◽  
Environmental Signals ◽  
Fluid Instabilities ◽  
Rayleigh Taylor Instability ◽  
Rapid Transport ◽  
Air Liquid Interface ◽  
Surface Production

ABSTRACTCellulose over-producing wrinkly spreader mutants ofPseudomonas fluorescensSBW25 have been the focus of much investigation, but conditions promoting the production of cellulose in ancestral SBW25, its effects and consequences have escaped investigation through lack ofin vitrophenotype. Here, using a custom built device, we reveal that in static broth microcosms ancestral SBW25 encounters environmental signals at the air-liquid interface that activate, via three diguanylate cyclase-encoding pathways (Wsp, Aws and Mws), production of cellulose. Secretion of the polymer at the meniscus leads to modification of the environment and growth of numerous micro-colonies that extend out from the surface. Accumulation of cellulose and associated microbial growth leads to Rayleigh-Taylor instability resulting in bioconvection and rapid transport of water-soluble products over tens of millimetres. Drawing upon data we build a mathematical model that recapitulates experimental results and captures the interactions between biological, chemical and physical processes.IMPORTANCEThis work reveals a new bacterial behaviour that manifests at the air-liquid interface, which depends on production of cellulose, and hints to hitherto unrecognised dimensions to bacterial life at surfaces. Additionally, the study links activation of known diguanylate cyclase-encoding pathways to cellulose expression and to signals encountered at the air-liquid interface. Further significance stems from recognition of the consequences of fluid instabilities arising from surface production of cellulose for transport of water-soluble products over large distances.

scholarly journals Paracellular Pathway-Mediated Mycoplasma hyopneumoniae Migration across Porcine Airway Epithelial Barrier under Air-Liquid Interface Conditions

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Haiyan Wang ◽  
Zhenzhen Zhang ◽  
Xing Xie ◽  
Beibei Liu ◽  
Yanna Wei ◽  
...  
Keyword(s):  
Mycoplasma Hyopneumoniae ◽  
Liquid Interface ◽  
Respiratory Pathogen ◽  
Epithelial Barrier ◽  
Airway Epithelial ◽  
Migration Ability ◽  
Content Type ◽  
Air Liquid Interface

ABSTRACT Mycoplasma hyopneumoniae is an important respiratory pathogen of pigs that causes persistent and secondary infections. However, the mechanisms by which this occurs are unclear. In this study, we established air-liquid interface culture systems for pig bronchial epithelial cells (ALI-PBECs) that were comparable to the conditions in the native bronchus in vivo. We used this ALI-PBECs model to study the infection and migration characteristics of M. hyopneumoniae in vitro. Based on the results, we confirmed that M. hyopneumoniae was able to adhere to ALI-PBECs and disrupt mucociliary function. Importantly, M. hyopneumoniae could migrate to the basolateral chamber through the paracellular route but not the transcellular pathway, and this was achieved by reversibly disrupting tight junctions (TJs) and increasing the permeability and damaging the integrity of the epithelial barrier. We examined the migration ability of M. hyopneumoniae using an ALI-PBECs model for the first time. The disruption of the epithelial barrier allowed M. hyopneumoniae to migrate to the basolateral chamber through the paracellular route, which may be related to immune evasion, extrapulmonary dissemination, and persistent infection of M. hyopneumoniae.

Zwischen Luft und Wasser – Air-Liquid-Interface-Kulturen als In-vitro-Modell des Respirationstrakts

2020 ◽  
Author(s):  
S. Runft ◽  
L. Burigk ◽  
A. Lehmbecker ◽  
K. Schöne ◽  
D. Waschke ◽  
...  
Keyword(s):  
Liquid Interface ◽  
Air Liquid Interface

scholarly journals Real-time measurement of cellular bioenergetics in fully differentiated human nasal epithelial cells grown at air-liquid-interface

2020 ◽  
Vol 318 (6) ◽  
pp. L1158-L1164
Author(s):  
Emily Mavin ◽  
Bernard Verdon ◽  
Sean Carrie ◽  
Vinciane Saint-Criq ◽  
Jason Powell ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Real Time ◽  
Aerobic Glycolysis ◽  
Airway Epithelial Cells ◽  
Respiratory Epithelium ◽  
Liquid Interface ◽  
Human Nasal Epithelial Cells ◽  
Respiratory Epithelial ◽  
Air Liquid Interface

Shifts in cellular metabolic phenotypes have the potential to cause disease-driving processes in respiratory disease. The respiratory epithelium is particularly susceptible to metabolic shifts in disease, but our understanding of these processes is limited by the incompatibility of the technology required to measure metabolism in real-time with the cell culture platforms used to generate differentiated respiratory epithelial cell types. Thus, to date, our understanding of respiratory epithelial metabolism has been restricted to that of basal epithelial cells in submerged culture, or via indirect end point metabolomics readouts in lung tissue. Here we present a novel methodology using the widely available Seahorse Analyzer platform to monitor real-time changes in the cellular metabolism of fully differentiated primary human airway epithelial cells grown at air-liquid interface (ALI). We show increased glycolytic, but not mitochondrial, ATP production rates in response to physiologically relevant increases in glucose availability. We also show that pharmacological inhibition of lactate dehydrogenase is able to reduce glucose-induced shifts toward aerobic glycolysis. This method is timely given the recent advances in our understanding of new respiratory epithelial subtypes that can only be observed in vitro through culture at ALI and will open new avenues to measure real-time metabolic changes in healthy and diseased respiratory epithelium, and in turn the potential for the development of novel therapeutics targeting metabolic-driven disease phenotypes.

scholarly journals Preliminary Study of In Vitro Three-Dimensional Skin Model Using an Ovine Collagen Type I Sponge Seeded with Co-Culture Skin Cells: Submerged versus Air-Liquid Interface Conditions

Polymers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 2784
Author(s):  
Mh Busra Fauzi ◽  
Zahra Rashidbenam ◽  
Aminuddin Bin Saim ◽  
Ruszymah Binti Hj Idrus
Keyword(s):  
Collagen Type ◽  
Three Dimensional ◽  
Collagen Type I ◽  
Liquid Interface ◽  
Type I ◽  
Skin Model ◽  
Skin Cells ◽  
Air Liquid Interface ◽  
In Vitro Skin Model

Three-dimensional (3D) in vitro skin models have been widely used for cosmeceutical and pharmaceutical applications aiming to reduce animal use in experiment. This study investigate capability of ovine tendon collagen type I (OTC-I) sponge suitable platform for a 3D in vitro skin model using co-cultured skin cells (CC) containing human epidermal keratinocytes (HEK) and human dermal fibroblasts (HDF) under submerged (SM) and air-liquid interface (ALI) conditions. Briefly, the extracted OTC-I was freeze-dried and crosslinked with genipin (OTC-I_GNP) and carbodiimide (OTC-I_EDC). The gross appearance, physico-chemical characteristics, biocompatibility and growth profile of seeded skin cells were assessed. The light brown and white appearance for the OTC-I_GNP scaffold and other groups were observed, respectively. The OTC-I_GNP scaffold demonstrated the highest swelling ratio (~1885%) and water uptake (94.96 ± 0.14%). The Fourier transformation infrared demonstrated amide A, B and I, II and III which represent collagen type I. The microstructure of all fabricated sponges presented a similar surface roughness with the presence of visible collagen fibers and a heterogenous porous structure. The OTC-I_EDC scaffold was more toxic and showed the lowest cell attachment and proliferation as compared to other groups. The micrographic evaluation revealed that CC potentially formed the epidermal- and dermal-like layers in both SM and ALI that prominently observed with OTC-I_GNP compared to others. In conclusion, these results suggest that OTC_GNP could be used as a 3D in vitro skin model under ALI microenvironment.

scholarly journals Role ofCaulobacterCell Surface Structures in Colonization of the Air-Liquid Interface

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Aretha Fiebig
Keyword(s):  
Cell Surface ◽  
Caulobacter Crescentus ◽  
Three Dimensional ◽  
Liquid Interface ◽  
Type Iv Pili ◽  
Surface Structures ◽  
Aquatic Environments ◽  
Content Type ◽  
Type Iv ◽  
Air Liquid Interface

ABSTRACTIn aquatic environments,Caulobacterspp. can be found at the boundary between liquid and air known as the neuston. I report an approach to study temporal features ofCaulobacter crescentuscolonization and pellicle biofilm development at the air-liquid interface and have defined the role of cell surface structures in this process. At this interface,C. crescentusinitially forms a monolayer of cells bearing a surface adhesin known as the holdfast. When excised from the liquid surface, this monolayer strongly adheres to glass. The monolayer subsequently develops into a three-dimensional structure that is highly enriched in clusters of stalked cells known as rosettes. As this pellicle film matures, it becomes more cohesive and less adherent to a glass surface. A mutant strain lacking a flagellum does not efficiently reach the surface, and strains lacking type IV pili exhibit defects in organization of the three-dimensional pellicle. Strains unable to synthesize the holdfast fail to accumulate at the boundary between air and liquid and do not form a pellicle. Phase-contrast images support a model whereby the holdfast functions to trapC. crescentuscells at the air-liquid boundary. Unlike the holdfast, neither the flagellum nor type IV pili are required forC. crescentusto partition to the air-liquid interface. While it is well established that the holdfast enables adherence to solid surfaces, this study provides evidence that the holdfast has physicochemical properties that allow partitioning of nonmotile mother cells to the air-liquid interface and facilitate colonization of this microenvironment.IMPORTANCEIn aquatic environments, the boundary at the air interface is often highly enriched with nutrients and oxygen. Colonization of this niche likely confers a significant fitness advantage in many cases. This study provides evidence that the cell surface adhesin known as a holdfast enablesCaulobacter crescentusto partition to and colonize the air-liquid interface. Additional surface structures, including the flagellum and type IV pili, are important determinants of colonization and biofilm formation at this boundary. Considering that holdfast-like adhesins are broadly conserved inCaulobacterspp. and other members of the diverse classAlphaproteobacteria, these surface structures may function broadly to facilitate colonization of air-liquid boundaries in a range of ecological contexts, including freshwater, marine, and soil ecosystems.

scholarly journals In well-differentiated primary human bronchial epithelial cells, TGF-β1 and TGF-β2 induce expression of furin

2020 ◽  
Author(s):  
Michael J O'Sullivan ◽  
Jennifer A Mitchel ◽  
Chimwemwe Mwase ◽  
Maureen McGill ◽  
Phyllis Kanki ◽  
...  
Keyword(s):  
Liquid Interface ◽  
Immunofluorescence Staining ◽  
Cellular Mechanisms ◽  
Specific Expression ◽  
Bronchial Epithelial ◽  
Cellular Entry ◽  
Well Differentiated ◽  
Air Liquid Interface ◽  
Tgf Β1

Background: The COVID-19 pandemic is an ongoing threat to public health. Since the identification of COVID-19, the disease caused by SARS-CoV-2, no drugs have been developed to specifically target SARS-CoV-2. To develop effective and safe treatment options, a better understanding of cellular mechanisms underlying SARS-CoV-2 infection is required. To fill this knowledge gap, researchers require reliable experimental systems that express the host proteins necessary for the cellular entry of SARS-CoV-2. These proteins include the viral receptor, ACE2 and the proteases TMPRSS2 and furin. A number of studies have reported cell-type specific expression of the genes encoding these molecules. However, less is known about the protein expression of these molecules. Methods: We assessed the suitability of primary human bronchial epithelial (HBE) cells maintained in air-liquid interface (ALI) as an experimental system for studying SARS-CoV-2 infection in vitro. During cellular differentiation, we measured the expression of ACE2, TMPRSS2, and furin over progressive ALI days by RT-qPCR, western blot and immunofluorescence staining. We also explored the effect of the fibrotic cytokine TGF-b on the expression of these proteins in well-differentiated HBE cells. Results/Discussion: Like ACE2, TMPRSS2 and furin proteins are localized in differentiated ciliated cells as confirmed by immunofluorescence staining. These data suggest that well-differentiated HBE cells maintained in air-liquid interface is a reliable in vitro system for investigating cellular mechanisms of SARS-CoV-2 infection. We further identified that profibrotic mediators, TGF-β1 and TGF-β2, increase the expression of furin, which is a protease required for the cellular entry of SARS-CoV-2.

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