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The Endoplasmic Reticulum Stress Sensor Inositol-Requiring Enzyme 1α Augments Bacterial Killing through Sustained Oxidant Production.

Abuaita BH, Burkholder KM, Boles BR, O'Riordan MX - MBio (2015)

Bottom Line: In contrast, dead MRSA showed early colocalization with ROS but was a poor activator of IRE1 and did not trigger sustained ROS generation.Taken together, these results suggest that IRE1-mediated persistent ROS generation might act as a fail-safe mechanism to kill bacterial pathogens that evade the initial macrophage oxidative burst.Our study highlights a key role for IRE1 in promoting macrophage bactericidal capacity and reveals a fail-safe mechanism that leads to the concentration of antimicrobial effector molecules in the macrophage phagosome.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, Michigan, USA.

No MeSH data available.


Related in: MedlinePlus

Sec 22B controls sustained ROS accumulation in phagosomes. (A) Flow cytometry analysis results for global ROS production in NT-control- and Sec22B KD-infected macrophages. (Left) Representative histogram plots are shown, with the percentage of ROS+ cells. (Right) Geometric mean fluorescence intensity of ROS production. (B) Live cell fluorescent images of NT-control and Sec22B KD macrophages infected with MRSA-mCherry (MOI, 20) and stained with a ROS fluorescence indicator at 8h pi. (C) The percentage of cells with ROS+ phagosomes was quantified from NT-control and Sec22B KD macrophages. The percentage of cells was determined from the number of cells with at least one enriched area of ROS colocalized with MRSA from at least 100 ROS+ infected cells. (D) The percentage of cells with ROS+ phagosomes was quantified from NT-control, STX4A KD, or STX5A KD macrophages as described for panel C. Graphs represent mean results from ≥3 independent experiments ± standard deviations. *, P < 0.05; **, P < 0.01.
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fig6: Sec 22B controls sustained ROS accumulation in phagosomes. (A) Flow cytometry analysis results for global ROS production in NT-control- and Sec22B KD-infected macrophages. (Left) Representative histogram plots are shown, with the percentage of ROS+ cells. (Right) Geometric mean fluorescence intensity of ROS production. (B) Live cell fluorescent images of NT-control and Sec22B KD macrophages infected with MRSA-mCherry (MOI, 20) and stained with a ROS fluorescence indicator at 8h pi. (C) The percentage of cells with ROS+ phagosomes was quantified from NT-control and Sec22B KD macrophages. The percentage of cells was determined from the number of cells with at least one enriched area of ROS colocalized with MRSA from at least 100 ROS+ infected cells. (D) The percentage of cells with ROS+ phagosomes was quantified from NT-control, STX4A KD, or STX5A KD macrophages as described for panel C. Graphs represent mean results from ≥3 independent experiments ± standard deviations. *, P < 0.05; **, P < 0.01.

Mentions: We envisioned at least two possibilities to explain how Sec22B might promote bacterial killing. First, Sec22B could be required for global induction of ROS, perhaps via delivery of bacterial ligands from the phagosome to the ER (26). Second, Sec22B could promote local production or accumulation of ROS in the phagosome, perhaps by delivering the machinery of ROS production from the ER to the phagosome or by altering phagolysosome fusion (12). To test the first possibility, we assessed whether Sec22B contributes to global ROS production. NT-control and Sec22B KD cells were infected with MRSA, and total ROS was measured by flow cytometry at 1 and 8h pi. There was no decrease in the amount of global ROS produced upon MRSA infection between NT-control and Sec22B KD macrophages (Fig. 6A). We therefore tested the second possibility by determining whether Sec22B was required for IRE1-dependent accumulation of phagosomal ROS, which we previously observed (Fig. 6B). Sec22B KD and NT-control cells were infected with MRSA, and the percentage of cells with ROS+ MRSA-containing phagosomes was measured. Similar to wild-type RAW 264.7 cells, approximately 40% of NT-control macrophages contained at least one ROS+ MRSA-containing phagosome at 8h pi (Fig. 6C). Silencing Sec22B reduced the number of cells with one or more ROS+ phagosome to approximately 10%, even though total ROS production was not affected. Transient silencing of STX4A or STX5A also decreased the number of macrophages with at least one ROS+ MRSA-containing phagosome at 8h pi (Fig. 6D). Our data suggest the possibility that Sec22B, STX4A, and STX5A contribute to IRE1-dependent killing by directly or indirectly enabling ROS accumulation in the bacterial phagosome.


The Endoplasmic Reticulum Stress Sensor Inositol-Requiring Enzyme 1α Augments Bacterial Killing through Sustained Oxidant Production.

Abuaita BH, Burkholder KM, Boles BR, O'Riordan MX - MBio (2015)

Sec 22B controls sustained ROS accumulation in phagosomes. (A) Flow cytometry analysis results for global ROS production in NT-control- and Sec22B KD-infected macrophages. (Left) Representative histogram plots are shown, with the percentage of ROS+ cells. (Right) Geometric mean fluorescence intensity of ROS production. (B) Live cell fluorescent images of NT-control and Sec22B KD macrophages infected with MRSA-mCherry (MOI, 20) and stained with a ROS fluorescence indicator at 8h pi. (C) The percentage of cells with ROS+ phagosomes was quantified from NT-control and Sec22B KD macrophages. The percentage of cells was determined from the number of cells with at least one enriched area of ROS colocalized with MRSA from at least 100 ROS+ infected cells. (D) The percentage of cells with ROS+ phagosomes was quantified from NT-control, STX4A KD, or STX5A KD macrophages as described for panel C. Graphs represent mean results from ≥3 independent experiments ± standard deviations. *, P < 0.05; **, P < 0.01.
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fig6: Sec 22B controls sustained ROS accumulation in phagosomes. (A) Flow cytometry analysis results for global ROS production in NT-control- and Sec22B KD-infected macrophages. (Left) Representative histogram plots are shown, with the percentage of ROS+ cells. (Right) Geometric mean fluorescence intensity of ROS production. (B) Live cell fluorescent images of NT-control and Sec22B KD macrophages infected with MRSA-mCherry (MOI, 20) and stained with a ROS fluorescence indicator at 8h pi. (C) The percentage of cells with ROS+ phagosomes was quantified from NT-control and Sec22B KD macrophages. The percentage of cells was determined from the number of cells with at least one enriched area of ROS colocalized with MRSA from at least 100 ROS+ infected cells. (D) The percentage of cells with ROS+ phagosomes was quantified from NT-control, STX4A KD, or STX5A KD macrophages as described for panel C. Graphs represent mean results from ≥3 independent experiments ± standard deviations. *, P < 0.05; **, P < 0.01.
Mentions: We envisioned at least two possibilities to explain how Sec22B might promote bacterial killing. First, Sec22B could be required for global induction of ROS, perhaps via delivery of bacterial ligands from the phagosome to the ER (26). Second, Sec22B could promote local production or accumulation of ROS in the phagosome, perhaps by delivering the machinery of ROS production from the ER to the phagosome or by altering phagolysosome fusion (12). To test the first possibility, we assessed whether Sec22B contributes to global ROS production. NT-control and Sec22B KD cells were infected with MRSA, and total ROS was measured by flow cytometry at 1 and 8h pi. There was no decrease in the amount of global ROS produced upon MRSA infection between NT-control and Sec22B KD macrophages (Fig. 6A). We therefore tested the second possibility by determining whether Sec22B was required for IRE1-dependent accumulation of phagosomal ROS, which we previously observed (Fig. 6B). Sec22B KD and NT-control cells were infected with MRSA, and the percentage of cells with ROS+ MRSA-containing phagosomes was measured. Similar to wild-type RAW 264.7 cells, approximately 40% of NT-control macrophages contained at least one ROS+ MRSA-containing phagosome at 8h pi (Fig. 6C). Silencing Sec22B reduced the number of cells with one or more ROS+ phagosome to approximately 10%, even though total ROS production was not affected. Transient silencing of STX4A or STX5A also decreased the number of macrophages with at least one ROS+ MRSA-containing phagosome at 8h pi (Fig. 6D). Our data suggest the possibility that Sec22B, STX4A, and STX5A contribute to IRE1-dependent killing by directly or indirectly enabling ROS accumulation in the bacterial phagosome.

Bottom Line: In contrast, dead MRSA showed early colocalization with ROS but was a poor activator of IRE1 and did not trigger sustained ROS generation.Taken together, these results suggest that IRE1-mediated persistent ROS generation might act as a fail-safe mechanism to kill bacterial pathogens that evade the initial macrophage oxidative burst.Our study highlights a key role for IRE1 in promoting macrophage bactericidal capacity and reveals a fail-safe mechanism that leads to the concentration of antimicrobial effector molecules in the macrophage phagosome.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, Michigan, USA.

No MeSH data available.


Related in: MedlinePlus