scholarly journals Beclin 1 Is Required for Starvation-Enhanced, but Not Rapamycin-Enhanced, LC3-Associated Phagocytosis of Burkholderia pseudomallei in RAW 264.7 Cells

2012 ◽  
Vol 81 (1) ◽  
pp. 271-277 ◽  
Author(s):  
Xuelei Li ◽  
Mark Prescott ◽  
Ben Adler ◽  
John D. Boyce ◽  
Rodney J. Devenish
Keyword(s):  
Burkholderia Pseudomallei ◽  
Defense Mechanism ◽  
Beclin 1 ◽  
Raw 264.7 Cells ◽  
Early Event ◽  
Raw 264.7 ◽  
Content Type ◽  
Innate Defense ◽  
Infected Cells ◽  
Interfering Rna

LC3-associated phagocytosis (LAP) ofBurkholderia pseudomalleiby murine macrophage (RAW 264.7) cells is an intracellular innate defense mechanism. Beclin 1, a protein with several roles in autophagic processes, is known to be recruited to phagosomal membranes as a very early event in LAP. We sought to determine whether knockdown of Beclin 1 by small interfering RNA (siRNA) would affect recruitment of LC3 and subsequent LAP of infectingB. pseudomallei. Both starvation and rapamycin treatment can induce Beclin 1-dependent autophagy. Therefore, we analyzed the consequences of Beclin 1 knockdown for LAP in infected cells that had been either starved or treated with rapamycin by determining the levels of bacterial colocalization with LC3 and intracellular survival. Concurrently, we confirmed the location of bacteria as either contained in phagosomes or free in the cytoplasm. We found that both rapamycin and starvation treatment enhanced LAP ofB. pseudomalleibut that the rapamycin response is Beclin 1 independent whereas the starvation response is Beclin 1 dependent.

scholarly journals Burkholderia pseudomallei Type III Secretion System Cluster 3 ATPase BsaS, a Chemotherapeutic Target for Small-Molecule ATPase Inhibitors

2015 ◽  
Vol 83 (4) ◽  
pp. 1276-1285 ◽  
Author(s):  
Lan Gong ◽  
Shu-Chin Lai ◽  
Puthayalai Treerat ◽  
Mark Prescott ◽  
Ben Adler ◽  
...  
Keyword(s):  
Small Molecule ◽  
Type Iii Secretion ◽  
Burkholderia Pseudomallei ◽  
Deletion Mutant ◽  
Secretion System ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Bacterial Survival ◽  
Atpase Inhibitor ◽  
Content Type

Melioidosis is an infectious disease of high mortality for humans and other animal species; it is prevalent in tropical regions worldwide. The pathogenesis of melioidosis depends on the ability of its causative agent, the Gram-negative bacteriumBurkholderia pseudomallei, to enter and survive in host cells.B. pseudomalleican escape from the phagosome into the cytosol of phagocytic cells where it replicates and acquires actin-mediated motility, avoiding killing by the autophagy-dependent process, LC3 (microtubule-associated protein light chain 3)-associated phagocytosis (LAP). The type III secretion system cluster 3 (TTSS3) facilitates bacterial escape from phagosomes, although the mechanism has not been fully elucidated. Given the recent identification of small-molecule inhibitors of the TTSS ATPase, we sought to determine the potential of the predicted TTSS3 ATPase, encoded bybsaS, as a target for chemotherapeutic treatment of infection. AB. pseudomalleibsaSdeletion mutant was generated and used as a control against which to assess the effect of inhibitor treatment. Infection of RAW 264.7 cells with wild-type bacteria and subsequent treatment with the ATPase inhibitor compound 939 resulted in reduced intracellular bacterial survival, reduced escape from phagosomes, and increased colocalization with both LC3 and the lysosomal marker LAMP1 (lysosome-associated membrane protein 1). These changes were similar to those observed for infection of RAW 264.7 cells with thebsaSdeletion mutant. We propose that treatment with the ATPase inhibitor compound 939 decreased intracellular bacterial survival through a reduced ability of bacteria to escape from phagosomes and increased killing via LAP. Therefore, small-molecule inhibitors of the TTSS3 ATPase have potential as therapeutic treatments against melioidosis.

scholarly journals NOD2/c-Jun NH2-Terminal Kinase Triggers Mycoplasma ovipneumoniae-Induced Macrophage Autophagy

2020 ◽  
Vol 202 (20) ◽  
Author(s):  
Haixia Luo ◽  
Xixi Wu ◽  
Zhaokun Xu ◽  
Xiujing Hao ◽  
Yongyu Wang ◽  
...  
Keyword(s):  
Muramyl Dipeptide ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Jnk Pathway ◽  
Content Type ◽  
Autophagy Induction ◽  
Pathway Activation ◽  
A Cell ◽  
Interfering Rna ◽  
Mycoplasma Ovipneumoniae

ABSTRACT Mycoplasma ovipneumoniae belongs to Mycoplasma, a genus containing the smallest self-replicating microorganisms, and causes infectious pleuropneumonia in goats and sheep. Nucleotide-binding oligomerization domain-containing protein (NOD2), an intracellular pattern recognition receptor, interacts with muramyl dipeptide (MDP) to recognize bacterial peptidoglycans and is involved in autophagy induction. However, there have been no reports about NOD recognition of mycoplasmas or M. ovipneumoniae-induced autophagy. In this study, we sought to determine the role of NOD2 in M. ovipneumoniae-induced autophagy using Western blotting, immunofluorescence, real-time PCR (RT-PCR), and color-changing unit (CCU) analysis. M. ovipneumoniae infection markedly increased NOD2 but did not increase NOD1 expression in RAW 264.7 cells. Treating RAW 264.7 cells with MDP significantly increased colocalization of M. ovipneumoniae and LC3, whereas treatment with NOD inhibitor, NOD-IN-1, decreased colocalization of M. ovipneumoniae and LC3. Furthermore, suppressing NOD2 expression with small interfering RNA (siRNA)-NOD2 failed to trigger M. ovipneumoniae-induced autophagy by detecting autophagy markers Atg5, beclin1, and LC3-II. In addition, M. ovipneumoniae infection significantly increased the phosphorylated c-Jun NH2-terminal kinase (p-JNK)/JNK, p-Bcl-2/Bcl-2, beclin1, Atg5, and LC3-II ratios in RAW 264.7 cells. Treatment with JNK inhibitor, SP600126, or siRNA-NOD2 did not increase this reaction. These findings suggested that M. ovipneumoniae infection activated NOD2, and both NOD2 and JNK pathway activation promoted M. ovipneumoniae-induced autophagy. This study provides new insight into the NOD2 reorganization mechanism and the pathogenesis of M. ovipneumoniae infection. IMPORTANCE M. ovipneumoniae, which lacks a cell wall, causes infectious pleuropneumonia in goats and sheep. In the present study, we focused on the interaction between NOD and M. ovipneumoniae, as well as its association with autophagy. We showed for the first time that NOD2 was activated by M. ovipneumoniae even when peptidoglycans were not present. We also observed that both NOD2 and JNK pathway activation promoted M. ovipneumoniae-induced autophagy.

scholarly journals Staphylococcus aureus Biofilm-Conditioned Medium Impairs Macrophage-Mediated Antibiofilm Immune Response by Upregulating KLF2 Expression

2019 ◽  
Vol 87 (4) ◽  
Author(s):  
Talib Alboslemy ◽  
Bing Yu ◽  
Tara Rogers ◽  
Min-Ho Kim
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Response ◽  
Conditioned Medium ◽  
Innate Immune Response ◽  
Immune Defense ◽  
Innate Immune ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Content Type ◽  
Cell Functions

ABSTRACT Staphylococcus aureus infections associated with the formation of biofilms on medical implants or host tissue play a critical role in the persistence of chronic infections. One critical mechanism of biofilm infection that leads to persistent infection lies in the capacity of biofilms to evade the macrophage-mediated innate immune response. It is now increasingly apparent that microorganisms exploit the negative regulatory mechanisms of the pattern recognition receptor (PRR)-mediated inflammatory response to subvert host cell functions by using various virulence factors. However, the detailed molecular mechanism, along with the identity of a target molecule, underlying the evasion of the macrophage-mediated innate immune response against S. aureus infection associated with biofilm formation remains to be elucidated. Here, using an in vitro culture model of murine macrophage-like RAW 264.7 cells, we demonstrate that S. aureus biofilm-conditioned medium significantly attenuated the capacity for macrophage bactericidal and proinflammatory responses. Importantly, the responses were associated with attenuated activation of NF-κB and increased expression of Kruppel-like factor 2 (KLF2) in RAW 264.7 cells. Small interfering RNA (siRNA)-mediated silencing of KLF2 in RAW 264.7 cells could restore the activation of NF-κB toward the bactericidal activity and generation of proinflammatory cytokines in the presence of S. aureus biofilm-conditioned medium. Collectively, our results suggest that factors secreted from S. aureus biofilms might exploit the KLF2-dependent negative regulatory mechanism to subvert macrophage-mediated innate immune defense against S. aureus biofilms.

scholarly journals Key Residues of Outer Membrane Protein OprI Involved in Hexamer Formation and Bacterial Susceptibility to Cationic Antimicrobial Peptides

2015 ◽  
Vol 59 (10) ◽  
pp. 6210-6222 ◽  
Author(s):  
Ting-Wei Chang ◽  
Chiu-Feng Wang ◽  
Hsin-Jye Huang ◽  
Iren Wang ◽  
Shang-Te Danny Hsu ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Antimicrobial Peptides ◽  
Outer Membrane ◽  
Conformational Changes ◽  
Outer Membrane Protein ◽  
Defense Mechanism ◽  
Vital Role ◽  
Content Type ◽  
Innate Defense ◽  
Link Type

ABSTRACTAntimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading pathogens. Our previous studies have shown that the outer membrane protein, OprI fromPseudomonas aeruginosaor its homologue, plays a vital role in the susceptibility of Gram-negative bacteria to cationic α-helical AMPs (Y. M. Lin, S. J. Wu, T. W. Chang, C. F. Wang, C. S. Suen, M. J. Hwang, M. D. Chang, Y. T. Chen, Y. D. Liao, J Biol Chem 285:8985–8994, 2010,http://dx.doi.org/10.1074/jbc.M109.078725; T. W. Chang, Y. M. Lin, C. F. Wang, Y. D. Liao, J Biol Chem287:418–428, 2012,http://dx.doi.org/10.1074/jbc.M111.290361). Here, we obtained two forms of recombinant OprI: rOprI-F, a hexamer composed of three disulfide-bridged dimers, was active in AMP binding, while rOprI-R, a trimer, was not. All the subunits predominantly consisted of α-helices and exhibited rigid structures with a melting point centered around 76°C. Interestingly, OprI tagged withEscherichia colisignal peptide was expressed in a hexamer, which was anchored on the surface ofE. coli, possibly through lipid acids added at the N terminus of OprI and involved in the binding and susceptibility to AMP as nativeP. aeruginosaOprI. Deletion and mutation studies showed that Cys1 and Asp27 played a key role in hexamer formation and AMP binding, respectively. The increase of OprI hydrophobicity upon AMP binding revealed that it undergoes conformational changes for membrane fusion. Our results showed that OprI on bacterial surfaces is responsible for the recruitment and susceptibility to amphipathic α-helical AMPs and may be used to screen antimicrobials.

scholarly journals Inhibition of Hydrogen Sulfide Production by Gene Silencing Attenuates Inflammatory Activity by Downregulation of NF-κB and MAP Kinase Activity in LPS-Activated RAW 264.7 Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Alireza Badiei ◽  
Nethaji Muniraj ◽  
Stephen Chambers ◽  
Madhav Bhatia
Keyword(s):  
Hydrogen Sulfide ◽  
Kinase Activity ◽  
Inflammatory Mediator ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Activation Of Transcription ◽  
Hydrogen Sulfide Production ◽  
Cellular Signal ◽  
Interfering Rna ◽  
Transcription Factor Nuclear Factor

Hydrogen sulfide is an endogenous inflammatory mediator produced by the activity of cystathionineγ-lyase (CSE) in macrophages. The objective of this study was to explore the mechanism by which hydrogen sulfide acts as an inflammatory mediator in lipopolysaccharide- (LPS-) induced macrophages. In this study, we used small interfering RNA (siRNA) to inhibit CSE expression in macrophages. We found that CSE silencing siRNA could reduce the LPS-induced activation of transcription factor nuclear factor-κB (NF-κB) significantly. Phosphorylation and activation of extra cellular signal-regulated kinase 1/2 (ERK1/2) increased in LPS-induced macrophages. We showed that phosphorylation of ERK in LPS-induced RAW 264.7 cells reached a peak 30 min after activation. Our findings show that silencing CSE gene by siRNA reduces phosphorylation and activation of ERK1/2 in LPS-induced RAW 264.7 cells. These findings suggest that siRNA reduces the inflammatory effects of hydrogen sulfide through the ERK-NF-κB signalling pathway and hydrogen sulfide plays its inflammatory role through ERK-NF-κB pathway in these cells.

scholarly journals Burkholderia pseudomallei Type III Secretion System Mutants Exhibit Delayed Vacuolar Escape Phenotypes in RAW 264.7 Murine Macrophages

2008 ◽  
Vol 76 (7) ◽  
pp. 2991-3000 ◽  
Author(s):  
Mary N. Burtnick ◽  
Paul J. Brett ◽  
Vinod Nair ◽  
Jonathan M. Warawa ◽  
Donald E. Woods ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Burkholderia Pseudomallei ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Triple Mutant ◽  
Necrosis Factor Alpha ◽  
Murine Macrophages ◽  
Type Iii

ABSTRACT Burkholderia pseudomallei is a facultative intracellular pathogen capable of surviving and replicating within eukaryotic cells. Recent studies have shown that B. pseudomallei Bsa type III secretion system 3 (T3SS-3) mutants exhibit vacuolar escape and replication defects in J774.2 murine macrophages. In the present study, we characterized the interactions of a B. pseudomallei bsaZ mutant with RAW 264.7 murine macrophages. Following uptake, the mutant was found to survive and replicate within infected RAW 264.7 cells over an 18-h period. In addition, high levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES, but not IL-1α and IL-1β, were detected in culture supernatants harvested from infected monolayers. The subcellular location of B. pseudomallei within infected RAW 264.7 cells was determined, and as expected, the bsaZ mutant demonstrated early-vacuolar-escape defects. Interestingly, however, experiments also indicated that this mutant was capable of delayed vacuolar escape. Consistent with this finding, evidence of actin-based motility and multinucleated giant cell formation were observed between 12 and 18 h postinfection. Further studies demonstrated that a triple mutant defective in all three B. pseudomallei T3SSs exhibited the same phenotype as the bsaZ mutant, indicating that functional T3SS-1 and T3SS-2 did not appear to be responsible for the delayed escape phenotype in RAW 264.7 cells. Based upon these findings, it appears that B. pseudomallei may not require T3SS-1, -2, and -3 to facilitate survival, delayed vacuolar escape, and actin-based motility in activated RAW 264.7 macrophages.

scholarly journals Active Escape of Orientia tsutsugamushi from Cellular Autophagy

2012 ◽  
Vol 81 (2) ◽  
pp. 552-559 ◽  
Author(s):  
Youngho Ko ◽  
Ji-Hye Choi ◽  
Na-Young Ha ◽  
Ik-Sang Kim ◽  
Nam-Hyuk Cho ◽  
...  
Keyword(s):  
Defense Mechanism ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Orientia Tsutsugamushi ◽  
Content Type ◽  
Cellular Autophagy ◽  
A Cell ◽  
Infected Cells ◽  
Obligate Intracellular Pathogen

ABSTRACTOrientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular pathogen. After entry into host cells, the bacterium rapidly escapes from the endosomal pathway and replicates in the cytosol of eukaryotic host cells. Here we show thatO. tsutsugamushiinfection efficiently promotes cellular autophagy, a cell-autonomous defense mechanism of innate immunity. However, most of the internalized bacteria barely colocalized with the induced autophagosomes, even when stimulated with rapamycin, a chemical inducer of autophagy. Treatment of infected cells with tetracycline suppressed bacterial evasion from autophagy and facilitatedO. tsutsugamushitargeting to autophagosomes, suggesting that the intracellular pathogen may be equipped with a bacterial factor or factors that block autophagic recognition. Finally, we also found that chemical modulators of cellular autophagy or genetic knockout of theatg3gene does not significantly affect the intracellular growth ofO. tsutsugamushiin vitro. These results suggest thatO. tsutsugamushihas evolved to block autophagic microbicidal defense by evading autophagic recognition even though it activates the autophagy pathway during the early phase of infection.

scholarly journals Coxiella burnetiiSubverts p62/Sequestosome 1 and Activates Nrf2 Signaling in Human Macrophages

2018 ◽  
Vol 86 (5) ◽  
Author(s):  
Caylin G. Winchell ◽  
Amanda L. Dragan ◽  
Katelynn R. Brann ◽  
Frances I. Onyilagha ◽  
Richard C. Kurten ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Content Type ◽  
Sequestosome 1 ◽  
Type Iv ◽  
Host Signaling ◽  
Infected Cells ◽  
Pv Generation ◽  
Interfering Rna

ABSTRACTCoxiella burnetiiis the causative agent of human Q fever, a debilitating flu-like illness that can progress to chronic disease presenting as endocarditis. Following inhalation,C. burnetiiis phagocytosed by alveolar macrophages and generates a lysosome-like replication compartment termed the parasitophorous vacuole (PV). A type IV secretion system (T4SS) is required for PV generation and is one of the pathogen's few known virulence factors. We previously showed thatC. burnetiiactively recruits autophagosomes to the PV using the T4SS but does not alter macroautophagy. In the current study, we confirmed that the cargo receptor p62/sequestosome 1 (SQSTM-1) localizes near the PV in primary human alveolar macrophages infected with virulentC. burnetii. p62 and LC3 typically interact to select cargo for autophagy-mediated degradation, resulting in p62 degradation and LC3 recycling. However, inC. burnetii-infected macrophages, p62 was not degraded when cells were starved, suggesting that the pathogen stabilizes the protein. In addition, phosphorylated p62 levels increased, indicative of activation, during infection. Small interfering RNA experiments indicated that p62 is not absolutely required for intracellular growth, suggesting that the protein serves a signaling role during infection. Indeed, the Nrf2-Keap1 cytoprotective pathway was activated during infection, as evidenced by sustained maintenance of Nrf2 levels and translocation of the protein to the nucleus inC. burnetii-infected cells. Collectively, our studies identify a new p62-regulated host signaling pathway exploited byC. burnetiiduring intramacrophage growth.

scholarly journals Titanium dioxide nanoparticles induce in vitro autophagy

2018 ◽  
Vol 38 (1) ◽  
pp. 56-64 ◽  
Author(s):  
X Dai ◽  
R Liu ◽  
N Li ◽  
J Yi
Keyword(s):  
Titanium Dioxide ◽  
Messenger Rna ◽  
Titanium Dioxide Nanoparticles ◽  
Beclin 1 ◽  
Raw 264.7 Cells ◽  
Phagocytic Index ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Concentration Dependent Manner

Aim: Concerns about the possible toxicity to environment and human health of titanium dioxide nanoparticles (TiO2 NPs) are increasing. The aim of this study was to investigate the relationship between toxicology and autophage in vitro. Methods: RAW 264.7 cells were exposed to five concentrations (50, 100, 200, 300, and 400 μg/mL) and two particle size of TiO2 NPs (30 and 100 nm) for 24 h. Results: The results showed that TiO2 NPs decreased cell viability, phagocytic rate, and phagocytic index in a concentration-dependent manner, thereby inducing autophagy. TiO2 NPs-induced autophagy was indicated by monodansyl cadaverine staining and transmission electron microscopy. TiO2 NPs-induced messenger RNA expression of autophagy-related proteins LC3 and Beclin-1 was also significantly increased compared with those of the unexposed control cells. LC3 and Beclin-1 protein expression levels were markedly increased with the increase of TiO2 NPs concentrations. Conclusion: These results suggest the possibility that TiO2 NPs-induced toxicology probably plays a key role in autophagy in RAW 264.7 cells, and further exhaustive research on the harmful effects of these NPs in relevant organisms is needed for their safe application.

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