scholarly journals Identification of a Novel Adhesion Molecule Involved in the Virulence of Legionella pneumophila

2005 ◽  
Vol 73 (7) ◽  
pp. 4272-4280 ◽  
Author(s):  
Bin Chang ◽  
Fumiaki Kura ◽  
Junko Amemura-Maekawa ◽  
Nobuo Koizumi ◽  
Haruo Watanabe
Keyword(s):  
Cell Line ◽  
Adhesion Molecule ◽  
Legionella Pneumophila ◽  
Alveolar Epithelial Cell ◽  
Host Cells ◽  
Epithelial Cell Line ◽  
Human Macrophage ◽  
Convalescent Phase ◽  
Legionnaires Disease

ABSTRACT Legionella pneumophila is an intracellular bacterium, and its successful parasitism in host cells involves two reciprocal phases: transmission and intracellular replication. In this study, we sought genes that are involved in virulence by screening a genomic DNA library of an L. pneumophila strain, 80-045, with convalescent-phase sera of Legionnaires' disease patients. Three antigens that reacted exclusively with the convalescent-phase sera were isolated. One of them, which shared homology with an integrin analogue of Saccharomyces cerevisiae, was named L. pneumophila adhesion molecule homologous with integrin analogue of S. cerevisiae (LaiA). The laiA gene product was involved in L. pneumophila adhesion to and invasion of the human lung alveolar epithelial cell line A549 during in vitro coculture. However, its presence did not affect multiplication of L. pneumophila within a U937 human macrophage cell line. Furthermore, after intranasal infection of A/J mice, the laiA mutant was eliminated from lungs and caused reduced mortality compared to the wild isolate. Thus, we conclude that the laiA gene encodes a virulence factor that is involved in transmission of L. pneumophila 80-045 and may play a role in Legionnaires' disease in humans.

scholarly journals Transcriptional and Proteomic Characterization of Telomere-Induced Senescence in a Human Alveolar Epithelial Cell Line

2021 ◽  
Vol 8 ◽  
Author(s):  
Daniel I. Sullivan ◽  
Mao Jiang ◽  
Angela M. Hinchie ◽  
Mark G. Roth ◽  
Harinath Bahudhanapati ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Alveolar Epithelial Cell ◽  
Epithelial Cell Line ◽  
Telomere Dysfunction ◽  
Alveolar Epithelial ◽  
Novel Proteins ◽  
Human Alveolar Epithelial Cell ◽  
Species Specific

Cellular senescence due to telomere dysfunction has been hypothesized to play a role in age-associated diseases including idiopathic pulmonary fibrosis (IPF). It has been postulated that paracrine mediators originating from senescent alveolar epithelia signal to surrounding mesenchymal cells and contribute to disease pathogenesis. However, murine models of telomere-induced alveolar epithelial senescence fail to display the canonical senescence-associated secretory phenotype (SASP) that is observed in senescent human cells. In an effort to understand human-specific responses to telomere dysfunction, we modeled telomere dysfunction-induced senescence in a human alveolar epithelial cell line. We hypothesized that this system would enable us to probe for differences in transcriptional and proteomic senescence pathways in vitro and to identify novel secreted protein (secretome) changes that potentially contribute to the pathogenesis of IPF. Following induction of telomere dysfunction, a robust senescence phenotype was observed. RNA-seq analysis of the senescent cells revealed the SASP and comparisons to previous murine data highlighted differences in response to telomere dysfunction. We conducted a proteomic analysis of the senescent cells using a novel biotin ligase capable of labeling secreted proteins. Candidate biomarkers selected from our transcriptional and secretome data were then evaluated in IPF and control patient plasma. Four novel proteins were found to be differentially expressed between the patient groups: stanniocalcin-1, contactin-1, tenascin C, and total inhibin. Our data show that human telomere-induced, alveolar epithelial senescence results in a transcriptional SASP that is distinct from that seen in analogous murine cells. Our findings suggest that studies in animal models should be carefully validated given the possibility of species-specific responses to telomere dysfunction. We also describe a pragmatic approach for the study of the consequences of telomere-induced alveolar epithelial cell senescence in humans.

scholarly journals Identification of Legionella pneumophila-Specific Genes by Genomic Subtractive Hybridization with Legionella micdadei and Identification of lpnE, a Gene Required for Efficient Host Cell Entry

2006 ◽  
Vol 74 (3) ◽  
pp. 1683-1691 ◽  
Author(s):  
Hayley J. Newton ◽  
Fiona M. Sansom ◽  
Vicki Bennett-Wood ◽  
Elizabeth L. Hartland
Keyword(s):  
Cell Line ◽  
Legionella Pneumophila ◽  
Alveolar Epithelial Cell ◽  
Subtractive Hybridization ◽  
Open Reading Frames ◽  
Epithelial Cell Line ◽  
Specific Gene ◽  
Gene Encoding ◽  
Tetratricopeptide Repeats ◽  
Alveolar Epithelial

ABSTRACT Legionella pneumophila is a ubiquitous environmental organism and a facultative intracellular pathogen of humans. To identify genes that may contribute to the virulence of L. pneumophila, we performed genomic subtractive hybridization between L. pneumophila serogroup 1 strain 02/41 and L. micdadei strain 02/42. A total of 144 L. pneumophila-specific clones were sequenced, revealing 151 genes that were absent in L. micdadei strain 02/42. Low-stringency Southern hybridization was used to determine the distribution of 41 sequences, representing 40 open reading frames (ORFs) with a range of putative functions among L. pneumophila isolates of various serogroups as well as strains of Legionella longbeachae, L. micdadei, Legionella gormanii, and Legionella jordanis. Twelve predicted ORFs were L. pneumophila specific, including the gene encoding the dot/icm effector, lepB, as well as several genes predicted to play a role in lipopolysaccharide biosynthesis and cell wall synthesis and several sequences with similarity to virulence-associated determinants. A further nine predicted ORFs were in all L. pneumophila serotypes tested and an isolate of L. gormanii. These included icmD, the 5′ end of a pilMNOPQ locus, and two genes known to be upregulated during growth within macrophages, cadA2 and ceaA. Disruption of an L. pneumophila-specific gene (lpg2222 locus tag) encoding a putative protein with eight tetratricopeptide repeats resulted in reduced entry into the macrophage-like cell line, THP-1, and the type II alveolar epithelial cell line, A549. The gene was subsequently renamed lpnE, for “L. pneumophila entry.” In summary, this investigation has revealed important genetic differences between L. pneumophila and other Legionella species that may contribute to the phenotypic and clinical differences observed within this genus.

Campylobacter fetusadheres to and enters INT 407 cells

2002 ◽  
Vol 48 (11) ◽  
pp. 995-1007 ◽  
Author(s):  
Lori L Graham
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Reference Strain ◽  
Immunofluorescence Microscopy ◽  
Host Cells ◽  
Clinical Isolates ◽  
Epithelial Cell Line ◽  
Infection Period ◽  
Campylobacter Fetus

Campylobacter fetus is a Gram-negative bacterial pathogen of humans and ungulates and is normally transmitted via ingestion of contaminated food or water with infection resulting in mild to severe enteritis. However, despite clinical evidence that C. fetus infection often involves transient bacteremic states from which systemic infection may develop and the frequent isolation of C. fetus from extra-intestinal sites, this organism displays very poor invasiveness in in vitro models of infection. In this study, immunofluorescence microscopy and gentamicin protection assays were used to investigate the ability of six clinical isolates and one reference strain of C. fetus to adhere to and invade the human intestinal epithelial cell line, INT 407. During an initial 4-h infection period, all C. fetus strains were detected intracellularly using both techniques, though adherence and internalization levels were very low when determined from gentamicin protection assays. Microscopy results indicated that during a 4-h infection period, four of the five clinical strains tested were adherent to 41.3–87.3% of INT 407 cells observed and that 25.2–34.6% of INT 407 cells contained intracellular C. fetus. The C. fetus reference strain displayed the lowest levels of adherence and internalization. A modified infection assay revealed thatC. fetus adherence did not necessarily culminate in internalization. Despite the large percentage of INT 407 cells with adherent bacteria, the percentage of INT 407 cells with intracellular bacteria remained unchanged when incubation was extended from 4 h to 20 h. However, microscopy of INT 407 cells 24 h postinfection (p.i.) revealed that infected host cells contained clusters of densely packed C. fetus cells. Gentamicin protection assays revealed that intracellular C. fetus cells were not only viable 24 h p.i. but also that C. fetus had increased in number approximately three- to fourfold between 4 and 24 h p.i., indicative of intracellular replication. Investigation of the role of the host cell cytoskeleton revealed that pretreatment of host cells with cytochalasin D, colchicine, vinblastine, taxol, or dimethyl sulfoxide (DMSO) did not impact upon C. fetus adherence or internalization of INT 407 cells. Microscopy indicated neither rearrangement nor colocalization of either microtubules or microfilaments in INT 407 cells in response to C. fetus adherence or internalization. Together, these data indicate that clinical isolates of C. fetus are capable of adhering, entering, and surviving within the nonphagocytic epithelial cell line, INT 407.Key words: Campylobacter fetus, INT 407, immunofluorescence microscopy, S layer.

Role of the LuxR family transcriptional regulator Lpg2524 in the survival ofLegionella pneumophilain water

2017 ◽  
Vol 63 (6) ◽  
pp. 535-545 ◽  
Author(s):  
Laam Li ◽  
Sébastien P. Faucher
Keyword(s):  
Cell Density ◽  
Legionella Pneumophila ◽  
High Cell Density ◽  
Acanthamoeba Castellanii ◽  
Transcriptional Regulator ◽  
Human Macrophage ◽  
High Cell ◽  
Legionnaires Disease

The water-borne Gram-negative bacterium Legionella pneumophila (Lp) is the causative agent of Legionnaires’ disease. Lp is typically transmitted to humans from water systems, where it grows inside amoebae. Survival of Lp in water is central to its transmission to humans. A transcriptomic study previously identified many genes induced by Lp in water. One such gene, lpg2524, encodes a putative LuxR family transcriptional regulator. It was hypothesized that this gene could be involved in the survival of Lp in water. Deletion of lpg2524 does not affect the growth of Lp in rich medium, in the amoeba Acanthamoeba castellanii, or in human macrophage-like THP-1 cells, showing that Lpg2524 is not required for growth in vitro and in vivo. Nevertheless, deletion of lpg2524 results in a faster colony-forming unit (CFU) reduction in an artificial freshwater medium, Fraquil, indicating that Lpg2524 is important for Lp to survive in water. Overexpression of Lpg2524 also results in a survival defect, suggesting that a precise level of this transcriptional regulator is essential for its function. However, our result shows that Lpg2524 is dispensable for survival in water when Lp is at a high cell density (109CFU/mL), suggesting that its regulon is regulated by another regulator activated at high cell density.

scholarly journals Anti-Fibrosis Effects of Magnesium Lithospermate B in Experimental Pulmonary Fibrosis: By Inhibiting TGF-βRI/Smad Signaling

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1715
Author(s):  
Xin Luo ◽  
Qiangqiang Deng ◽  
Yaru Xue ◽  
Tianwei Zhang ◽  
Zhitao Wu ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Cell Line ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Salvia Miltiorrhiza ◽  
A549 Cells ◽  
Epithelial Cell Line ◽  
Human Lung Fibroblast ◽  
Magnesium Lithospermate B

Pulmonary fibrosis is a severe and irreversible interstitial pulmonary disease with high mortality and few treatments. Magnesium lithospermate B (MLB) is a hydrosoluble component of Salvia miltiorrhiza and has been reported to have antifibrotic effects in other forms of tissue fibrosis. In this research, we studied the effects of MLB on pulmonary fibrosis and the underlying mechanisms. Our results indicated that MLB treatment (50 mg/kg) for seven days could attenuate bleomycin (BLM)-induced pulmonary fibrosis by reducing the alveolar structure disruption and collagen deposition in the C57 mouse model. MLB was also found to inhibit transforming growth factor-beta (TGF-β)-stimulated myofibroblastic transdifferentiation of human lung fibroblast cell line (MRC-5) cells and collagen production by human type II alveolar epithelial cell line (A549) cells, mainly by decreasing the expression of TGF-β receptor I (TGF-βRI) and regulating the TGF-β/Smad pathway. Further studies confirmed that the molecular mechanisms of MLB in BLM-induced pulmonary fibrosis mice were similar to those observed in vitro. In summary, our results demonstrated that MLB could alleviate experimental pulmonary fibrosis both in vivo and in vitro, suggesting that MLB has great potential for pulmonary fibrosis treatment.

scholarly journals Exploitation of conserved eukaryotic host cell farnesylation machinery by an F-box effector of Legionella pneumophila

2010 ◽  
Vol 207 (8) ◽  
pp. 1713-1726 ◽  
Author(s):  
Christopher T.D. Price ◽  
Tasneem Al-Quadan ◽  
Marina Santic ◽  
Snake C. Jones ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Biological Function ◽  
Host Cells ◽  
Dependent Manner ◽  
Protein Farnesyltransferase ◽  
Type Iv ◽  
Eukaryotic Proteins ◽  
Bacterial Effectors

Farnesylation involves covalent linkage of eukaryotic proteins to a lipid moiety to anchor them into membranes, which is essential for the biological function of Ras and other proteins. A large cadre of bacterial effectors is injected into host cells by intravacuolar pathogens through elaborate type III–VII translocation machineries, and many of these effectors are incorporated into the pathogen-containing vacuolar membrane by unknown mechanisms. The Dot/Icm type IV secretion system of Legionella pneumophila injects into host cells the F-box effector Ankyrin B (AnkB), which functions as platforms for the docking of polyubiquitinated proteins to the Legionella-containing vacuole (LCV) to enable intravacuolar proliferation in macrophages and amoeba. We show that farnesylation of AnkB is indispensable for its anchoring to the cytosolic face of the LCV membrane, for its biological function within macrophages and Dictyostelium discoideum, and for intrapulmonary proliferation in mice. Remarkably, the protein farnesyltransferase, RCE-1 (Ras-converting enzyme-1), and isoprenyl cysteine carboxyl methyltransferase host farnesylation enzymes are recruited to the LCV in a Dot/Icm-dependent manner and are essential for the biological function of AnkB. In conclusion, this study shows novel localized recruitment of the host farnesylation machinery and its anchoring of an F-box effector to the LCV membrane, and this is essential for biological function in vitro and in vivo.

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