Intracellular Trafficking Modulation by Ginsenoside Rg3 Inhibits Brucella abortus Uptake and Intracellular Survival within RAW 264.7 Cells

Abstract Background Nanoparticles have been used for biomedical applications, including drug delivery, diagnosis, and imaging based on their unique properties derived from small size and large surface-to-volume ratio. However, concerns regarding unexpected toxicity due to the localization of nanoparticles in the cells are growing. Herein, we quantified the number of cell-internalized nanoparticles and monitored their cellular localization, which are critical factors for biomedical applications of nanoparticles. Methods This study investigates the intracellular trafficking of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)] in various live single cells, such as HEK293, NIH3T3, and RAW 264.7 cells, using site-specific direct stochastic optical reconstruction microscopy (dSTORM). The time-dependent subdiffraction-limit spatial resolution of the dSTORM method allowed intracellular site-specific quantification and tracking of MNPs@SiO2(RITC). Results The MNPs@SiO2(RITC) were observed to be highly internalized in RAW 264.7 cells, compared to the HEK293 and NIH3T3 cells undergoing single-particle analysis. In addition, MNPs@SiO2(RITC) were internalized within the nuclei of RAW 264.7 and HEK293 cells but were not detected in the nuclei of NIH3T3 cells. Moreover, because of the treatment of the MNPs@SiO2(RITC), more micronuclei were detected in RAW 264.7 cells than in other cells. Conclusion The sensitive and quantitative evaluations of MNPs@SiO2(RITC) at specific sites in three different cells using a combination of dSTORM, transcriptomics, and molecular biology were performed. These findings highlight the quantitative differences in the uptake efficiency of MNPs@SiO2(RITC) and ultra-sensitivity, varying according to the cell types as ascertained by subdiffraction-limit super-resolution microscopy. Graphical Abstract

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Identification of genes contributing to the intracellular replication of Brucella abortus within HeLa and RAW 264.7 cells

Veterinary Microbiology ◽

10.1016/j.vetmic.2012.02.019 ◽

2012 ◽

Vol 158 (3-4) ◽

pp. 322-328 ◽

Cited By ~ 9

Author(s):

Dong Hyeok Kim ◽

Jeong Ju Lim ◽

Jin Ju Lee ◽

Dae Geun Kim ◽

Hu Jang Lee ◽

...

Keyword(s):

Brucella Abortus ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Intracellular Replication

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Activation of NF-kB-Mediated TNF-Induced Antimicrobial Immunity Is Required for the Efficient Brucella abortus Clearance in RAW 264.7 Cells

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2017.00437 ◽

2017 ◽

Vol 7 ◽

Cited By ~ 16

Author(s):

Huynh T. Hop ◽

Alisha W. B. Reyes ◽

Tran X. N. Huy ◽

Lauren T. Arayan ◽

WonGi Min ◽

...

Keyword(s):

Brucella Abortus ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Antimicrobial Immunity

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Adenosine receptor Adora2b antagonism attenuates Brucella abortus 544 infection in professional phagocyte RAW 264.7 cells and BALB/c mice

Veterinary Microbiology ◽

10.1016/j.vetmic.2020.108586 ◽

2020 ◽

Vol 242 ◽

pp. 108586

Author(s):

Alisha Wehdnesday Bernardo Reyes ◽

Son Hai Vu ◽

Tran Xuan Ngoc Huy ◽

WonGi Min ◽

Hu Jang Lee ◽

...

Keyword(s):

Brucella Abortus ◽

Adenosine Receptor ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Professional Phagocyte

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Immunization With a Combination of Four Recombinant Brucella abortus Proteins Omp16, Omp19, Omp28, and L7/L12 Induces T Helper 1 Immune Response Against Virulent B. abortus 544 Infection in BALB/c Mice

Frontiers in Veterinary Science ◽

10.3389/fvets.2020.577026 ◽

2021 ◽

Vol 7 ◽

Author(s):

Tran Xuan Ngoc Huy ◽

Trang Thi Nguyen ◽

Alisha Wehdnesday Bernardo Reyes ◽

Son Hai Vu ◽

WonGi Min ◽

...

Keyword(s):

Brucella Abortus ◽

Preventive Therapy ◽

Raw 264.7 Cells ◽

Control Group ◽

Raw 264.7 ◽

Potential Candidate ◽

T Helper ◽

T Helper 1 ◽

Protective Efficiency ◽

Ifn Γ

Protective efficiency of a combination of four recombinant Brucella abortus (B. abortus) proteins, namely outer membrane protein (Omp) 16, Omp19, Omp28, and 50S ribosomal protein L7/L12 was evaluated as a combined subunit vaccine (CSV) against B. abortus infection in RAW 264.7 cell line and murine model. The immunoreactivity of these four recombinant proteins as well as pCold-TF vector reacted with Brucella-positive serum individually, but not with Brucella-negative serum by immunoblotting assay. CSV-treated RAW 264.7 cells significantly induced production of IFN-γ and IL-12 while decreased IL-10 production at the late stage of infection compared to PBS-treated control cells. In addition, the enhancement of nitric oxide production together with cytokines secretion profile in CSV-treated cells proved that CSV notably activated bactericidal mechanisms in macrophages. Consistently, mice immunized with CSV strongly elicited production of pro-inflammatory cytokines TNF-α, IL-6 and MCP-1 compared to PBS control group. Moreover, the concentration of IFN-γ was >IL-10 and titers of IgG2a were also heightened compared to IgG1 in CSV-immunized mice which suggest that CSV induced predominantly T helper 1 T cell. These results suggest that the CSV used in the present study is a potential candidate as a preventive therapy against brucellosis.

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The effects of red ginseng saponin fraction-A (RGSF-A) on phagocytosis and intracellular signaling in Brucella abortus infected RAW 264.7 cells

FEMS Microbiology Letters ◽

10.1093/femsle/fnv070 ◽

2015 ◽

Vol 362 (11) ◽

Cited By ~ 3

Author(s):

Lauren Togonon Arayan ◽

Hannah Leah Simborio ◽

Alisha Wehdnesday Bernardo Reyes ◽

Huynh Tan Hop ◽

WonGi Min ◽

...

Keyword(s):

Brucella Abortus ◽

Intracellular Signaling ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Red Ginseng ◽

Ginseng Saponin ◽

Saponin Fraction

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Nocodazole treatment interrupted Brucella abortus invasion in RAW 264.7 cells, and successfully attenuated splenic proliferation with enhanced inflammatory response in mice

Microbial Pathogenesis ◽

10.1016/j.micpath.2016.11.028 ◽

2017 ◽

Vol 103 ◽

pp. 87-93 ◽

Cited By ~ 6

Author(s):

Alisha Wehdnesday Bernardo Reyes ◽

Huynh Tan Hop ◽

Lauren Togonon Arayan ◽

Tran Xuan Ngoc Huy ◽

Wongi Min ◽

...

Keyword(s):

Inflammatory Response ◽

Brucella Abortus ◽

Raw 264.7 Cells ◽

Raw 264.7

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Deletion of the transcriptional regulator GntR affects apoptosis and autophagy in Brucella abortus-infected RAW 264.7 cells

10.21203/rs.3.rs-91178/v1 ◽

2020 ◽

Author(s):

Zhiqiang Li ◽

Shujuan Wei ◽

Shuli Wang ◽

Li Xi ◽

Yanyan Cui ◽

...

Keyword(s):

Brucella Abortus ◽

Target Genes ◽

Transcriptional Regulator ◽

Infected Group ◽

Raw 264.7 Cells ◽

Confocal Laser ◽

Raw 264.7 ◽

Flow Cytometry Analysis ◽

Cell Processes ◽

The Relationship

Abstract Background: Brucellosis is an important zoonotic disease caused by the pathogen Brucella. Regulating apoptosis and autophagy is the prerequisite for the intracellular survival of Brucella. GntR is an important transcriptional regulator of Brucella that can regulate the expression of many target genes, and then play a regulatory role in many cell processes, including regulation apoptosis and autophagy. Therefore, understanding the relationship between GntR and apoptosis and autophagy is crucial to comprehending the pathogenic mechanism of Brucella. Methods: In the present study, we described the influence of GntR on apoptosis and autophagy after the infection of RAW 264.7 cells with Brucella. We constructed the GntR mutant strain (2308ΔGntR) of Brucella abortus 2308 (S2308). Following the infection of the RAW 264.7 cells with S2308 and 2308ΔGntR, apoptosis and autophagy were detected. Results: Western blot analysis and flow cytometry analysis indicated that the apoptosis rate of the 2308ΔGntR-infected group was remarkably higher than that of the S2308-infected group. Confocal laser microscopy experiments indicated the presence of the P62 protein as punctate aggregates in the 2308ΔGntR group. Conclusion: These results showed that 2308ΔGntR promoted apoptosis and inhibited autophagy in the RAW 264.7 cells during Brucella infection.

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