scholarly journals Construction and validation of EGFP-expressingStaphylococcus aureusclinical strains for adhesion and internalization assays on epithelial cells

2019 ◽  
Author(s):  
Sana Charaoui-Boukerzaza ◽  
M. Fedy Morgene ◽  
Josselin Rigaill ◽  
Estelle Audoux ◽  
Zhiguo He ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Epithelial Cells ◽  
Mammalian Cells ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Egfp Expression ◽  
Confocal Laser ◽  
Wild Type ◽  
Clinical Strains ◽  
One Step

ABSTRACTBackgroundStaphylococcus aureusis both a major pathogen and a commensal bacterium in humans. It is able to adhere at the surface of epithelial cells of the anterior nares and can trigger its internalization inside these non-professional phagocytic cells. To better understand the interactions of clinical isolates with keratinocytes in the anterior nares, we developed and validated a one-step protocol expressing enhanced green fluorescent protein (EGFP) inS. aureusclinical strains with the aim to study adhesion to and internalization into mammalian cells.MethodsTwentyS. aureusclinical isolates belonging to clonal complexes 5, 8, 30, 45, 398 were selected for one-step transformation protocol with the EGFP-encoding plasmid pBSU101. EGFP expression was analysed by flow cytometry and confocal microscopy. Wild type and isogenic EGFP-expressing strains were compared for adhesion and internalization levels by using the HaCaT cell model.ResultsTransformation was achieved in all theS. aureusstrains regardless of their genetic background. The flow cytometry analysis showed that the mean proportion of EGFP-expressing bacteria was 97.2% (± 2.1) after 4h of incubation. Adhesion and internalization levels were similar in wild-type and isogenic EGFP-expressingS. aureusstrains. Confocal laser scanning microscopy confirmed that EGFP-expressingS. aureusbacteria could be easily identified inside HaCaT keratinocytes.ConclusionThis study reports an efficient protocol for expressing EGFP inS. aureusclinical strains and demonstrates that these EGFP-expressing strains are suitable for adhesion and internalization assays using HaCaT cells, which allows to perform static and dynamicin vitrostudies ofS. aureuscolonization.

scholarly journals Essential Role for the Legionella pneumophila Rep Helicase Homologue in Intracellular Infection of Mammalian Cells

2000 ◽  
Vol 68 (12) ◽  
pp. 6970-6978 ◽  
Author(s):  
Omar S. Harb ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Alveolar Epithelial Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wild Type ◽  
Intracellular Replication ◽  
Simultaneous Exposure ◽  
U937 Macrophage

ABSTRACT We have previously isolated 32 mutants of Legionella pneumophila that are defective in the infection of mammalian cells but not protozoa. The mutated loci have been designated macrophage-specific infectivity (mil) loci. In this study we characterized the mil mutant GK11. This mutant was incapable of growth within U937 macrophage-like cells and WI-26 alveolar epithelial cells. This defect in intracellular replication correlated with a defect in cytopathogenicity to these cells. Sequence analysis of the GK11 locus revealed it to be highly similar torep helicase genes of other bacteria. Since helicase mutants of Escherichia coli are hypersensitive to thymine starvation, we examined the sensitivity of GK11 to thymineless death (TLD). In the absence of thymine and thymidine, mutant GK11 did not undergo TLD but was defective for in vitro growth, and the defect was partially restored when these compounds were added to the growth medium. In addition, supplementation with thymidine or thymine partially restored the ability of GK11 to grow within and kill U937 macrophage-like cells. The data suggested that the low levels of thymine or thymidine in the L. pneumophila phagosome contributed to the defect of GK11 within macrophages. Using confocal laser scanning microscopy, we determined the effect of the mutation in the Rep helicase homologue on the intracellular trafficking of GK11 within macrophages. In contrast to the wild-type strain, phagosomes harboring GK11 colocalized with several late endosomal/lysosomal markers, including LAMP-1, LAMP-2, and cathepsin D. In addition, only 50% of the GK11 phagosomes colocalized with the endoplasmic reticulum marker BiP 4 h postinfection. Colocalization of BiP with GK11 phagosomes was absent 6 h postinfection, while 90% of the wild-type phagosomes colocalized with this marker at both time points. We propose that the low level of thymine within the L. pneumophila phagosome in combination with simultaneous exposure to multiple stress stimuli results in deleterious mutations that cannot be repaired in therep helicase homologue mutant, rendering it defective in intracellular replication.

scholarly journals Salivary Histatin 1 and 2 Are Targeted to Mitochondria and Endoplasmic Reticulum in Human Cells

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 795 ◽  
Author(s):  
Dandan Ma ◽  
Wei Sun ◽  
Kamran Nazmi ◽  
Enno C. I. Veerman ◽  
Floris J. Bikker ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Metabolic Activity ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Epithelial Cell Line ◽  
Intracellular Space

Human salivary histatin 1 (Hst1) and Hst2 exhibit a series of cell-activating properties (e.g., promoting adhesion, spreading, migration and metabolic activity of mammalian cells). In contrast, Hst5 shows an anti-fungal property but no cell-activating properties. Previous findings suggest that their uptake and association with subcellular targets may play a determinant role in their functions. In this study, we studied the uptake dynamics and subcellular targets of Hst1, Hst2 and Hst5 in epithelial cells (HO1N1 human buccal carcinoma epithelial cell line). Confocal laser scanning microscopy (CLSM) revealed that fluorescently labeled Hst1 (F-Hst1) was taken up into the intracellular space of epithelial cells. Then, 60 min post-incubation, the total fluorescence of cell-associated F-Hst1, as measured using flow cytometry, was significantly higher compared to those of F-Hst2 and F-Hst5. In contrast, virtually no association occurred using the negative control—scrambled F-Hst1 (F-Hstscr). CLSM images revealed that F-Hst1, 2 and 5 co-localized with mitotrackerTM-labeled mitochondria. In addition, F-Hst1 and F-Hst2 but neither F-Hst5 nor F-Hst1scr co-localized with the ER-trackerTM-labeled endoplasmic reticulum. No co-localization of Hst1, 2 and 5 with lysosomes or the Golgi apparatus was observed. Furthermore, Hst1 and Hst2 but not Hst5 or Hst1scr significantly promoted the metabolic activity of both human epithelial cell lines, HaCaT human keratinocytes and primary human gingival fibroblasts.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

scholarly journals Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

2019 ◽  
Vol 75 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Odel Soren ◽  
Ardeshir Rineh ◽  
Diogo G Silva ◽  
Yuming Cai ◽  
Robert P Howlin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Nitric Oxide Donor ◽  
Confocal Laser ◽  
Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

scholarly journals One-step preparation of antimicrobial silicone materials based on PDMS and salicylic acid: insights from spatially and temporally resolved techniques

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Luca Barbieri ◽  
Ioritz Sorzabal Bellido ◽  
Alison J. Beckett ◽  
Ian A. Prior ◽  
Jo Fothergill ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Laser Scanning ◽  
Pathogenic Bacteria ◽  
Pharmaceutical Products ◽  
Laser Scanning Microscopy ◽  
Wound Dressings ◽  
Confocal Laser ◽  
Vis Spectroscopy ◽  
Wide Range ◽  
One Step

AbstractIn this work, we introduce a one-step strategy that is suitable for continuous flow manufacturing of antimicrobial PDMS materials. The process is based on the intrinsic capacity of PDMS to react to certain organic solvents, which enables the incorporation of antimicrobial actives such as salicylic acid (SA), which has been approved for use in humans within pharmaceutical products. By combining different spectroscopic and imaging techniques, we show that the surface properties of PDMS remain unaffected while high doses of the SA are loaded inside the PDMS matrix. The SA can be subsequently released under physiological conditions, delivering a strong antibacterial activity. Furthermore, encapsulation of SA inside the PDMS matrix ensured a diffusion-controlled release that was tracked by spatially resolved Raman spectroscopy, Attenuated Total Reflectance IR (ATR-IR), and UV-Vis spectroscopy. The biological activity of the new material was evaluated directly at the surface and in the planktonic state against model pathogenic bacteria, combining confocal laser scanning microscopy, electron microscopy, and cell viability assays. The results showed complete planktonic inhibition for clinically relevant strains of Staphylococcus aureus and Escherichia coli, and a reduction of up to 4 orders of magnitude for viable sessile cells, demonstrating the efficacy of these surfaces in preventing the initial stages of biofilm formation. Our approach adds a new option to existing strategies for the antimicrobial functionalisation of a wide range of products such as catheters, wound dressings and in-dwelling medical devices based on PDMS.

scholarly journals The PTS Components in Klebsiella pneumoniae Affect Bacterial Capsular Polysaccharide Production and Macrophage Phagocytosis Resistance

2021 ◽  
Vol 9 (2) ◽  
pp. 335
Author(s):  
Novaria Sari Dewi Panjaitan ◽  
Yu-Tze Horng ◽  
Chih-Ching Chien ◽  
Hung-Chi Yang ◽  
Ren-In You ◽  
...  
Keyword(s):  
Survival Rate ◽  
Klebsiella Pneumoniae ◽  
Laser Scanning ◽  
Bacterial Resistance ◽  
Capsular Polysaccharide ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Intracellular Bacteria ◽  
Wild Type ◽  
Macrophage Phagocytosis

Capsular polysaccharide (CPS) is a crucial virulence factor for Klebsiella pneumoniae infection. We demonstrated an association of CPS production with two phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs). Deficiency of crr, encoding enzyme IIA of PTS, in K. pneumoniae enhanced the transcriptional activities of galF, wzi and gnd, which are in the cps gene cluster, leading to high CPS production. A crr mutant exhibited a higher survival rate in 1% hydrogen peroxide than the wild-type. The crr mutant showed less sensitivity to engulfment by macrophage (RAW 264.7) than the wild-type by observing the intracellular bacteria using confocal laser scanning microscopy (CLSM) and by calculating the colony-forming units (CFU) of intracellular bacteria. After long-term incubation, the survival rate of the intracellular crr mutant was higher than that of the wild-type. Deficiency of crr enhanced the transcriptional activities of etcABC which encodes another putative enzyme II complex of a PTS. Deletion of etcABC in the crr mutant reduced CPS production and the transcriptional activities of galF compared to those of the crr mutant. These results indicated that one PTS component, Crr, represses CPS production by repressing another PTS component, EtcABC, in K. pneumoniae. In addition, PTS plays a role in bacterial resistance to macrophage phagocytosis.

scholarly journals PGC-1-Related Coactivator, a Novel, Serum-Inducible Coactivator of Nuclear Respiratory Factor 1-Dependent Transcription in Mammalian Cells

2001 ◽  
Vol 21 (11) ◽  
pp. 3738-3749 ◽  
Author(s):  
Ulf Andersson ◽  
Richard C. Scarpulla
Keyword(s):  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Peroxisome Proliferator ◽  
Nuclear Respiratory Factor ◽  
Nuclear Respiratory Factor 1 ◽  
Cell Extracts

ABSTRACT The thermogenic peroxisome proliferator-activated receptor γ (PPAR-γ) coactivator 1 (PGC-1) has previously been shown to activate mitochondrial biogenesis in part through a direct interaction with nuclear respiratory factor 1 (NRF-1). In order to identify related coactivators that act through NRF-1, we searched the databases for sequences with similarities to PGC-1. Here, we describe the first characterization of a 177-kDa transcriptional coactivator, designated PGC-1-related coactivator (PRC). PRC is ubiquitously expressed in murine and human tissues and cell lines; but unlike PGC-1, PRC was not dramatically up-regulated during thermogenesis in brown fat. However, its expression was down-regulated in quiescent BALB/3T3 cells and was rapidly induced by reintroduction of serum, conditions where PGC-1 was not detected. PRC activated NRF-1-dependent promoters in a manner similar to that observed for PGC-1. Moreover, NRF-1 was immunoprecipitated from cell extracts by antibodies directed against PRC, and both proteins were colocalized to the nucleoplasm by confocal laser scanning microscopy. PRC interacts in vitro with the NRF-1 DNA binding domain through two distinct recognition motifs that are separated by an unstructured proline-rich region. PRC also contains a potent transcriptional activation domain in its amino terminus adjacent to an LXXLL motif. The spatial arrangement of these functional domains coincides with those found in PGC-1, supporting the conclusion that PRC and PGC-1 are structurally and functionally related. We conclude that PRC is a functional relative of PGC-1 that operates through NRF-1 and possibly other activators in response to proliferative signals.

Cytokinesis arrest and redistribution of actin-cytoskeleton regulatory components in cells expressing the Rho GTPase CDC42Hs

1996 ◽  
Vol 109 (2) ◽  
pp. 367-377 ◽  
Author(s):  
H. Dutartre ◽  
J. Davoust ◽  
J.P. Gorvel ◽  
P. Chavrier
Keyword(s):  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Rho Gtpase ◽  
Laser Scanning Microscopy ◽  
Regulatory Subunit ◽  
Confocal Laser ◽  
Complex Architectures ◽  
Immunofluorescence Labelling

In mammalian cells, Rho GTPases control the reorganisation of the actin cytoskeleton in response to growth factors. In the cytoplasm, the polymerisation of actin filaments and their organisation into complex architectures is orchestrated by numerous proteins which act either directly, by interacting with actin, or by producing secondary messengers which serve as mediators between signal transduction pathways and the microfilament organisation. We sought to determine whether the intracellular distribution of some of these regulatory components may be controlled by the Rho GTPase CDC42Hs. With this aim, we have established HeLa-derived human cell lines in which expression of a constitutively activated mutant of CDC42Hs is inducible. Morphological analysis by immunofluorescence labelling and confocal laser scanning microscopy revealed a massive reorganisation of F-actin in cortical microspikes as well as podosome-like structures located at the ventral face of the cells. Concomitantly, the cells became giant and multinucleate indicating that cytokinesis was impaired. The actin bundling protein T-plastin, the vasodilatator-stimulated phosphoprotein (VASP), a profilin ligand, as well as the 85 kDa regulatory subunit of the phosphoinosite 3-kinase redistributed with F-actin into the CDC42Hs-induced structures.

Analysis of ploidy level of Artemisia annua L. based on flow cytometry and confocal laser scanning microscopy

2021 ◽  
Vol 50 (1) ◽  
pp. 29-35
Author(s):  
Shu-Gen Wei ◽  
Ling-Yun Wan ◽  
Ying Wei ◽  
Li-Li He ◽  
Jin-E Fu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Artemisia Annua ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Ploidy Levels ◽  
Artemisia Annua L

Eighty nine Artemisia samples treated with different concentrations of colchicine were used as breeding samples, with diploid Artemisia as the control. The ploidy levels of samples were determined by flow cytometry and confocal laser scanning microscopy (CLSM). An analysis of the flow cytometry results identified three suspected tetraploid plants and seven suspected triploid plants. The results of this study may be useful for breeding new Artemisia lines.

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