Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ΔF508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery

Abdulrahman, Basant; Khweek, Arwa Abu; Akhter, Anwari; Caution, Kyle; Tazi, Mia; Hassan, Hesham; Zhang, Yucheng; Rowland, Patrick D.; Malhotra, Sankalp; Aeffner, Famke; Davis, Ian C.; Valvano, Miguel A.; Amer, Amal O.

doi:10.1074/jbc.m112.411728

Cited by 57 publications

(99 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…B. cenocepacia ΔT6SS was kindly provided by Dr. Valvano at Queen’s University, Belfast, UK. All bacterial strains were grown overnight in LB medium at 37°C and 200 rpm as previously described [5,14,41,65]. …”

Section: Methodsmentioning

confidence: 99%

“…For the generation of primary bone marrow-derived macrophages from mice, tibias and femurs were flushed with IMDM medium (Thermo Fisher Scientific, 12,440,053) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, 16,000,044), 50% L cell-conditioned medium [66,67], 0.6x MEM non-essential amino acids (Thermo Fisher Scientific, 11,140,050) and 0.1% penicillin and streptomycin (Thermo Fisher Scientific, 15,140,122) as previously described [5,16,25,30,41,65]. Cells were cultivated at least 6 days at 37°C in a humidified atmosphere containing 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

“…To stain intracellular B. cenocepacia strain MH1K, live macrophages were incubated with 1 μg/mL Hoechst in PBS for 20 min and subsequently fixed with 4% paraformaldehyde. Images were captured using a laser scanning confocal fluorescence microscope with a 60X objective (Olympus Fluoview FV10i) as previously described [25,41,65]. Intensities of target proteins were measured using ImageJ Software.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

CASP4/caspase-11 promotes autophagosome formation in response to bacterial infection

Krause¹,

Caution²,

Badr³

et al. 2018

Autophagy

Self Cite

View full text Add to dashboard Cite

CASP4/caspase-11-dependent inflammasome activation is important for the clearance of various Gram-negative bacteria entering the host cytosol. Additionally, CASP4 modulates the actin cytoskeleton to promote the maturation of phagosomes harboring intracellular pathogens such as Legionella pneumophila but not those enclosing nonpathogenic bacteria. Nevertheless, this non-inflammatory role of CASP4 regarding the trafficking of vacuolar bacteria remains poorly understood. Macroautophagy/autophagy, a catabolic process within eukaryotic cells, is also implicated in the elimination of intracellular pathogens such as Burkholderia cenocepacia. Here we show that CASP4-deficient macrophages exhibit a defect in autophagosome formation in response to B. cenocepacia infection. The absence of CASP4 causes an accumulation of the small GTPase RAB7, reduced colocalization of B. cenocepacia with LC3 and acidic compartments accompanied by increased bacterial replication in vitro and in vivo. Together, our data reveal a novel role of CASP4 in regulating autophagy in response to B. cenocepacia infection.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

CASP4/caspase-11 promotes autophagosome formation in response to bacterial infection

Krause¹,

Caution²,

Badr³

et al. 2018

Autophagy

Self Cite

View full text Add to dashboard Cite

show abstract

“…5 The function of the CFTR channel in macrophages remains unclear although our recent work demonstrates that defective CFTR function is accompanied by an impaired innate immune response to specific infections. [6][7][8][9][10][11] In CF patients with the most common mutation, F508del, the mutant form of the protein fails to traffic properly to the plasma membrane. This leads to a critical lack of fluid exchange across the membrane.…”

Section: Introductionmentioning

confidence: 99%

“…15,16 Recent studies have implicated reduced autophagy activity in a number of physiological and pathophysiological processes such as aging, cancer, neurodegenerative diseases, innate immunity, and CF. 10,11,[17][18][19] Autophagy functions to yield energy and nutrients during stress or starvation of the cell. 20 In addition, autophagy can also restrict specific pathogens within macrophages and improve clearance of misfolded protein aggregates that cannot be managed by proteasomes.…”

Section: Introductionmentioning

confidence: 99%

Elevated Mirc1/Mir17-92 cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages

et al. 2016

Self Cite

View full text Add to dashboard Cite

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.

show abstract

An increase in intracellular p62/NBR1 and persistence of Burkholderia mallei and B. pseudomallei in infected mice linked to autophagy deficiency

Saikh

Dankmeyer

Zeng

et al. 2018

Immunity Inflam & Disease

View full text Add to dashboard Cite

Introduction Burkholderia mallei ( B. mallei ) and Burkholderia pseudomallei ( B. pseudomallei ), causative agents of glanders and melioidosis, respectively, are invasive intracellular pathogens that actively multiply in phagocytic and non‐phagocytic cells. Activation of cell‐autonomous autophagy mechanism eliminate intracellular pathogens in which p62 a cytosolic cargo protein is selectively degraded, and an accumulation of this marker occurs if autophagy is deficient. Recurrent, relapsed and reinfection of B. pseudomallei in melioidosis patients in endemic area indicative of lack of complete of clearance and persistence of the pathogen. Reasoning that abundance in the levels of p62 may provide an indication of the intracellular infection, we sought to examine whether increase in intracellular p62 and bacterial burden with Burkholderia infection are linked to autophagy deficiency. Methods In this study, we investigated cell culture and mouse models of disease to identify an association between autophagy biomarkers (p62/NBR1) accumulation and intracellular persistence of B. mallei and B. pseudomallei . Results We demonstrate, that elevated levels of intracellular p62/NBR1 correlated with bacterial persistence, while pre‐treatment with a pharmacological inducer of autophagy, rapamycin, reduced both intracellular p62, and bacterial survival. Our results showed an elevated p62 levels (2‐5 fold) in spleen and liver cells of Burkholderia ‐infected BALB/c mice, as well as in spleen cells of Burkholderia ‐infected C57BL/6 mice, suggesting that an increase in p62/NBR1 was due to an autophagy deficiency. Similar to p62, cytosolic LC3‐I levels were also elevated, while the characteristic conversion to the autophagosome‐associated membrane bound form LC3‐II was low in spleens of the infected mice further supporting the conclusion that autophagy was deficient. Conclusion Taken together, our results suggest that an increase in intracellular p62/NBR1 may be a potential host cell biomarker of B. mallei or B. pseudomallei infections, and identifying autophagy manipulation may potentially aid to therapeutic approach for complete clearance of the pathogen.

show abstract

Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ΔF508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery

Cited by 57 publications

References 51 publications

CASP4/caspase-11 promotes autophagosome formation in response to bacterial infection

CASP4/caspase-11 promotes autophagosome formation in response to bacterial infection

Elevated Mirc1/Mir17-92 cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages

An increase in intracellular p62/NBR1 and persistence of Burkholderia mallei and B. pseudomallei in infected mice linked to autophagy deficiency

Contact Info

Product

Resources

About