Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier

Kuehn, Anna; Kletting, Stephanie; Carvalho-Wodarz, Cristiane de Souza; Repnik, Urška; Griffiths, Gareth; Fischer, Ulrike; Meese, Eckart; Huwer, Hanno; Wirth, Dagmar; May, Tobias; Schneider-Daum, Nicole; Lehr, Claus Michael

doi:10.14573/altex.1511131

Cited by 72 publications

(107 citation statements)

References 38 publications

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“…The immortalisation and characterisation of the muGob (Cl10) cells will be described elsewhere (Truschel et al, in preparation). Briefly, murine intestinal organoids were plated and infected with different lentiviruses encoding the CI-SCREEN gene library (Kuehn et al, 2016). After transduction, the clonal cell line muGob (Cl10) was established, which has integrated the following recombinant genes of the CI-SCREEN library: Id1, Id2, Id3, Myc, Fos, E7, Core, Rex (Zfp42).…”

Section: Invasion and Adhesion Assaysmentioning

confidence: 99%

Flagellin phase-dependent swimming on epithelial cell surfaces contributes to productiveSalmonellagut colonisation

Horstmann

Zschieschang

Truschel³

et al. 2017

Cellular Microbiology

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The flagellum is a sophisticated nanomachine and an important virulence factor of many pathogenic bacteria. Flagellar motility enables directed movements towards host cells in a chemotactic process, and near-surface swimming on cell surfaces is crucial for selection of permissive entry sites. The long external flagellar filament is made of tens of thousands subunits of a single protein, flagellin, and many Salmonella serovars alternate expression of antigenically distinct flagellin proteins, FliC and FljB. However, the role of the different flagellin variants during gut colonisation and host cell invasion remains elusive. Here, we demonstrate that flagella made of different flagellin variants display structural differences and affect Salmonella's swimming behaviour on host cell surfaces. We observed a distinct advantage of bacteria expressing FliC-flagella to identify target sites on host cell surfaces and to invade epithelial cells. FliC-expressing bacteria outcompeted FljB-expressing bacteria for intestinal tissue colonisation in the gastroenteritis and typhoid murine infection models. Intracellular survival and responses of the host immune system were not altered. We conclude that structural properties of flagella modulate the swimming behaviour on host cell surfaces, which facilitates the search for invasion sites and might constitute a general mechanism for productive host cell invasion of flagellated bacteria.

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Section: Invasion and Adhesion Assaysmentioning

confidence: 99%

Flagellin phase-dependent swimming on epithelial cell surfaces contributes to productiveSalmonellagut colonisation

Horstmann

Zschieschang

Truschel³

et al. 2017

Cellular Microbiology

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“…With regard to the establishment of cell culture models, it is most important to obtain cells with a physiological and functional phenotype which should be as close as possible to primary cells. In this line, a functionality of immortalized cells displaying similar sets of integrated transgenes has already been demonstrated for different cell types, including human alveolar epithelial cells and murine hepatocytes (Kuehn et al, ; Lipps et al, ). Furthermore, MSC immortalization is increasingly performed using combinations of different factors to improve the characteristics of the resulting cell lines and overcome limitations of hTERT‐immortalized MSC (Liu et al, ; Tang et al, ; Tátrai et al, ).…”

Section: Discussionmentioning

confidence: 96%

“…Experiments were performed in at least three independent replicates using passage (P) 3 MSC. K5 iMSC were smaller in diameter (a), showed a higher colony forming unit potential (b), reached a higher population doubling level within the observed subculture period, indicating more rapid proliferation (c), and migrated more efficiently including human alveolar epithelial cells and murine hepatocytes (Kuehn et al, 2016;Lipps et al, 2018). Furthermore, MSC immortalization is increasingly performed using combinations of different factors to improve the characteristics of the resulting cell lines and overcome limitations of hTERT-immortalized MSC (Liu et al, 2013;Tang et al, 2013;Tátrai et al, 2012).…”

Section: Migration Of Cells In the Scratch Assay Was Observed In K5 Imscmentioning

confidence: 99%

Generation and characterization of a functional human adipose‐derived multipotent mesenchymal stromal cell line

Burk

Holland²,

Lauermann

et al. 2019

Biotech & Bioengineering

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Multipotent mesenchymal stromal cells (MSC) and MSC-derived products have emerged as promising therapeutic tools. To fully exploit their potential, further mechanistic studies are still necessary and bioprocessing needs to be optimized, which requires an abundant supply of functional MSC for basic research. To address this need, here we used a novel technology to establish a human adipose-derived MSC line with functional characteristics representative of primary MSC. Primary MSC were isolated and subjected to lentiviral transduction with a library of expansion genes. Clonal cell lines were generated and evaluated on the basis of their morphology, immunophenotype, and proliferation potential. One clone (K5 iMSC) was then selected for further characterization. This clone had integrated a specific transgene combination including genes involved in stemness and maintenance of adult stem cells. Favorably, the K5 iMSC showed cell characteristics resembling juvenile MSC, as they displayed a shorter cell length and enhanced migration and proliferation compared with the nonimmortalized original primary MSC (p < 0.05). Still, their immunophenotype and differentiation potential corresponded to the original primary MSC and the MSC definition criteria, and cytogenetic analyses revealed no clonal aberrations. We conclude that the technology used is applicable to generate functional MSC lines for basic research and possible future bioprocessing applications. K E Y W O R D S bioprocessing, cell line, immortalization, mesenchymal stromal cells, multipotent mesenchymal stromal cell (MSC)

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“…The immortalization and characterization of the muGob (Cl11) cells will be described elsewhere (Truschel et al, in preparation). Briefly, murine intestinal organoids were plated and infected with different lentiviruses encoding the CI-SCREEN gene library 61 . After transduction, the clonal cell line muGob (Cl11) was established, which has integrated the following recombinant genes of the CI-SCREEN library: Id1, Id2, Id3, Myc, Fos, E7, Core, Rex (Zfp42).…”

Section: Methodsmentioning

confidence: 99%

Flagella methylation promotes bacterial adhesion and host cell invasion

Horstmann

Lunelli

Cazzola

et al. 2019

Preprint

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35The flagellum is the motility device of many bacteria and the long external filament is 36 made of several thousand copies of a single protein, flagellin. While posttranslational 37 modifications of flagellin are common among bacterial pathogens, the role of lysine 38 methylation remained unknown. Here, we show that both flagellins of Salmonella 39 enterica, FliC and FljB, are methylated at surface-exposed lysine residues. A 40 Salmonella mutant deficient in flagellin methylation was outcompeted for gut 41 colonization in a gastroenteritis mouse model. In support, methylation of flagellin 42 promoted invasion of epithelial cells in vitro. Lysine methylation increased the surface 43 hydrophobicity of flagellin and enhanced flagella-dependent adhesion of Salmonella to 44 phosphatidylcholine vesicles and epithelial cells. In summary, posttranslational flagellin 45 methylation constitutes a novel mechanism how flagellated bacteria facilitate adhesion 46 to hydrophobic host cell surfaces and thereby contributes to efficient gut colonization 47 and successful infection of the host. 48 49 50 51 52 53 54 Introduction: 57 The Gram-negative enteropathogen Salmonella enterica uses a variety of strategies to 58 successfully enter and replicate within a host. In this respect, bacterial motility enables 59 the directed movement of the bacteria towards nutrients or the target site of infection. A 60 rotary nanomachine, the flagellum, mediates motility of many bacteria, including 61 Salmonella enterica 1 . Flagella also play a central role in other infection processes, 62 involving biofilm formation, immune system modulation and adhesion 2-4 . 63 The eukaryotic plasma membrane plays an important role in the interaction of 64 flagellated bacteria with host cells during the early stages of infection 5 . The flagella of S. 65 enterica, Escherichia coli and Pseudomonas aeruginosa can function as adhesion 66 molecules 6-8 mediating the contact to various lipidic plasma membrane components, 67 including cholesterol, phospholipids, sulfolipids and the gangliosides GM1 and aGM1 9-68 12 . 69 Structurally, the flagellum consists out of three main parts: the basal body embedded 70 within the inner and outer membranes of the bacterium, a flexible linking structure -the 71 hook, and the long, external filament, which functions as the propeller of the motility 72 device 13 . The filament is formed by more than 20,000 subunits of a single protein, 73 flagellin. Many S. enterica serovars express either of two distinct flagellins, FliC or FljB, 74 in a process called flagellar phase variation 14,15 . FliC-expressing bacteria display a 75 distinct motility behavior on host cell surfaces and a competitive advantage in 76 colonization of the intestinal epithelia compared to FljB-expressing bacteria 16 . However, 77while the structure of FliC has been determined previously 17 , the structure of FljB 78 remained unknown. 130We next aligned the amino acid sequences of FljB and FliC up-and downstream of the 131 identified ɛ-N-methyl-lysine residues (...

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Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier

Cited by 72 publications

References 38 publications

Flagellin phase-dependent swimming on epithelial cell surfaces contributes to productiveSalmonellagut colonisation

Flagellin phase-dependent swimming on epithelial cell surfaces contributes to productiveSalmonellagut colonisation

Generation and characterization of a functional human adipose‐derived multipotent mesenchymal stromal cell line

Flagella methylation promotes bacterial adhesion and host cell invasion

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