scholarly journals Rapid removal of phagosomal ferroportin in macrophages contributes to nutritional immunity

2021 ◽  
Vol 5 (2) ◽  
pp. 459-474
Author(s):  
Ronald S. Flannagan ◽  
Tayler J. Farrell ◽  
Steven M. Trothen ◽  
Jimmy D. Dikeakos ◽  
David E. Heinrichs
Keyword(s):  
Fluorescent Protein ◽  
Critical Role ◽  
Cellular Level ◽  
Protein Fusion ◽  
Extracellular Milieu ◽  
Nutritional Immunity ◽  
Protein Receptor ◽  
Serovar Typhimurium ◽  
Green Fluorescent ◽  
Phagosomal Membrane

Abstract Nutrient sequestration is an essential facet of host innate immunity. Macrophages play a critical role in controlling iron availability through expression of the iron transport protein ferroportin (FPN), which extrudes iron from the cytoplasm to the extracellular milieu. During phagocytosis, the limiting phagosomal membrane, which derives from the plasmalemma, can be decorated with FPN and, if functional, will move iron from the cytosol into the phagosome lumen. This serves to feed iron to phagocytosed microbes and would be counterproductive to the many other known host mechanisms working to starve microbes of this essential metal. To understand how FPN is regulated during phagocytosis, we expressed FPN as a green fluorescent protein–fusion protein in macrophages and monitored its localization during uptake of various phagocytic targets, including Staphylococcus aureus, Salmonella enterica serovar Typhimurium, human erythrocytes, and immunoglobulin G opsonized latex beads. We find that FPN is rapidly removed, independently of Vps34 and PI(3)P, from early phagosomes and does not follow recycling pathways that regulate transferrin receptor recycling. Live-cell video microscopy showed that FPN movement on the phagosome is dynamic, with punctate and tubular structures forming before FPN is trafficked back to the plasmalemma. N-ethylmaleimide–sensitive factor, which disrupts soluble NSF attachment protein receptor (SNARE)–mediated membrane fusion and trafficking, prevented FPN removal from the phagosome. Our data support the hypothesis that removal of FPN from the limiting phagosomal membrane will, at the cellular level, ensure that iron cannot be pumped into phagosomes. We propose this as yet another mechanism of host nutritional immunity to subvert microbial growth.

scholarly journals Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1590 ◽  
Author(s):  
Magda Wąchalska ◽  
Małgorzata Graul ◽  
Patrique Praest ◽  
Rutger D. Luteijn ◽  
Aleksandra W. Babnis ◽  
...  
Keyword(s):  
Antigen Processing ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Peptide Transporter ◽  
Virus Species ◽  
Protein Fusion ◽  
Fluorescent Properties ◽  
Bovine Herpesvirus 1 ◽  
Green Fluorescent

Transporter associated with antigen processing (TAP), a key player in the major histocompatibility complex class I-restricted antigen presentation, makes an attractive target for viruses that aim to escape the immune system. Mechanisms of TAP inhibition vary among virus species. Bovine herpesvirus 1 (BoHV-1) is unique in its ability to target TAP for proteasomal degradation following conformational arrest by the UL49.5 gene product. The exact mechanism of TAP removal still requires elucidation. For this purpose, a TAP-GFP (green fluorescent protein) fusion protein is instrumental, yet GFP-tagging may affect UL49.5-induced degradation. Therefore, we constructed a series of TAP-GFP variants using various linkers to obtain an optimal cellular fluorescent TAP platform. Mel JuSo (MJS) cells with CRISPR/Cas9 TAP1 or TAP2 knockouts were reconstituted with TAP-GFP constructs. Our results point towards a critical role of GFP localization on fluorescent properties of the fusion proteins and, in concert with the type of a linker, on the susceptibility to virally-induced inhibition and degradation. The fluorescent TAP platform was also used to re-evaluate TAP stability in the presence of other known viral TAP inhibitors, among which only UL49.5 was able to reduce TAP levels. Finally, we provide evidence that BoHV-1 UL49.5-induced TAP removal is p97-dependent, which indicates its degradation via endoplasmic reticulum-associated degradation (ERAD).

scholarly journals Weak Transcription of thecry1AcGene in Nonsporulating Bacillus thuringiensis Cells

2012 ◽  
Vol 78 (18) ◽  
pp. 6466-6474 ◽  
Author(s):  
Hui Yang ◽  
Pinshu Wang ◽  
Qi Peng ◽  
Rong Rong ◽  
Chunxia Liu ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Bacillus Thuringiensis ◽  
Fluorescent Protein ◽  
Cellular Level ◽  
Protein Fusion ◽  
Reporter System ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Green Fluorescent ◽  
Sigma E

ABSTRACTThecry1Acgene ofBacillus thuringiensissubsp.kurstakiHD-73 (B. thuringiensisHD-73) is a typical example of a sporulation-dependent crystal gene and is controlled by sigma E and sigma K during sporulation. To monitor the production and accumulation of Cry1Ac at the cellular level, we developed a green fluorescent protein-based reporter system. The production of Cry1Ac was monitored inspo0A,sigE, andsigKmutants, and these mutants were able to express the Cry1Ac-green fluorescent protein fusion protein. In nonsporulatingB. thuringiensisHD-73 cells, low-level expression ofcry1Acwas also observed. Reverse transcription-PCR and Western blotting results confirmed that thecry1Acpromoter has low activity in nonsporulatingB. thuringiensiscells. A beta-galactosidase assay demonstrated that the transcription of thecry1Acgene during exponential and transition phases is positively regulated by Spo0A. Additional bioassay results indicated thatspo0AandsigEmutants containing thecry1Ac-gfpfusion exhibited insecticidal activity againstPlutella xylostellalarvae.

scholarly journals Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

2011 ◽  
Vol 77 (23) ◽  
pp. 8310-8317 ◽  
Author(s):  
Joshua D. Morris ◽  
Jessica L. Hewitt ◽  
Lawrence G. Wolfe ◽  
Nachiket G. Kamatkar ◽  
Sarah M. Chapman ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Nile Red ◽  
Temporal Distribution ◽  
Time Lapse ◽  
Content Type ◽  
Rhamnolipid Production ◽  
Serovar Typhimurium ◽  
Surface Motility ◽  
Green Fluorescent

ABSTRACTMany bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacteriumPseudomonas aeruginosa. First, we quantify the temporal distribution ofP. aeruginosacells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming ofP. aeruginosaandSalmonella entericaserovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of severalP. aeruginosastrains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

scholarly journals Bistable Expression of CsgD in Biofilm Development of Salmonella enterica Serovar Typhimurium

2009 ◽  
Vol 192 (2) ◽  
pp. 456-466 ◽  
Author(s):  
Nina Grantcharova ◽  
Verena Peters ◽  
Claudia Monteiro ◽  
Katherina Zakikhany ◽  
Ute Römling
Keyword(s):  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Population Based ◽  
Quantitative Expression ◽  
Serovar Typhimurium ◽  
Cellular Aggregation ◽  
Green Fluorescent ◽  
Biofilm Development ◽  
Biofilm Structure

ABSTRACT Bacterial persistence in the environment and in the infected host is often aided by the formation of exopolymer-enclosed communities known as biofilms. Heterogeneous gene expression takes place in microcompartments formed within the complex biofilm structure. This study describes cell differentiation within an isogenic bacterial cell population based on the example of biofilm formation by Salmonella enterica serovar Typhimurium. We analyzed the expression of the major biofilm regulator CsgD at the single-cell level with a chromosomal CsgD-green fluorescent protein (GFP) translational fusion. In individual cells, CsgD-GFP expression is mostly found in the cytoplasm. Quantitative expression analysis and results from three different models of S. Typhimurium biofilms demonstrated that CsgD is expressed in a bistable manner during biofilm development. CsgD expression is, however, monomodal when CsgD is expressed in larger amounts due to a promoter mutation or elevated levels of the secondary signaling molecule c-di-GMP. High levels of CsgD-GFP are associated with cellular aggregation in all three biofilm models. Furthermore, the subpopulation of cells expressing large amounts of CsgD is engaged in cellulose production during red, dry, and rough (rdar) morphotype development and in microcolony formation under conditions of continuous flow. Consequently, bistability at the level of CsgD expression leads to a corresponding pattern of task distribution in S. Typhimurium biofilms.

scholarly journals Regulation of RamA by RamR in Salmonella enterica Serovar Typhimurium: Isolation of a RamR Superrepressor

2012 ◽  
Vol 56 (11) ◽  
pp. 6037-6040 ◽  
Author(s):  
Vito Ricci ◽  
Stephen J. W. Busby ◽  
Laura J. V. Piddock
Keyword(s):  
Transcription Factor ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Promoter Sequence ◽  
Palindromic Sequence ◽  
Content Type ◽  
Gfp Reporter ◽  
Serovar Typhimurium ◽  
Green Fluorescent

ABSTRACTRamA is a transcription factor involved in regulating multidrug resistance inSalmonella entericaserovar Typhimurium SL1344. Green fluorescent protein (GFP) reporter fusions were exploited to investigate the regulation of RamA expression by RamR. We show that RamR represses theramApromoter by binding to a palindromic sequence and describe a superrepressor RamR mutant that binds to theramApromoter sequence more efficiently, thus exhibiting aramAinactivated phenotype.

scholarly journals Fluorescence-Based Reporter for Gauging Cyclic Di-GMP Levels in Pseudomonas aeruginosa

2012 ◽  
Vol 78 (15) ◽  
pp. 5060-5069 ◽  
Author(s):  
Morten T. Rybtke ◽  
Bradley R. Borlee ◽  
Keiji Murakami ◽  
Yasuhiko Irie ◽  
Morten Hentzer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Subsequent Development ◽  
Cellular Level ◽  
Host Immune System ◽  
Chronic Infections ◽  
Content Type ◽  
Genes Encoding ◽  
Green Fluorescent

ABSTRACTThe increased tolerance toward the host immune system and antibiotics displayed by biofilm-formingPseudomonas aeruginosaand other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP inP. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsivecdrApromoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP inP. aeruginosastrains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment ofP. aeruginosawith the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology ofP. aeruginosa.

A novel linker histone-like protein is associated with cytoplasmic filaments inCaenorhabditis elegans

2002 ◽  
Vol 115 (14) ◽  
pp. 2881-2891
Author(s):  
Monika A. Jedrusik ◽  
Stefan Vogt ◽  
Peter Claus ◽  
Ekkehard Schulze
Keyword(s):  
Rna Interference ◽  
Fluorescent Protein ◽  
Muscle Growth ◽  
Egg Laying ◽  
Globular Domain ◽  
Protein Fusion ◽  
Linker Histones ◽  
C Elegans ◽  
Fusion Protein Expression ◽  
Green Fluorescent

The histone H1 complement of Caenorhabditis elegans contains a single unusual protein, H1.X. Although H1.X possesses the globular domain and the canonical three-domain structure of linker histones, the amino acid composition of H1.X is distinctly different from conventional linker histones in both terminal domains. We have characterized H1.X in C. elegans by antibody labeling, green fluorescent protein fusion protein expression and RNA interference. Unlike normal linker histones, H1.X is a cytoplasmic as well as a nuclear protein and is not associated with chromosomes. H1.X is most prominently expressed in the marginal cells of the pharynx and is associated with a peculiar cytoplasmic cytoskeletal structure therein, the tonofilaments. Additionally H1.X::GFP is expressed in the cytoplasm of body and vulva muscle cells, neurons, excretory cells and in the nucleoli of embryonic blastomeres and adult gut cells. RNA interference with H1.X results in uncoordinated and egg laying defective animals, as well as in a longitudinally enlarged pharynx. These phenotypes indicate a cytoplasmic role of H1.X in muscle growth and muscle function.

scholarly journals Functional Characterization of a Drought-Responsive Invertase Inhibitor from Maize (Zea mays L.)

2019 ◽  
Vol 20 (17) ◽  
pp. 4081 ◽  
Author(s):  
Lin Chen ◽  
Xiaohong Liu ◽  
Xiaojia Huang ◽  
Wei Luo ◽  
Yuming Long ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Protein Fusion ◽  
Drought Treatment ◽  
Vacuolar Invertase ◽  
Maize Leaves ◽  
Green Fluorescent ◽  
Proteinaceous Inhibitor

Invertases (INVs) play essential roles in plant growth in response to environmental cues. Previous work showed that plant invertases can be post-translationally regulated by small protein inhibitors (INVINHs). Here, this study characterizes a proteinaceous inhibitor of INVs in maize (Zm-INVINH4). A functional analysis of the recombinant Zm-INVINH4 protein revealed that it inhibited both cell wall and vacuolar invertase activities from maize leaves. A Zm-INVINH4::green fluorescent protein fusion experiment indicated that this protein localized in the apoplast. Transcript analysis showed that Zm-INVINH4 is specifically expressed in maize sink tissues, such as the base part of the leaves and young kernels. Moreover, drought stress perturbation significantly induced Zm-INVINH4 expression, which was accompanied with a decrease of cell wall invertase (CWI) activities and an increase of sucrose accumulation in both base parts of the leaves 2 to 7 days after pollinated kernels. In summary, the results support the hypothesis that INV-related sink growth in response to drought treatment is (partially) caused by a silencing of INV activity via drought-induced induction of Zm-INVINH4 protein.

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