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. 2018 Apr 20:8:103.
doi: 10.3389/fcimb.2018.00103. eCollection 2018.

Activation of Host IRE1α-Dependent Signaling Axis Contributes the Intracellular Parasitism of Brucella melitensis

Affiliations

Activation of Host IRE1α-Dependent Signaling Axis Contributes the Intracellular Parasitism of Brucella melitensis

Aseem Pandey et al. Front Cell Infect Microbiol. .

Abstract

Brucella spp. are intracellular vacuolar pathogens that causes brucellosis, a worldwide zoonosis of profound importance. We previously demonstrated that the activity of host unfolded protein response (UPR) sensor IRE1α (inositol-requiring enzyme 1) and ER-associated autophagy confer susceptibility to Brucella melitensis and Brucella abortus intracellular replication. However, the mechanism by which host IRE1α regulates the pathogen intracellular lifestyle remains elusive. In this study, by employing a diverse array of molecular approaches, including biochemical analyses, fluorescence microscopy imaging, and infection assays using primary cells derived from Ern1 (encoding IRE1) conditional knockout mice, we address this gap in our understanding by demonstrating that a novel IRE1α to ULK1, an important component for autophagy initiation, signaling axis confers susceptibility to Brucella intracellular parasitism. Importantly, deletion or inactivation of key signaling components along this axis, including IRE1α, BAK/BAX, ASK1, and JNK as well as components of the host autophagy system ULK1, Atg9a, and Beclin 1, resulted in striking disruption of Brucella intracellular trafficking and replication. Host kinases in the IRE1α-ULK1 axis, including IRE1α, ASK1, JNK1, and/or AMPKα as well as ULK1, were also coordinately phosphorylated in an IRE1α-dependent fashion upon the pathogen infection. Taken together, our findings demonstrate that the IRE1α-ULK1 signaling axis is subverted by the bacterium to promote intracellular parasitism, and provide new insight into our understanding of the molecular mechanisms of intracellular lifestyle of Brucella.

Keywords: Brucella melitensis; ULK1; autophagy; inositol-requiring enzyme 1 (IRE1); intracellular trafficking and replication; unfolded protein response (UPR).

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Figures

Figure 1
Figure 1
Activation of IRE1α contributes to Bm16M intracellular replication. Host cells were infected with Bm16M for various length of time. Samples were lysed for Western blot with the indicated antibodies or for CFU assays. (A,B) Bm16M infection activates host IRE1α in BMDMs (A) and quantification of the relative p-IRE1α level (mean of three blots for each time point) (B). (C,D) Inhibition of IRE1α activity with its specific inhibitor KIRA6 reduced IRE1α activation during Bm16M infection (C) and quantification of the relative p-IRE1α level (D). NI, no infection. (E) The intracellular replication of Bm16M was analyzed by gentamicin protection analysis in RAW264.7 macrophages treated with KIRA six before and during infection. Data represent means ± standard deviation (SD) from three independent experiments with triplicate wells examined for each treatment. **p < 0.01 and ***p < 0.001, respectively.
Figure 2
Figure 2
IRE1α downstream signaling components BAX/BAK and ASK1 contribute to Bm16M intracellular parasitism. The indicated drug-treated or untreated host cells were infected with Bm16M. At the indicated h.p.i., infected host cells were lysed for CFU or Western blot analysis. (A) The intracellular replication (right panel) was measured in MEFs harboring double deletion of host BAX and BAK genes (BAX/BAK DKO MEFs) as demonstrated in the left panel. (B) Quantification of Bm16M surrounded by the ER marker calreticulin (Crc-positive BCVs) in control and BAK/BAX DKO MEFs at the indicated time points post-infection. (C,D) IRE1α-dependent activation of host ASK1 during Bm16M infection of Ern1mut/mut and Ern1wt/wt BMDMs (C) and quantification of the relative p-ASK1 level (mean of three blots for each time point) (D). NI, no infection. (E) Bm16M intracellular replication was measured in cells where ASK1 activity was inhibited by supplementation with NDQI1, a selective inhibitor of ASK1 kinase activity. Data represent means ± SD from three independent experiments with triplicate wells examined for each treatment. *p < 0.05, **p < 0.01, and ***p < 0.001, respectively.
Figure 3
Figure 3
Activation of host JNK1/2 in IRE1α signaling cascade confers Bm16M infection. The indicated drug-treated or untreated host cells were infected with Bm16M. At the indicated h.p.i., infected host cells were lysed for Western blot or CFU analysis. (A,B) Activation of host JNK1/2 is IRE1α-dependent during Bm16M infection of Ern1mut/mut and Ern1wt/wt BMDMs (A) and quantification of the relative p-JNK1 (B, upper panel) and p-JNK2 (B, lower panel) levels (mean of three blots for each time point). Back and blue asterisks: significances when compared to the control of no infection of Ern1mut/mut cells and to the infected Ern1mut/mut cells at the same time points, respectively. (C) Bm16M infection (upper panel) was measured in host cells in which host JNK was ablated (lower panel, MEFs). (D) Determination of Bm16M entry into (left panel, 1 h.p.i.) and replication (right panel, 48 h.p.i.) in murine J774.A1 macrophages in which JNK activity was inactivated by pre-treatment with SP600125. (E) Inhibition of JNK activity reduced Bm16M intracellular replication in J774.A1 macrophages. (F,G) Representative images demonstrating the effects of inhibition of JNK activity in J774.A1 macrophages by SP600125 before (F) or after (G) Bm16M entry into host cells on intracellular replication of the pathogen at 48 h.p.i. NI, no infection. Rp-JNK1 and Rp-JNK2: relative phosphorylation levels of p-JNK1 and p-JNK2, respectively. SC: SP600125 concentration (μM); SP: SP600125. Data represent means ± SD from three independent experiments with triplicate wells examined for each treatment. *p < 0.05, **p < 0.01, and ***p < 0.001, respectively.
Figure 4
Figure 4
Host ULK1 activity contributes to Brucella intracellular trafficking and replication. Host cells were infected with Bm16M for the indicated lengths of times. Samples were lysed for Western blot or CFU assays, or fixed and processed for immunofluorescence with the indicated antibodies. (A,B) ULK1 activation during Bm16M infection (A) and quantification of the relative p-ULK1 level (mean of three blots for each time point) (B). (C,D) Activation of host ULK1 is IRE1α-dependent during Brucella infection (C) and quantification of the relative p-ULK1 level (D). (E) Bm16M intracellular replication was measured in ULK-depleted RAW264.7 macrophages. (F,G) Bm16M trafficking to and replication in calreticulin-positive compartments in control and ULK1-depleted host cells at 24 h.p.i. (F) and quantification of the Crc-positive BCVs in the infected cells at the indicated time points post-infection (G). (H,I) Trafficking of Bm16M cells to lysosomes (marked by Cathepsin D) in control and ULK1-depleted RAW264.7 macrophages (H) and quantification of BCVs in these infected cells at the indicated time points post-infection (I). Crc, calreticulin; CtD, cathepsin D. Data represent means ± SD from three independent experiments with triplicate wells examined for each treatment. **p < 0.01 and ***p < 0.001, respectively.
Figure 5
Figure 5
Host Atg9a supports Bm16M intracellular replication. (A) Bm16M intracellular replication in Atg9a−/− MEF cells (upper right inset: Western blot demonstration of the absence of the Atg9a protein in the cells). (B) Bm16M intracellular replication was measured in Atg9a-depleted RAW264.7 macrophages (upper right inset: depletion of Atg9a in the cells detected by Western blot). Data represent means ± SD from three independent experiments with triplicate wells examined for each treatment. **p < 0.01 and ***p < 0.001, respectively.
Figure 6
Figure 6
Host Atg proteins Beclin 1 and LC3 contribute to Bm16M intracellular parasitism. Host cells were infected with Bm16M. At the indicated h.p.i., the infected cells were lysed for CFU assays or fixed and processed for immunofluorescence with the indicated antibodies. (A) Bm16M infection of BMDMs in host cells in which Beclin 1 was ablated. (B,C) Bm16M trafficking to Beclin 1-positive compartments in Ern1wt/wt and Ern1mut/mut BMDMs at 48 h.p.i. (B) and quantification of Beclin 1-positive BCVs in the infected cells at the indicated time points post-infection (C). (D) Accumulation of host LC3 near BCVs during Bm16M intracellular replication (24 h.p.i.) in RAW264.7 macrophages. Sections of the merged panels (right and bottom) showing the tight contact of Bm16M and LC3. Data represent means ± SD from three independent experiments with triplicate wells examined for each treatment. *p < 0.05, **p < 0.01, and ***p < 0.001, respectively.
Figure 7
Figure 7
Host AMPKα activation during Bm16M infection. The indicated host cells were infected with Bm16M. At the indicated time points post-infection, infected cells were lysed for Western blot or CFU analysis. (A,B) Activation of host AMPKα during Bm16M infection of control and Ern1mut/mut BMDMs (A) and quantification of the relative p-AMPKα level (mean of three blots for each time point) (B). NI, no infection. (C) Bm16M intracellular replication in BMDMs derived from control or conditional AMPKα KO mice. The inset demonstrates deficiency of host AMPKα in BMDMs from conditional KO mice. Data represent means ± SD from three independent experiments with triplicate wells examined for each treatment. ***p < 0.001.
Figure 8
Figure 8
Proposed model describing host IRE1α-ULK1 signaling axis mediating Brucella intracellular lifestyle. Internalized Brucella activates host UPR sensor IRE1α that drives the activation of IRE1α-associated kinases, including ASK1, JNK, and/or AMPKα. The activities of these IRE1α-associated signaling proteins drive the activation or assembly of downstream proteins, including ULK1, Beclin 1 and Atg9a, as well as drive the remodeling of cellular membranes to support Brucella intracellular parasitism.

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