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Plasma Membrane Profiling Reveals Upregulation of ABCA1 by Infected Macrophages Leading to Restriction of Mycobacterial Growth.

Long J, Basu Roy R, Zhang YJ, Antrobus R, Du Y, Smith DL, Weekes MP, Javid B - Front Microbiol (2016)

Bottom Line: The plasma membrane represents a critical interface between the internal and extracellular environments, and harbors multiple proteins key receptors and transporters that play important roles in restriction of intracellular infection.We quantified 559 PM proteins in BCG-infected THP-1 cells.One significantly upregulated cell-surface protein was the cholesterol transporter ABCA1.

View Article: PubMed Central - PubMed

Affiliation: Collaboration Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University Beijing, China.

ABSTRACT
The plasma membrane represents a critical interface between the internal and extracellular environments, and harbors multiple proteins key receptors and transporters that play important roles in restriction of intracellular infection. We applied plasma membrane profiling, a technique that combines quantitative mass spectrometry with selective cell surface aminooxy-biotinylation, to Bacille Calmette-Guérin (BCG)-infected THP-1 macrophages. We quantified 559 PM proteins in BCG-infected THP-1 cells. One significantly upregulated cell-surface protein was the cholesterol transporter ABCA1. We showed that ABCA1 was upregulated on the macrophage cell-surface following infection with pathogenic mycobacteria and knockdown of ABCA1 resulted in increased mycobacterial survival within macrophages, suggesting that it may be a novel mycobacterial host-restriction factor.

No MeSH data available.


Related in: MedlinePlus

Pharmacological depletion of cholesterol in THP-1 macrophages results in decreased intra-cellular mycobacterial survival. Activated macrophages were pre-treated with simvastatin or mevalonate or both and infected with M. abscessus (MOI 5:1). Survival of intra-cellular bacteria was determined 44 h following infection compared with immediately following phagocytosis (hence survival rates >100%) – see Materials and Methods. KD = ABCA1 knock-down by lentiviral transduction, Mock = empty vector. ∗p < 0.05, ∗∗p < 0.01 by Student’s t-test.
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Figure 5: Pharmacological depletion of cholesterol in THP-1 macrophages results in decreased intra-cellular mycobacterial survival. Activated macrophages were pre-treated with simvastatin or mevalonate or both and infected with M. abscessus (MOI 5:1). Survival of intra-cellular bacteria was determined 44 h following infection compared with immediately following phagocytosis (hence survival rates >100%) – see Materials and Methods. KD = ABCA1 knock-down by lentiviral transduction, Mock = empty vector. ∗p < 0.05, ∗∗p < 0.01 by Student’s t-test.

Mentions: To determine whether the phenotype associated with ABCA1 depletion was due to its effects on cellular cholesterol depletion, we used the pharmacological agents simvastatin and mevalonate (Parihar et al., 2013). Mevalonate is a water-soluble precursor of cholesterol and its synthesis by HMG-CoA reductase is inhibited by simvastatin. Simvastatin depletes cellular cholesterol stores, which would be rescued by the addition of mevalonate, and simvastatin treatment has recently been shown to potentiate TB treatment (Dutta et al., 2016). Treating THP-1 cells infected with M. abscessus with simvastatin following knock-down of ABCA1 or empty vector led to a substantial decrease in mycobacterial survival (Figure 5), with no difference in mock-treated or ABCA1 knock-down cells. This suggested that, with the cellular depletion of cholesterol by simvastatin, ABCA1 inhibition had no additive effect. Whereas addition of mevalonate rescued the simvastatin phenotype and mevalonate alone increased intra-cellular mycobacterial survival, regardless of ABCA1 knock-down (Figure 5). Taken together, these data are highly suggestive that ABCA1 upregulation following mycobacterial infection is a host response to restrict mycobacterial growth by intra-cellular cholesterol depletion.


Plasma Membrane Profiling Reveals Upregulation of ABCA1 by Infected Macrophages Leading to Restriction of Mycobacterial Growth.

Long J, Basu Roy R, Zhang YJ, Antrobus R, Du Y, Smith DL, Weekes MP, Javid B - Front Microbiol (2016)

Pharmacological depletion of cholesterol in THP-1 macrophages results in decreased intra-cellular mycobacterial survival. Activated macrophages were pre-treated with simvastatin or mevalonate or both and infected with M. abscessus (MOI 5:1). Survival of intra-cellular bacteria was determined 44 h following infection compared with immediately following phagocytosis (hence survival rates >100%) – see Materials and Methods. KD = ABCA1 knock-down by lentiviral transduction, Mock = empty vector. ∗p < 0.05, ∗∗p < 0.01 by Student’s t-test.
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Related In: Results  -  Collection

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Figure 5: Pharmacological depletion of cholesterol in THP-1 macrophages results in decreased intra-cellular mycobacterial survival. Activated macrophages were pre-treated with simvastatin or mevalonate or both and infected with M. abscessus (MOI 5:1). Survival of intra-cellular bacteria was determined 44 h following infection compared with immediately following phagocytosis (hence survival rates >100%) – see Materials and Methods. KD = ABCA1 knock-down by lentiviral transduction, Mock = empty vector. ∗p < 0.05, ∗∗p < 0.01 by Student’s t-test.
Mentions: To determine whether the phenotype associated with ABCA1 depletion was due to its effects on cellular cholesterol depletion, we used the pharmacological agents simvastatin and mevalonate (Parihar et al., 2013). Mevalonate is a water-soluble precursor of cholesterol and its synthesis by HMG-CoA reductase is inhibited by simvastatin. Simvastatin depletes cellular cholesterol stores, which would be rescued by the addition of mevalonate, and simvastatin treatment has recently been shown to potentiate TB treatment (Dutta et al., 2016). Treating THP-1 cells infected with M. abscessus with simvastatin following knock-down of ABCA1 or empty vector led to a substantial decrease in mycobacterial survival (Figure 5), with no difference in mock-treated or ABCA1 knock-down cells. This suggested that, with the cellular depletion of cholesterol by simvastatin, ABCA1 inhibition had no additive effect. Whereas addition of mevalonate rescued the simvastatin phenotype and mevalonate alone increased intra-cellular mycobacterial survival, regardless of ABCA1 knock-down (Figure 5). Taken together, these data are highly suggestive that ABCA1 upregulation following mycobacterial infection is a host response to restrict mycobacterial growth by intra-cellular cholesterol depletion.

Bottom Line: The plasma membrane represents a critical interface between the internal and extracellular environments, and harbors multiple proteins key receptors and transporters that play important roles in restriction of intracellular infection.We quantified 559 PM proteins in BCG-infected THP-1 cells.One significantly upregulated cell-surface protein was the cholesterol transporter ABCA1.

View Article: PubMed Central - PubMed

Affiliation: Collaboration Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University Beijing, China.

ABSTRACT
The plasma membrane represents a critical interface between the internal and extracellular environments, and harbors multiple proteins key receptors and transporters that play important roles in restriction of intracellular infection. We applied plasma membrane profiling, a technique that combines quantitative mass spectrometry with selective cell surface aminooxy-biotinylation, to Bacille Calmette-Guérin (BCG)-infected THP-1 macrophages. We quantified 559 PM proteins in BCG-infected THP-1 cells. One significantly upregulated cell-surface protein was the cholesterol transporter ABCA1. We showed that ABCA1 was upregulated on the macrophage cell-surface following infection with pathogenic mycobacteria and knockdown of ABCA1 resulted in increased mycobacterial survival within macrophages, suggesting that it may be a novel mycobacterial host-restriction factor.

No MeSH data available.


Related in: MedlinePlus