scholarly journals Macrophage Proteome Analysis at Different Stages of Mycobacterium avium Subspecies paratuberculosis Infection Reveals a Mechanism of Pathogen Dissemination

Proteomes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 20
Author(s):  
Ida L. Phillips ◽  
Lia Danelishvili ◽  
Luiz E. Bermudez
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Mycobacterium Avium ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Global Analysis ◽  
Natural Infection ◽  
The Body ◽  
Enteric Infection ◽  
Phagocytic Cells ◽  
Macrophage Infection

Johne’s disease is a chronic and usually fatal enteric infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP) and is responsible for hundreds of millions of dollars in losses for the agricultural industry. Natural infection typically begins with bacterial uptake and translocation through the epithelium of the small intestine, followed by ingestion by tissue macrophages and dissemination via the lymphatic or blood system throughout the body. To gain insights into the host responses and adaptation of MAP within phagocytic cells, we utilized the previously developed cell culture passage model, and mass spectrometric-based quantitative proteomic approach. Using the cell culture system, which mimics an in vivo interaction of MAP with intestinal epithelium and tissue macrophages, bacteria were passed through the bovine epithelial cells and, subsequently, used for macrophage infection (termed indirect infection), while uninfected cells and macrophage infection initiated with the culture grown bacteria (termed direct infection) served as controls. Approximately 3900 proteins were identified across all studied groups. The comparison within the subset of proteins that showed synthesis for more than two-fold in the direct infection over the uninfected control revealed an enrichment for the pro-inflammatory pathways such as the NF-κB and cytokine/chemokine signaling, positive regulation of defense response, cell activation involved in the immune response and adaptive immune system. While these responses were absent in the indirect infection, cellular pathways such as cell cycle, healing, regulation of cell adhesion, ensemble of core extracellular matrix proteins, cell surface integrins and proteins mediating the integrin signaling were remarkably high within the indirect infection. In addition to global analysis of the macrophage proteome, we further validated the proteomics data and confirmed that MAP passage through epithelial cells modulates the expression and signaling of integrins in phagocytes. In this study, we demonstrate that predominant expression of integrins in the indirectly infected macrophages allows phagocytic cells to initiate stronger binding and efficient translocation through the endothelial cells, suggesting the important role of integrins in the spread of MAP infection.

scholarly journals Acanthamoeba castellanii as a Screening Tool for Mycobacterium avium Subspecies paratuberculosis Virulence Factors with Relevance in Macrophage Infection

2020 ◽  
Vol 8 (10) ◽  
pp. 1571
Author(s):  
Ida L. Phillips ◽  
Jamie L. Everman ◽  
Luiz E. Bermudez ◽  
Lia Danelishvili
Keyword(s):  
Virulence Factors ◽  
Mycobacterium Avium ◽  
Screening Tool ◽  
Gene Knockout ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Acanthamoeba Castellanii ◽  
Mammalian Host ◽  
Intracellular Growth ◽  
Macrophage Infection ◽  
Selection Of

The high prevalence of Johne’s disease has driven a continuous effort to more readily understand the pathogenesis of the etiological causative bacterium, Mycobacterium avium subsp. paratuberculosis (MAP), and to develop effective preventative measures for infection spread. In this study, we aimed to create an in vivo MAP infection model employing an environmental protozoan host and used it as a tool for selection of bacterial virulence determinants potentially contributing to MAP survival in mammalian host macrophages. We utilized Acanthamoeba castellanii (amoeba) to explore metabolic consequences of the MAP-host interaction and established a correlation between metabolic changes of this phagocytic host and MAP virulence. Using the library of gene knockout mutants, we identified MAP clones that can either enhance or inhibit amoeba metabolism and we discovered that, for most part, it mirrors the pattern of MAP attenuation or survival during infection of macrophages. It was found that MAP mutants that induced an increase in amoeba metabolism were defective in intracellular growth in macrophages. However, MAP clones that exhibited low metabolic alteration in amoeba were able to survive at a greater rate within mammalian cells, highlighting importance of both category of genes in bacterial pathogenesis. Sequencing of MAP mutants has identified several virulence factors previously shown to have a biological relevance in mycobacterial survival and intracellular growth in phagocytic cells. In addition, we uncovered new genetic determinants potentially contributing to MAP pathogenicity. Results of this study support the use of the amoeba model system as a quick initial screening tool for selection of virulence factors of extremely slow-grower MAP that is challenging to study.

scholarly journals Development of Microfluidic, Serum-Free Bronchial Epithelial Cells-on-a-Chip to Facilitate a More Realistic In vitro Testing of Nanoplastics

2021 ◽  
Vol 3 ◽  
Author(s):  
Govind Gupta ◽  
Srikanth Vallabani ◽  
Romain Bordes ◽  
Kunal Bhattacharya ◽  
Bengt Fadeel
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Cellular Uptake ◽  
Culture Conditions ◽  
Bronchial Epithelial Cells ◽  
The Body ◽  
Bronchial Epithelial ◽  
Serum Free ◽  
Static Conditions

Most cell culture models are static, but the cellular microenvironment in the body is dynamic. Here, we established a microfluidic-based in vitro model of human bronchial epithelial cells in which cells are stationary, but nutrient supply is dynamic, and we used this system to evaluate cellular uptake of nanoparticles. The cells were maintained in fetal calf serum-free and bovine pituitary extract-free cell culture medium. BEAS-2B, an immortalized, non-tumorigenic human cell line, was used as a model and the cells were grown in a chip within a microfluidic device and were briefly infused with amorphous silica (SiO2) nanoparticles or polystyrene (PS) nanoparticles of similar primary sizes but with different densities. For comparison, tests were also performed using static, multi-well cultures. Cellular uptake of the fluorescently labeled particles was investigated by flow cytometry and confocal microscopy. Exposure under dynamic culture conditions resulted in higher cellular uptake of the PS nanoparticles when compared to static conditions, while uptake of SiO2 nanoparticles was similar in both settings. The present study has shown that it is feasible to grow human lung cells under completely animal-free conditions using a microfluidic-based device, and we have also found that cellular uptake of PS nanoparticles aka nanoplastics is highly dependent on culture conditions. Hence, traditional cell cultures may not accurately reflect the uptake of low-density particles, potentially leading to an underestimation of their cellular impact.

scholarly journals Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model

Microbiology ◽  
2015 ◽  
Vol 161 (7) ◽  
pp. 1420-1434 ◽  
Author(s):  
Jamie L. Everman ◽  
Torsten M. Eckstein ◽  
Jonathan Roussey ◽  
Paul Coussens ◽  
John P. Bannantine ◽  
...  
Keyword(s):  
Cell Culture ◽  
Mycobacterium Avium ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Culture Passage ◽  
Inflammatory Phenotype ◽  
Cell Culture Passage

scholarly journals The novel Dbl homology/BAR domain protein, MsgA, of Talaromyces marneffei regulates yeast morphogenesis during growth inside host cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Harshini Weerasinghe ◽  
Hayley E. Bugeja ◽  
Alex Andrianopoulos
Keyword(s):  
Pathogenic Fungi ◽  
Host Cells ◽  
Microbial Pathogens ◽  
Mammalian Host ◽  
Host Immune System ◽  
Nucleotide Exchange ◽  
Phagocytic Cells ◽  
Macrophage Infection ◽  
Bar Domain ◽  
Talaromyces Marneffei

AbstractMicrobial pathogens have evolved many strategies to evade recognition by the host immune system, including the use of phagocytic cells as a niche within which to proliferate. Dimorphic pathogenic fungi employ an induced morphogenetic transition, switching from multicellular hyphae to unicellular yeast that are more compatible with intracellular growth. A switch to mammalian host body temperature (37 °C) is a key trigger for the dimorphic switch. This study describes a novel gene, msgA, from the dimorphic fungal pathogen Talaromyces marneffei that controls cell morphology in response to host cues rather than temperature. The msgA gene is upregulated during murine macrophage infection, and deletion results in aberrant yeast morphology solely during growth inside macrophages. MsgA contains a Dbl homology domain, and a Bin, Amphiphysin, Rvs (BAR) domain instead of a Plekstrin homology domain typically associated with guanine nucleotide exchange factors (GEFs). The BAR domain is crucial in maintaining yeast morphology and cellular localisation during infection. The data suggests that MsgA does not act as a canonical GEF during macrophage infection and identifies a temperature independent pathway in T. marneffei that controls intracellular yeast morphogenesis.

scholarly journals CHANGES IN FINE STRUCTURE DURING SILK PROTEIN PRODUCTION IN THE AMPULLATE GLAND OF THE SPIDER ARANEUS SERICATUS

1969 ◽  
Vol 42 (1) ◽  
pp. 284-295 ◽  
Author(s):  
Allen L. Bell ◽  
David B. Peakall
Keyword(s):  
Fine Structure ◽  
Epithelial Cells ◽  
Protein Production ◽  
Distal Portion ◽  
Silk Gland ◽  
The Body ◽  
Single Type ◽  
Secretory Cells ◽  
The Web ◽  
Tunica Intima

The ampullate silk gland of the spider, Araneus sericatus, produces the silk fiber for the scaffolding of the web. The fine structure of the various parts of the gland is described. The distal portion of the duct consist of a tube of epithelial cells which appear to secrete a substance which forms the tunica intima of the duct wall. At the proximal end of the duct there is a region of secretory cells. The epithelium of the sac portion contains five morphologically distinct types of granules. The bulk of the synthesis of silk occurs in the tail of the gland, and in this region only a single type of secretory droplet is seen in the epithelium. Protein synthesis can be stimulated by the injection of 1 mg/kg acetylcholine into the body fluids. 10 min after injection, much of the protein stored in the cytoplasm of the epithelial cells has been secreted into the lumen. 20 min after stimulation, the ergastoplasmic sacs form large whorls in the cytoplasm. Protein, similar in electron-opacity to protein found in the lumen, begins to form in that portion of the cytoplasm which is enclosed by the whorls. The limiting membrane of these droplets is formed by ergastoplasmic membranes which lose their ribosomes. No Golgi material has been found in these cells. Protein appears to be manufactured in the cytoplasm of the tail cells in a form which is ready for secretion.

scholarly journals Detection of Mycobacterium avium Subspecies Paratuberculosis in Pooled Fecal Samples by Fecal Culture and Real-Time PCR in Relation to Bacterial Density

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1605
Author(s):  
Annika Wichert ◽  
Esra Einax ◽  
Natalie Hahn ◽  
Anne Klassen ◽  
Karsten Donat
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Detection Probability ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Positive Association ◽  
Significant Loss ◽  
Positive Sample ◽  
Bacterial Density ◽  
Fecal Samples ◽  
Real Time Quantitative Pcr

Within paratuberculosis control programs Mycobacterium avium subsp. paratuberculosis (MAP)-infected herds have to be detected with minimum effort but with sufficient reliability. We aimed to evaluate a combination of random sampling (RS) and pooling for the detection of MAP-infected herds, simulating repeated RS in imitated dairy herds (within-herd prevalence 1.0%, 2.0%, 4.3%). Each RS consisted of taking 80 out of 300 pretested fecal samples, and five or ten samples were repeatedly and randomly pooled. All pools containing at least one MAP-positive sample were analyzed by culture and real-time quantitative PCR (qPCR). The pool detection probability was 47.0% or 45.9% for pools of size 5 or 10 applying qPCR and slightly lower using culture. Combining these methods increased the pool detection probability. A positive association between bacterial density in pools and pool detection probability was identified by logistic regression. The herd-level detection probability ranged from 67.3% to 84.8% for pools of size 10 analyzed by both qPCR and culture. Pools of size 10 can be used without significant loss of sensitivity compared with pools of size 5. Analyzing randomly sampled and pooled fecal samples allows the detection of MAP-infected herds, but is not recommended for one-time testing in low prevalence herds.

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