Brucella abortus Triggers a cGAS-Independent STING Pathway To Induce Host Protection That Involves Guanylate-Binding Proteins and Inflammasome Activation

The innate immune system is important as the first line of defense to sense invading pathogens (1). Nucleic acids represent critical pathogen signatures that trigger a host proinflammatory immune response (2). Much progress has been made in understanding how DNA and RNA trigger host defense countermeasures; however, several aspects of how cytosolic nucleic acids are sensed remain unclear. Microbial nucleic acids commonly find their way into subcellular compartments of the infected cells to be sensed by innate receptors. Several DNA sensors have been identified that recognize cytosolic DNA. These include RNA polymerase III (3), DNA-dependent activator of IFN-regulatory factors (4), Lrrfip1 (5), Ifi204 (6), Mre11 (7), Ddx41 (8), LSm14A (9), and AIM2 (10), among others. The large numbers of DNA sensors identified in the host suggest that they may play redundant roles during infection.

One important DNA sensor is stimulator of IFN genes (STING), a signaling molecule associated with the endoplasmic reticulum that is critical to control the transcription of numerous host defense genes, including type I IFNs in response to invading DNA viruses, bacteria, or transfected DNA (11). STING leads to activation of TBK1, which phosphorylates IFN regulatory factor (IRF)-3, a transcription factor required for the induction of IFN-β expression (12). Actually, STING functions as a direct sensor of bacterial-derived cyclic dinucleotides (CDNs) as well as an adaptor molecule in DNA recognition (13). Recent studies have demonstrated that cytosolic DNA species trigger STING activation after binding to the enzyme cyclic GMP–AMP synthase (cGAS) generating the second-messenger cyclic GMP–AMP (cGAMP) (14, 15). CDNs produced by cGAS bind to STING in the endoplasmic reticulum, inducing a conformational change in the molecule that leads to relocation of STING and TBK1 to the perinuclear region of the cell, resulting in activation (16).

During intracellular bacterial infections, activation of STING can be accomplished via two different mechanisms. First, STING can directly recognize bacterial CDNs and thus function as a primary pattern recognition receptor (13). Alternatively, DNA sensing via cGAS triggers the synthesis of cGAMP, which then engages STING as a secondary receptor (15). Listeria monocytogenes actively secretes the bacterial second messenger c-di-AMP that binds directly to STING and activates the production of IFN-β in mice (17). However, in contrast to L. monocytogenes, Mycobacterium tuberculosis, Legionella pneumophila, and Chlamydia trachomatis appear to activate this same STING-dependent pathway but via the DNA sensor cGAS (18, 19).

Brucella abortus is a Gram-negative facultative intracellular bacterium that causes brucellosis, with pathological manifestations of arthritis, endocarditis, and meningitis in humans, and in cattle it leads to abortion and infertility, resulting in serious economic losses to the livestock industry (20). This pathogen resists killing by neutrophils and replicates inside macrophages and dendritic cells, maintaining a long-lasting interaction with host cells. Recently, we have identified Brucella DNA as a major bacterial component that induces type I IFN and IL-1β. Our study revealed that Brucella DNA operates through a mechanism dependent on STING and AIM2 (21, 22). However, currently it is unclear whether cGAS participates as a Brucella DNA sensor and what the contribution of cGAS and STING are in protective immunity and in possible cooperation with AIM2.

In this study, we demonstrate that the DNA sensor STING detects B. abortus infection and triggers a type I IFN response and IRF-1–dependent signaling cascade, leading to the upregulation of several genes, including the guanylate-binding proteins (GBPs). Furthermore, GBPs were critical to induce inflammasome activation and IL-1β secretion, and GBP^chr3 knockout (KO) mice were more susceptible to Brucella infection compared with wild-type (WT) animals. Importantly, cGAS and STING were required to induce type I IFN responses; however, STING but not cGAS played the major role in controlling bacterial infection in macrophages and in vivo.

Wild-type C57BL/6 and 129 Sv/Ev mice were purchased from the Federal University of Minas Gerais. STING^−/−, cGAS^−/−, AIM2^−/−, MAVS^−/−, GBP2^−/−, GBP^chr3−/−, IRF-1^−/−, and IFNAR^−/− mice were described previously (10, 11, 23–26). The animals were maintained at the Federal University of Minas Gerais and used at 6–8 wk of age. All animal experiments were preapproved by the Institutional Animal Care and Use Committee of the Federal University of Minas Gerais (CETEA no. 128/2014).

Bacteria used in this study included B. abortus virulent strain S2308 obtained from our laboratory collection and a variant that constitutively expresses GFP (Brucella-GFP). The Brucella cyclic dimeric GMP (c-di-GMP) guanylate cyclase mutant strain (Δ1520) was generated in our laboratory using the constructs previously described (27). Before being used for cell infection or DNA extraction, bacteria were grown in Brucella broth medium (BD Pharmingen, San Diego, CA) for 3 d at 37°C under constant agitation.

To measure relative levels of intracellular c-di-GMP, a luciferase (lux) reporter vector containing the Vc2 riboswitch was used as previously demonstrated (28). Briefly, the c-di-GMP–responsive riboswitch was cloned from 348 bp upstream of the Vibrio cholerae VC1722 gene through the VC1722 start codon and inserted into pBBR1MCS-4 (GenBank U25060), upstream of a promoterless luxCDABE operon. To confirm the levels of c-di-GMP produced, the parental WT strain and Δ1520 mutant were transformed with this plasmid containing the c-di-GMP–responsive riboswitch driving lux expression. Bioluminescence was assayed on a Veritas microplate luminometer 100 (Promega) 24 h after transfection.

Additionally, we used a chemical c-di-GMP inhibitor, termed Ebselen. Mouse (C57BL/6) bone marrow–derived macrophages (BMDMs) were plated on six-well tissue culture plates at 5 × 10⁵ per well in 2 ml per well culture media (RPMI 1640 plus 10% FBS) and cultured overnight at 37°C in a 5% CO₂ incubator. The next day, cells were pretreated with Ebselen (50 μM) for 30 min, then they were uninfected (medium) or infected with a multiplicity of infection (MOI) of 100 of B. abortus (strain 2308) for 24 h in the presence or absence of Ebselen. Supernatant was then harvested and assayed for mouse IFN-β using the Legend Max mouse IFN-β ELISA kit (BioLegend) following the manufacturer’s instructions.

BMDMs were generated and cultured in DMEM medium as described (22). Briefly, bone marrow cells were differentiated for 10 d in DMEM (Life Technologies, Carlsbad, CA) containing 10% FBS (HyClone), 1% HEPES (Life Technologies), 1% penicillin G sodium (100 U/ml), and streptomycin sulfate (100 mg/ml), 10% L929 cell–conditioned medium, as the source of M-CSF for macrophages at 37°C in 5% CO₂. A day prior to stimulation of infection, macrophages were harvested and seeded onto 24-well plates at the density of 5 × 10⁵ cells per well (for cytokine and Western blot analysis) or 1 × 10⁵ cells per well over a sterile coverslip (for microscopy analysis). BMDMs were infected with B. abortus virulent strain 2308 or B. abortus Δ1520 with the indicated MOIs (see figure legends). WT and STING^−/− murine embryonic fibroblasts (MEFs) were provided by Dr. G.N. Barber (University of Miami). Cells were maintained in high-glucose DMEM (Life Technologies) supplemented with 10% FBS (Life Technologies), 10 mmol/l glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies/Invitrogen) at 37°C in 5% CO₂/95% air in a humidified incubator. MEFs were seeded on 24-well plates containing sterile coverslips at 1 × 10⁴ cells per well a day before the experiment and kept on normal growth medium.

B. abortus was grown for 3 d at 37°C under constant agitation and the DNA was purified using the Illustra bacteria genomic Prep Mini Spin kit (GE Healthcare) according to the manufacturer’s instructions. Transient transfections of BMDMs and MEFs were carried out using a Lipofectamine 2000 (Invitrogen) ratio (in milliliters) of 1:0.25, following the manufacturer’s directions. Cells were cultured in DMEM and transfected with Brucella DNA (1 μg per well), dsDNA90 (3 μg/ml), and 2′,3′-cGAMP (3 μg per well) (InvivoGen).

Mice were infected via i.p. injection of 1 × 10⁶ CFU of virulent B. abortus strain S2308. Animals were kept for different periods of time and then killed for CFU counting and pathology. Cultured cells were infected in vitro with virulent B. abortus strain 2308, B. abortus–GFP, or B. abortus Δ1520 in DMEM supplemented with 1% FBS (macrophages) or 10% FBS (MEFs). Different MOIs of bacteria were used as challenge for different analysis, as described below.

Five mice from each group of C57BL/6, STING^−/−, cGAS^−/−, AIM2^−/−, GBP2^−/−, and GBP^chr3−/− were infected with B. abortus as described above and killed at the mentioned time intervals. To count residual Brucella CFU, the spleen collected from each animal was macerated in 10 ml of saline, serially diluted, and plated in duplicate on Brucella broth agar. After 3 d of incubation at 37°C, the number of CFU was determined as described previously (29).

BMDMs were stimulated by Brucella infection (MOI of 100:1) or DNA transfection as described above. Where indicated, cells were treated with 100 U/ml IFN-β 18 h prior the course of infection or were untreated. After 17 h, supernatants from cell culture were harvested and assayed for the production of murine IL-1β, CXCL10/IP-10, IL-6, and TNF-α by ELISA (R&D Systems), in accordance with the manufacturer’s instructions. Human CXCL10 was measured in the supernatants of hTERT transfected cells by ELISA (R&D Systems), in accordance with the manufacturer’s instructions.

BMDMs were lysed with M-PER protein extraction reagent (Thermo Scientific) supplemented with 1:100 protease inhibitor mixture (Sigma-Aldrich). Equal amounts of protein were loaded onto 12% SDS polyacrylamide gel, transferred to nitrocellulose membranes (Amersham Biosciences), and blocked for 1 h at room temperature with TBS containing 0.1% Tween 20 and 5% nonfat dry milk. The following primary Abs were incubated overnight at 4°C: rabbit mAb IRF-1 (no. 8478l; Cell Signaling Technology), rabbit mAb β-actin (no. 4970; Cell Signaling Technology), anti–Caspase-1 (p20, mouse mAb no. AG-20B-0042; Adipogen, and IL-1β (mouse mAb no. 3A6; Cell Signaling Technology). For testing small interfering RNA (siRNA) knockdown efficiency on hTERT cells, a similar procedure was performed using rabbit anti-STING polyclonal Ab at 1:5000 and rabbit anti-cGAS (no. D1D3G; Cell Signaling Technology) at 1:1000. Subsequently, membranes were incubated for 1 h at room temperature with anti-rabbit IgG HRP-conjugated (no. 7074; Cell Signaling Technology) or anti-mouse IgG HRP-conjugated (Cell Signaling Technology) Abs. Proteins were visualized using Luminol chemiluminescent HRP substrate (EMD Millipore) in an Amersham Imager 600 (GE Healthcare).

Transcripts were profiled for Brucella-infected and bacterial DNA–transfected BMDMs from STING KO and C57BL/6 mice. Total RNA was isolated from BMDMs with the RNeasy RNA extraction kit (Qiagen) and analyzed by Bioanalyzer RNA 6000 Nano (Agilent Technologies). Gene array analysis was examined by Illumina Sentrix BeadChip array (mouse WG6 version 2 for RNA extracted from BMDMs) (Affymetrix) at the Oncogenomics Core Facility, University of Miami. Microarray data based on the Affymetrix mouse 2.0 ST platform were normalized using the robust multichip averaging algorithm as implemented in the Bioconductor package Affy. The probes were annotated using the Bioconductor annotation package mogene20sttranscriptcluster.db. Fold change was used to compare each pair of microarray samples. The heat map was generated by R package ggplot2. Microarray analysis was performed at the Center of Computational Science, University of Miami. Gene Expression Omnibus accession number is GSE96071 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=uvcjkweipvyzhyb&acc=GSE96071).

RNA was extracted from BMDMs with TRIzol reagent (Invitrogen) to isolate total RNA in accordance with the manufacturer’s instructions. Reverse transcription of 2 μg of total RNA was performed using Illustra Ready-To-Go RT-PCR Beads (GE Healthcare) according to the manufacturer’s directions. Real-time RT-PCR was performed using 2× SYBR Green PCR master mix (Applied Biosystems) on an ABI 7900 real-time PCR instrument (Applied Biosystems). The appropriate primers were used to amplify a specific fragment corresponding to specific gene targets as follows: β-actin, forward, 5′-GGC TGT ATT CCC CTC CAT CG-3′, reverse, 5′-CCA GTT GGT AAC AAT GCC ATG T-3′; IFN-β, forward, 5′-GCC TTT GCC ATC CAA GAG ATG C-3′, reverse, 5′-ACA CTG TCT GCT GGT GGA GTT C-3′; CXCL10, forward, 5′-CCTGCCCACGTGTTGAGAT-3′, reverse, 5′-TGATGGTCTTAGATTCCGGATTC-3′; GBP2, forward, 5′-CTG CAC TAT GTG ACG GAG CTA-3′, reverse, 5′-CGG AAT CGT CTA CCC CAC TC-3′; GBP3, forward, 5′-CTG ACA GTA AAT CTG GAA GCC AT-3′, reverse, 5′-CCG TCC TGC AAG ACG ATT CA-3′; GBP4, forward, 5′-GGA GAA GCT AAC GAA GGA ACA A-3′, reverse, 5′-TTC CAC AAG GGA ATC ACC ATT TT-3′; GBP5, forward, 5′-CTG AAC TCA GAT TTT GTG CAG GA-3′, reverse, 5′-CAT CGA CAT AAG TCA GCA CCA G-3′; IRGB10, forward, 5′-TAA TGC CCT TCG GGG AAT AGG-3′, reverse, 5′-CTG GTT TGA AGT TAG TTG TCC CA-3′; MX1, forward, 5′-GGGGAGGAAATAGAGAAAATGAT-3′, reverse, 5′-GTTTACAAAGGGCTTGCTTGCT-3′; PYDC3, forward, 5′-GCCTGATGGAAGCTTGGGAA-3′, reverse, 5′-CTGGGGAGTCAGTGGTTCAC-3′; PYHIN1, forward, 5′-TCTGGACCCTCCAGTGTCTT-3′, reverse, 5′-ACCTTGCTGGTGACCATTTT-3′; CXCL11, forward, 5′-AGGAAGGTCACAGCCATAGC-3′, reverse, 5′-CGATCTCTGCCATTTTGACG-3′; TNFSF10, forward, 5′-CAACGAGCTGAAGCAGAT-3′, reverse, 5′-GGGTCCCAATAACTGTCATC-3′; NOS2, forward, 5′-AGC ACT TTG GGT GAC CAC CAG GA-3′, reverse, 5′-AGC TAA GTA TTA GAG CGG CGG CA-3′; ARG1, forward, 5′-TGA CAT CAA CAC TCC CCT GAC AAC-3′, reverse, 5′-GCC TTT TCT TCC TTC CCA GCA G-3′. All data are presented as relative expression units after normalization to the β-actin gene, and measurements were conducted in triplicate.

BMDMs were transfected with siRNA from siGENOME SMARTpools (Dharmacon) with the GenMute siRNA transfection reagent according to the manufacturer’s instructions (SignaGen). siGENOME SMARTpool siRNAs specific for mouse GBP2 (M-040199-00-0005), GBP3 (M-063076-01-0005), and GBP5 (M-054703-01-0005), were used in this study. A control siRNA pool was used (D-001206-14-05). Forty-eight hours after transfection, cells were infected with B. abortus (MOI of 100:1) for 24 h as described above. hTERT cells were transfected with siRNA from siGENOME SMARTpools siRNA STING (J-024333-20-0002), siRNA cGAS (L-015607-02-0005), or siRNA control (D-001810-10-05) at 80 μM siRNA and 1 μl of Lipofectamine RNAiMAX (Thermo Fisher) in 100 μl of Opti-MEM media. Culture medium was replaced 48 h after transfection with DMEM supplemented with 10% heat-inactivated FBS, penicillin-streptomycin, 20% 199 media, sodium pyruvate, and l-glutamine solution and the cells were incubated for an additional 24 h. After 72 h of the transfection process, hTERT cells were infected with B. abortus or mutant strain Δ1520 (MOI of 100) or transfected with bacterial DNA (1 μg) and the supernatant was collected after 17 h to measure human CXCL10 by ELISA.

A Click-iT 5-ethynyl-2-deoxyuridine (EdU) imaging kit (Molecular Probes) was used to provide specific labeling of bacterial DNA in infected macrophages. Brucella was grown as described above and EdU solution was added (20 μM) to the growth medium for 6 h before macrophage infection. Bacteria were then washed three times in PBS before being added to macrophages to prevent carryover of unbound EdU. Detection of incorporated EdU with Alexa Fluor 488 was performed in 24-h-infected macrophages following the manufacturer’s instructions (see below). Additionally, ProLong Gold with DAPI mounting medium (Invitrogen) was used to label eukaryotic as well bacterial DNA in all slides prepared for microscopy.

Intracellular localization of AIM2, STING, GBP2, IRF-3, and NF-κB–p65 was analyzed by immunofluorescence in macrophages or MEFs infected with Brucella-GFP, or transfected with Brucella DNA as follows: 1) localization of STING and nuclear translocation of transcription factors was analyzed in MEFs infected as described above with Brucella-GFP (MOI of 1000:1) or transfected with Brucella DNA for 1, 2, 3, and 4 h; 2) intracellular localization of AIM2 was analyzed in macrophages infected with EdU-incorporated Brucella (MOI of 100:1) for 24 h; 3) intracellular localization of GBP2 was analyzed in macrophages infected with Brucella-GFP (MOI of 100:1) for 24 h. At specific times, cells were washed twice with PBS and fixed in 4% paraformaldehyde (pH 7.4) at room temperature for 30 min. After fixation, coverslips were washed three times with PBS and kept at 4°C until immunofluorescence was performed. Permeabilization was done in PBS containing 0.3% Triton X-100 for 15 min, and cells were subsequently blocked for 1 h with 1% BSA in PBS at room temperature prior to incubation with anti–IRF-3, anti–NF-κB-p65, anti-GBP2, or anti-AIM2 primary Abs at 4°C overnight. Anti-rabbit conjugated with Alexa Fluor 546 was used for detection of primary Abs. Coverslips were mounted in slides using ProLong Gold with DAPI mounting medium (Invitrogen). Confocal microscopy analysis was performed in a Zeiss 880 confocal system. Three coverslips were analyzed per sample and images were taken using a ×40 objective for six random areas of each coverslip providing information for an average of 100 cells per coverslip. The differences in intensity of anti-GBP2 labeling in infected versus uninfected macrophages were quantified to assess alterations in GBP2 intracellular localization. Briefly, the intensity of gray levels (pseudocolored in red in the image) was measured using ImageJ from anti-GBP2 images in three circular areas of 3 μm diameter for each cell. This was done to obtain an estimation of mean intensity of GBP2 staining in at least 9 μm² of each cell. Regions of brightest Ab signal on each cell (perinuclear regions) were preferentially chosen. Abs were purchased from the following sources: anti-GBP2 (Proteintech), anti–IRF-3 (FL-425) (Santa Cruz Biotechnology), anti–NF-κB-p65 (Cell Signaling Technology), anti-AIM2 (Santa Cruz Biotechnology). Anti-mouse and anti-rabbit secondary Abs conjugated with Alexa Fluor 488 or Alexa Fluor 546 were purchased from Jackson ImmunoResearch Laboratories. Rabbit polyclonal Ab against STING was described previously (11). For evaluation of STING activation by B. abortus mutant strain Δ1520, MEFs or hTERT cells were infected with B. abortus S2308 or B. abortus Δ1520 (MOI of 1000:1) or transfected with dsDNA90 (3 μg/ml) or cGAMP (1 μg per well) for 4 h. Cells were processed for immunofluorescence as described above and incubated with anti-STING or anti-Brucella LPS (1:100) Abs for bacterial staining. Coverslips were mounted in slides using ProLong Gold with DAPI mounting medium (Invitrogen) and microscopy analysis was performed as described above.

BMDMs were infected as described above with Brucella-GFP (MOI of 10:1). Such lower bacterial load at the beginning of infection ensured that macrophages from more susceptible mouse strains would not have an overgrowth of intracellular Brucella and could be analyzed. Bacteria number was assessed in cells infected for 24, 48, and 72 h. Cells infected for only 24 h were kept in the absence of antibiotics during the whole experiment. After the first 24 h, gentamicin (10 μg/ml) was added to the medium of cells infected for longer periods to prevent secondary infections. Fixation and permeabilization of the cells were performed as for immunofluorescence. Staining of the actin cytoskeleton with rhodamine-phalloidin (0.04 μM in 0.3% Triton X-100 in PBS; Thermo Fisher) was performed to visualize cell shape. Preparation of slides and acquisition of microscopy data were performed as described for immunofluorescence. Counts of intracellular bacteria were performed manually by visualization of individual GFP-expressing Brucella.

For the measurement of viable intracellular bacteria using CFU, after transfection with siRNA and infection with B. abortus strain 2308 or B. abortus Δ1520 at different MOIs (see figure legends), cells were washed twice with PBS and then lysed for 10 min at room temperature in 800 μl of PBS containing 0.1% Triton X-100 under manual agitation. Lysates were diluted from 10 to 1000 times in PBS and plated on petri dishes containing Brucella broth agar. Petri dishes were incubated for 3–4 d at 37°C before CFU counting.

BMDMs (1 × 10⁶) from C57BL/6 and GPB^crh3 KO mice were derived as described previously, infected with B. abortus (MOI of 100:1) for 6 h at 37°C, and washed three times with phosphate buffer (1 M). The cells were then fixed with glutaraldehyde (2.5% in 1 M phosphate buffer) for 24 h at 4°C, washed three times with phosphate buffer (1 M), and the samples were sent to the Microscopy Center at the Federal University of Minas Gerais for dehydration, treatment with osmium tetroxide and uranyl acetate, and transmission electron microscopy (TEM) analysis. To evaluate the percentage of ruptured Brucella-containing vacuole (BCV) membranes, we evaluated 30 macrophages per group. Each macrophage was evaluated in relationship to the total number of bacteria in ×14,500 magnification. Then, the membrane integrity of each BCV was carefully evaluated in a higher magnification (×60,000). After analysis, we calculated the percentage of ruptured BCVs in relationship to the total number of bacteria counted.

C57BL/6, STING^−/−, and cGAS^−/−mice were infected with B. abortus (1 × 10⁶ CFU). Seven days after infection, the spleen cells were harvested and washed twice with sterile PBS. After washing, the cells were adjusted to 1 × 10⁶ cells in RPMI 1640 medium supplemented with 10% FBS, 150 U of penicillin G sodium, and 150 μg of streptomycin sulfate per well in a 96-well plate. All cells were stimulated with Con A (5 μg/ml), 1 μg of brefeldin A was added per well, and the cultures were incubated at 37°C for 4 h. After the incubation period, the cells were centrifuged at 1500 rpm for 7 min at 4°C and washed with PBS containing 1% BSA (PBS/BSA). Then, the cells were incubated with anti-CD16/CD32 (FcBlock) (1:30 diluted in PBS/BSA) for 20 min at 4°C, washed in PBS/BSA, and incubated for 20 min at 4°C with a mixture of the following Abs: hamster IgG anti-murine CD3 conjugated to biotin (clone 500A2; 1:200) and rat IgG2b anti-murine CD4 conjugated to allophycocyanin-Cy7 (clone GK 1.5; 1:200). All Abs were obtained from BD Biosciences (San Diego, CA). After that, splenocytes were washed again with PBS/BSA and incubated with streptavidin conjugated to PE-Cy5.5 (1:30) for 20 min at 4°C and fixed and permeabilized using BD Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer’s instructions. The cells were then incubated with rat IgG1 anti-murine IFN-γ conjugated to PE (clone XMG1.2; 1:30; BD Biosciences) or with rat IgG1 anti-murine IL-4 conjugated to PE (clone 11B11; 1:30; BD Biosciences) for 30 min at 4°C. Finally, the cells were washed three times, suspended in PBS buffer, and evaluated using Attune acoustic focusing equipment (Life Technologies). The results were analyzed using FlowJo software (Tree Star, Ashland, OR).

The results of this study were analyzed using ANOVA, as indicated, with GraphPad Prism 5 computer software (GraphPad Software). A p value < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).

To test the role of STING during B. abortus infection in vitro, primary murine BMDMs were isolated from STING KO and C57BL/6 mice. These cells were infected with virulent B. abortus S2308 or transfected with B. abortus S2308 DNA and microarray analysis was performed. Microarray analysis revealed that several type I IFN–related genes are downregulated in STING KO cells either infected with Brucella or transfected with bacterial DNA as observed in Fig. 1A and 1B. To confirm that the induction of these type I IFN–related mRNAs were STING-dependent genes, we performed quantitative PCR (qPCR) analysis. Macrophages from C57BL/6 mice transfected with Brucella DNA robustly activate an array of genes, including IFN-β, GBP3, GBP4, and GBP5 (Fig. 1C–F). However, the expression of these genes was dramatically reduced in STING KO cells. GBPs are known to colocalize with vacuolar bacterial pathogens such as Salmonella typhimurium and Mycobacterium bovis and to recruit antimicrobial effector mechanisms (30, 31). Additionally, other STING-dependent genes were also validated by qPCR such as CXCL11, Mx1, TNFSF10, PYDC3, and PYHIN1 (Supplemental Fig. 1). Taken together, our data demonstrated that both Brucella infection and bacterial DNA induce the expression of several innate immune genes in a STING-dependent fashion.

Given the importance of STING in regulating cytoplasmic DNA signaling events, we infected MEFs with Brucella-GFP or transfected with Brucella DNA and observed STING translocation through confocal microscopy. After 4 h of infection with Brucella and 2 h of transfection with bacterial DNA, STING rapidly underwent trafficking from the endoplasmic reticulum to the perinuclear-associated endosomal regions of the cell (Fig. 2A). This event usually accompanies STING phosphorylation and degradation, likely to avoid sustained STING-activated cytokine production (32). Additionally, after transfection of WT MEFs with Brucella DNA, IRF-3 and the p65 subunit of NF-κB became phosphorylated and translocated into the nucleus; however, these events did not occur in STING KO cells (Fig. 2B, 2C). These data indicate that the pathway of IRF-3 and NF-κB activation induced by bacterial DNA is STING-dependent.

To investigate the roles of STING and cGAS during cytosolic sensing of Brucella DNA or bacterial infection in inducing proinflammatory cytokine production, BMDMs were isolated from STING and cGAS KO mice. Brucella DNA or bacterial infection–triggered IFN-β expression was partially dependent on STING and cGAS (Fig. 3A). However, the level of IFN-β expression induced by transfected bacterial DNA was much higher than Brucella infection. Delivery of the STING ligand cGAMP via transfection bypassed the cGAS requirement for IFN-β expression and CXCL10 production in agreement with previous reports establishing that cGAS functions upstream of STING (15) (Supplemental Fig. 2). We also observed that CXCL10 production, a surrogate cytokine for type I IFN expression, was reduced in STING and cGAS KO BMDMs infected or transfected with Brucella DNA (Fig. 3B). Because STING activation is required for NF-κB translocation induced by bacterial DNA (Fig. 2), we also measured TNF-α and IL-6 production by STING and cGAS KO BMDMs transfected with bacterial DNA or infected and compared with WT cells. Either Brucella-infected or DNA-transfected macrophages from cGAS and STING KO mice produced a reduction in TNF-α and a modest decrease in IL-6 compared with WT cells (Fig. 3C, 3D). These data suggest that the STING pathway is required for full production of TNF-α and IL-6. However, other Brucella ligands or bacterial DNA may activate NF-κB in a cGAS/STING-independent pathway, for example through the TLR pathway. We have previously demonstrated that IFN-β can be partially produced by innate cells activated with Brucella components via TLR7 (33). Furthermore, we have shown in this study using MAVS KO macrophages that type I IFN expression depends in part on the RIG-I pathway independently of the STING/cGAS axis (Fig. 3I).

Additionally, we have demonstrated that inflammasome activation is important to induce protective immunity against Brucella infection (22). However, the molecular mechanisms that govern assembly of the DNA sensor AIM2 are less clear than those described for the NLRP3 inflammasome. Because type I IFN contributes to activation of the AIM2 inflammasome in response to Francisella novocida (34), we determined the role of STING in IL-1β secretion and caspase-1 activation during B. abortus infection. Macrophages from STING KO mice transfected with bacterial genomic DNA or infected with Brucella showed a reduction in IL-1β secretion and caspase-1 activation when compared with cGAS KO or WT cells (Fig. 3E, 3F). Therefore, only STING deficiency leads to a reduction in IL-1β and caspase-1 activation when compared with cGAS KO macrophages. These findings are consistent with a reduction of IL-1β but not pro–IL-1β observed in STING KO cells in the Western blot (Fig. 3F), suggesting that a lack of STING affects caspase-1 processing. Furthermore, addition of exogenous rIFN-β increased the levels of CXCL10, TNF-α, IL-6, and IL-1β in Brucella-infected macrophages, demonstrating the role of this molecule in modulating proinflammatory cytokine production.

Finally, NOS2 and Arg1 have been identified as markers for M1 and M2 macrophages, respectively (35). In this study, we observed that STING KO macrophages had an increase in Arg1 expression and a decrease in NOS2 expression compared with WT and cGAS KO cells (Fig. 3G, 3H). This profile is suggestive of M2-type macrophages (alternatively activated macrophages) that are typically associated with bacterial persistence.

Taken together, these results demonstrate that even though cGAS and STING are important sensors involved in the production of inflammatory cytokines, STING plays the predominant role during Brucella infection.

Bacterial CDN levels are regulated by the opposing activities of cyclases and phosphodiesterases. To determine the levels of c-di-GMP produced by Brucella WT strain versus c-di-GMP guanylate cyclase mutant (Δ1520), we transformed all strains with a plasmid containing a c-di-GMP–responsive riboswitch that drives lux expression as previously demonstrated (28). The Brucella Δ1520 mutant displayed a phenotype showing lower levels of c-di-GMP when compared with WT strain S2308 (Fig. 4B). Furthermore, we performed confocal microscopy analysis in WT MEFs infected with Brucella Δ1520 mutant and WT S2308 strains. Cells infected with WT bacteria or transfected with DNA or cGAMP showed a specific STING-activation profile characterized by an aggregate speck formation in the perinuclear region of the cell; however, we did not observe this activation phenotype in Brucella Δ1520 mutant–infected MEFs (Fig. 4A). Additionally, we infected BMDMs from STING KO and C57BL/6 mice with Brucella Δ1520 mutant or WT S2308 strains and measured IFN-β expression and IL-1β production. Our results demonstrated that the Brucella Δ1520 mutant, which produces lower levels of c-di-GMP, induced lower expression levels of IFN-β and IL-1β secretion compared with the virulent strain S2308 (Fig. 4C, 4D) and equivalent to production in the STING KO macrophages. To confirm the Brucella Δ1520 mutant findings, we tested a chemical inhibitor of c-di-GMP, termed Ebselen (36). Macrophages treated with Ebselen and infected with WT Brucella produced much less IFN-β compared with cells untreated and infected (Fig. 4E). The greater difference observed in type I IFN responses between the use of Ebselen compared with the Δ1520 mutant strain might be related to the fact that Ebselen inhibits the binding of c-di-GMP to receptors containing an RxxD domain including PelD and diguanylate cyclases with a broader action and potential off-target effects (36). In contrast, the Δ1520 mutant strain has a deletion on a single Brucella diguanylate cyclase. Taken together, these results suggest that c-di-GMP produced by Brucella is a key metabolite to induce type I IFN responses. Furthermore, we measured CFU counts of Brucella Δ1520 mutant and WT S2308 strains after 48 h of infection in macrophages. Brucella Δ1520 mutant showed reduced bacterial numbers compared with WT bacteria in either C57BL/6 or STING KO cells (Fig. 4F).

To confirm our findings in the human system, we used human fibroblast cells (hTERT). After infection of hTERT with Brucella Δ1520 mutant or WT S2308 strain, we observed STING activation by confocal microscopy in the cells infected with WT bacteria but not with the mutant strain (Fig. 5A). Additionally, we performed siRNA silencing of STING and cGAS in hTERT cells. The siRNA knockdown efficiency is demonstrated in Fig. 5B. The results observed in Fig. 5A were correlated to CXCL10 production that was reduced in siRNA control cells infected with Brucella Δ1520 mutant compared with WT bacteria (Fig. 5C). Additionally, cells treated with cGAS or STING siRNA produced diminished levels of CXCL10 compared with siRNA control when they were infected with either Brucella strain or with bacterial DNA.

Collectively, these results suggest that Brucella is able to produce its own second messenger to activate the STING pathway directly. In the present study, we provide strong evidence that the bacterial c-di-GMP is important in activating STING in mouse macrophages and human cells.

To determine whether STING or cGAS restricts Brucella growth in macrophages and in vivo, we infected WT, STING, and cGAS KO BMDMs and mice and quantified Brucella-GFP⁺ in vitro at 24, 48, and 72 h by confocal microscopy and in vivo at 1 and 3 wk postinfection. WT macrophages and cGAS KO infected with an MOI of 100:1 efficiently controlled intracellular replication, whereas STING KO BMDMs contained larger numbers of B. abortus at all time intervals studied (Fig. 6A, 6B). Because the cGAS/STING axis is important for stimulating type I IFN, we investigated whether these DNA sensors also play a role in host defense in vivo. We infected age- and sex-matched WT, cGAS, and STING KO mice i.p. with 1 × 10⁶ CFU of B. abortus virulent strain 2308. STING KO displayed a significantly higher bacterial burden at 1 and 3 wk postinfection compared with WT animals. However, cGAS KO had no defect in overall resistance to B. abortus, as we observed similar bacterial numbers in spleens of these mice compared with WT (Fig. 6C). Because Th1 responses are crucial for efficient control of B. abortus, we measured the frequency of CD4⁺ T cells producing IFN-γ or IL-4 in WT, cGAS, and STING KO mice after infection. As demonstrated in Fig. 6D, the frequency of CD4⁺ T cells producing IL-4 is higher in STING KO animals compared with WT and cGAS KO mice. Regarding the percentage of CD4⁺ T cells producing IFN-γ, similar levels were detected in all mouse strains tested (data not shown). Furthermore, the presence of markers associated with M2 macrophages shown previously also coincided with enhanced frequency of CD4⁺ T cells producing IL-4 and bacterial loads in STING KO mice compared with cGAS KO and WT control.

Taken together, these findings suggest that cGAS appears to be dispensable for bacterial resistance in vivo and in vitro. These results confirmed that STING is critical to host defense against B. abortus in vitro and in vivo.

Infection with B. abortus results in the formation of liver and spleen granulomas, where inflammatory cells aggregate to restrain bacterial growth. Because only STING KO mice were more susceptible to infection in vivo, we analyzed the role of STING in regulating liver pathology. At 1 and 6 wk postinfection, STING KO mice displayed a significant reduction in granuloma number and size when compared with WT counterparts (Supplemental Fig. 3). These data suggest that STING modulates host liver pathology at early and late stages of Brucella infection.

The transcription factor IRF-1 impacts adaptive immune responses by regulating MHC class I expression and influencing development of NK and T cells (37). Additionally, IRF-1 was identified by its binding to DNA sequences that are common to the promoters of IFN-α/β genes (38). Thus, IRFs were proposed to be the regulators of type I IFN responses. In this study, we evaluated the level of IRF-1 protein expression in macrophages of WT, STING, cGAS, and AIM2 KO animals following infection with Brucella or transfection with bacterial DNA. We observed a major increase in IRF-1 expression in WT cells after bacterial infection or DNA transfection compared with the negative control (Fig. 7A). However, this upregulation was partially dependent on the STING/cGAS pathway, as we observed reduced IRF-1 expression in cGAS and STING KO macrophages. Conversely, lack of AIM2 robustly augmented IRF-1 expression in macrophages infected with Brucella or DNA transfected, demonstrating that AIM2 regulates type I IFN responses. Furthermore, we tested whether IRF-1 expression was dependent on the IFNAR signaling pathway. As shown in Fig. 7B, IFNAR KO cells transfected with bacterial DNA or infected with Brucella had diminished levels of IRF-1 when compared with WT macrophages.

Because others demonstrated that IRF-1 KO animals are more susceptible to Brucella infection in vivo (39), we investigated the hypothesis that type I IFN signaling and IRF-1 affect the ability of macrophages to control Brucella replication. Therefore, we infected WT, IFNAR KO, and IRF-1 KO BMDMs with Brucella-GFP⁺ and quantified bacteria over time (Fig. 7C). Single-cell analysis revealed that WT macrophages restricted bacterial replication after 24 h of infection, whereas IFNAR and IRF-1 KO cells failed to control bacterial replication and harbored a significantly larger number of bacteria compared with WT BMDMs (Fig. 7D). The greater susceptibility to Brucella replication observed in IRF-1 KO cells suggests that IRF-1 is important in multiple antimicrobial defense mechanisms.

GBPs are IFN-inducible GTPases that exert antimicrobial effects (24). Additionally, GBPs encoded by genes on mouse chromosome 3 (GBP1, GBP2, GBP3, GBP5, and GBP7) promote recognition of the vacuolar bacterium S. typhimurium, leading to the escape of the bacteria into the cytosol (31). In this study, we detected by qPCR analysis the downregulation of GBP2 and GBP3 genes in the absence of IRF-1 and IFNAR in macrophages infected with Brucella (Fig. 8A). However, the residual expression of GBP2 and GBP3 in IRF-1 KO macrophages indicates that an alternative pathway independent of IRF-1 exists to induce GBP expression. To address that, we infected IFNAR KO macrophages with B. abortus and found that lack of type I IFN signaling robustly reduced GBP2 and GBP3 expression (Fig. 8A). Additionally, we observed that GBP2 and GBP3 expression is partially dependent on the STING pathway, probably via IFN-β production (Fig. 8B). Interestingly, reduction of GBP2 and GBP3 expression following Brucella infection in macrophages was only dependent on STING and not cGAS. Furthermore, we observed by confocal microscopy that Brucella infection induces the formation of GBP2 aggregates (37% increase compared with uninfected cells) located in close proximity to bacterial-containing compartments in macrophages (Fig. 8C, 8D). Additionally, we performed electron microscopy analysis and we detected that 74.2% of BCV was disrupted in C57BL/6 macrophages when compared with 38.5% in GBP^chr3 KO cells (Fig. 8E, 8F). This result suggests that the GBP machinery is important to target the BCV to release bacterial components to host cell cytosol.

Taken together, these findings also provide evidence that GBP2 and GBP3 are produced in response to B. abortus infection, and that GBP2 and GBP3 are under the control of type I IFN signaling at least partially via the STING pathway.

GBPs can target vacuolar bacteria such as Salmonella and Francisella and induce the recruitment of antimicrobial peptides to kill the bacteria (25, 31). To investigate whether GBPs contained on mouse chromosome 3 (GBP^chr3) or GBP2 alone directly affected the viability of bacteria in macrophages, we treated WT BMDMs with GBP2 and GBP pool (containing GBP2, GBP3, and GBP5) siRNA and infected them with B. abortus, and bacteria were quantified at 24 h postinfection. Analysis of CFU counts revealed that WT macrophages restricted bacterial replication whereas GBP pool– and GBP2 siRNA–treated cells failed to control Brucella growth intracellularly and harbored a significantly larger number of bacteria (Fig. 8G). We further analyzed the role of GBP2 and GBP^chr3 in controlling bacterial infection in vivo by infecting GBP2 and GBP^chr3 KO and WT mice and measuring CFU in spleen at 1 wk postinfection. As shown in Fig. 8H, only GBP^chr3 KO mice were more susceptible to Brucella infection when compared with GBP2 KO or WT animals. These findings demonstrated that GBPs mediate Brucella control in vitro and in vivo.

Because GBPs play a role in releasing bacteria from vacuoles and thus enable greater access to cytosolic sensors, we sought to address the role of GBPs in inflammasome activation during Brucella infection. Brucella-infected macrophages from C57BL/6 mice treated with siRNA for GBP2 or a GBP pool were assessed for pro–IL-1β in cell lysates, IL-1β secretion, and caspase-1 activation. GBP2 and GBP pool–treated macrophages displayed a significant reduction in cytokine release and caspase-1 activation compared with siRNA control-treated cells (Fig. 9A, 9B). Although GBP2 and GBP pool siRNA-treated cells had a reduced IL-1β secretion, the amount of pro–IL-1β was intact, indicating that pathogen recognition receptor and NF-κB responses were normal but an inflammasome activatory signal was affected. To confirm these findings, we used macrophages from GBP2 and GBP^chr3 KO mice and transfected them with bacterial DNA or infected them with Brucella and measured IL-1β secretion. The results demonstrated that GBP2 and GBP^chr3 KO cells also showed a reduced secretion of IL-1β when macrophages were either transfected with DNA or infected with the bacteria, corroborating our siRNA data (Supplemental Fig. 4). Taken together, these findings are consistent with a role for GBPs in releasing Brucella components into the cytosol for inflammasome detection.

AIM2 was found to recognize cytoplasmic dsDNA through its HIN-200 domain and ASC via its pyrin domain (10). Franciscella tularensis and L. monocytogenes DNA activate the AIM2 inflammasome by its interaction with ASC to induce caspase-1 (34, 40). For AIM2 to detect dsDNA, Brucella or its DNA must escape the BCV and enter the cytoplasm. To determine whether AIM2 inflammasome directly senses Brucella DNA intracellularly, we infected C57BL/6 macrophages and observed that labeled AIM2 colocalized with Brucella DNA that was previously stained using the Click-iT EdU imaging kit. Indeed, costaining of AIM2 and bacterial DNA from Brucella-infected macrophages revealed the formation of an inflammasome aggregation of AIM2 and Brucella DNA specks as result of cell activation (Fig. 9C). We also determined the role of AIM2 in regulating Brucella growth in vivo. Consistent with our previous in vitro findings using macrophages identifying a critical role for AIM2 and ASC to trigger caspase-1 activation and IL-1β secretion (22), AIM2 KO mice showed a reduced resistance to Brucella at 1, 3, and 6 wk postinfection as determined by bacterial numbers in the spleens (Fig. 9D). Bacterial load recovery was 0.7, 1.6, and 2.3 logs higher at respective weeks in AIM2 KO mice when compared with C57BL/6 animals. Taken together, these results strongly suggest that inflammasomes are important to induce resistance to B. abortus infection and implicate AIM2 receptors in this process. Additionally, we proposed a schematic model of how STING pathway connects with AIM2 inflammasome activation (Fig. 10).

Pathogenic bacteria can use many different strategies to enter and establish infection inside the host, and the immune system has mechanisms to detect and eliminate a broad range of these bacteria. Cytosolic detection leads to activation of potent antimicrobial effector pathways such as the inflammasome and the cytosolic surveillance pathway. The cGAS/STING axis is an important cytosolic surveillance pathway by which innate immune cells recognize both viral and bacterial pathogens that access the cytosol, but different bacteria have evolved different mechanisms to initiate this response (11, 23). Additionally, bacterial ligands must secure entry into the cytoplasm to activate cytosolic sensors; however, the mechanisms by which concealed ligands are liberated in the cytoplasm have remained unclear. Because the detection of Brucella infection by macrophages is so critical to the innate immune response, we sought to define the receptors that sense Brucella DNA in the cytosol and determine their role in the host response to infection. Previously, we have shown that AIM2 is important for sensing Brucella DNA and triggering IL-1β secretion and caspase-1 activation (22), but the role of the cGAS/STING axis during this bacterial infection remained to be determined.

In this study, we observed that Brucella infection or bacteria DNA transfection triggers the expression of innate immune genes such as IFN-β and GBPs in a STING-dependent manner. Furthermore, bacterial infection or DNA transfection induces STING activation in MEFs as observed by aggregated speck formation in the perinuclear region of the cell. IRF-3 and NF-κB translocation to the nucleus was also observed in DNA-transfected MEFs and shown to be STING-dependent. Given the potential positive and negative roles of type I IFN during bacterial infections, we sought to determine the overall role of cGAS and STING in B. abortus pathogenesis. Infection of cGAS and STING KO macrophages led to a partial decrease in type I IFN expression when compared with WT cells. Additionally, lack of STING had a partial effect on production of NF-κB–dependent cytokines, such as TNF-α and IL-6, suggesting that Brucella partially activates NF-κB through a STING-dependent pathway. However, STING but not cGAS control bacterial replication in vitro. The same phenotype was observed in infected KO mice in vivo, demonstrating that lack of cGAS had no defect in overall resistance to B. abortus. Higher bacterial burdens were detected in spleens of STING KO mice compared with cGAS KO and WT animals. Additionally, STING altered the formation of hepatic granulomas in vivo, which is an important host strategy to restrain bacterial growth. Furthermore, we observed a reduced secretion of IL-1β and caspase-1 activation and GBP2 and GBP3 expression in STING KO cells infected with B. abortus when compared with cGAS-deficient macrophages. Collectively, these findings suggest lack of cGAS is less critical to the activation of innate immune effector mechanisms related to protective immunity against Brucella.

In the context of animal models of bacterial infection, M1-type macrophages (classically activated macrophages) and NO production are often, but not exclusively, associated with host protection (41). Conversely, M2-type macrophages are typically associated with bacterial persistence. In this study, we observed that the presence of markers associated with M2 macrophages in STING KO mice compared with cGAS KO and WT controls, which also coincided with enhanced frequency of CD4⁺ T cells producing IL-4. Previously, Xavier et al. (42) demonstrated that M2 macrophages support increased levels of intracellular Brucella replication during chronic infection. Taken together, these findings suggest that STING activation seems to be involved in inhibiting the differentiation of M2 macrophages in Brucella-infected cells that parallels with enhanced bacterial replication in STING KO cells.

L. monocytogenes effectively short-circuits the cGAS cytosolic surveillance pathway by providing its own second messenger, whereas M. tuberculosis and L. pneumophila generate the type I IFN signal via DNA binding to cGAS. Interestingly, Watson et al. (18) demonstrated that cGAS also plays no role in controlling M. tuberculosis infection. Additionally, S. typhimurium predominantly activates type I IFN via a cGAS/STING-independent mechanism, likely via the TLR4/TRIF pathway (43). In this study, we provide evidence in mouse and human cells that production of Brucella c-di-GMP activates STING type I IFN signaling events. Furthermore, STING activation leads to GBP expression and inflammasome activation.

IRF-1 has been identified as a transactivator of IFN-β (44); therefore, we used BMDMs from IFNAR and IRF-1 KO mice to determine their relative roles in restricting Brucella replication in macrophages. By confocal microscopy, we observed more intracellular Brucella-GFP⁺ per macrophage in both IFNAR and IRF-1 KO cells compared with WT cells. In the present study, STING was required for full expression of IRF-1 after Brucella infection in macrophages. STING activation, type I IFN signaling, and the transcription factor IRF-1 were also required for robust expression of GBP2 and GBP3 following Brucella infection. Because we observed that GBP siRNA-treated macrophages produced a major reduction in IL-1β secretion, we therefore concluded that there is a specific requirement of STING and GBPs in Brucella-induced inflammasome activation.

Members of the IFN-inducible GTPase family are executioners of cell-autonomous immunity and have the ability to target the vacuolar membrane encapsulating intracellular parasites and bacteria (45, 46). To determine whether GBPs were directly involved in the activation of inflammasomes, we knocked down by siRNA GBP2 and the pool of GBP2, GBP3, and GBP5 in C57BL/6 macrophages infected with Brucella. In this study, cells not expressing GBP2 or the pool of GBPs produced much less IL-1β than did the cells transfected with control siRNA. Additionally, GBP2 and GBP pool knocked-down cells had a reduced ability to control Brucella replication intracellularly. However, Brucella infection observed in cells treated with the GBP pool was markedly augmented compared with cells treated GBP2 only siRNA, suggesting the GBP proteins work together nonredundantly to control Brucella. Furthermore, we observed during in vivo experiments that only GBP3^chr3 KO mice were more susceptible to Brucella at 1 wk postinfection, suggesting that GBPs other than GBP2 are more important to host protection. A recent study suggested that besides the membrane-destabilizing activity of GBPs, their bacteriolytic role could also result in release of microbe-associated molecular patterns such as DNA (47). GBP-mediated bacteriolysis may release bacterial DNA to activate AIM2 inflammasome and/or amplification of type I IFN production via the cGAS/STING pathway. In this study, we provide evidence by TEM experiments that GBPs from the chromosome 3 are able to target the Brucella-containing vacuole, disrupting its membrane and making bacterial products available to be sensed by cytosolic receptors. Additionally, we demonstrate that Brucella DNA is colocalized with AIM2 during infection of macrophages. Furthermore, we extended our findings to an in vivo setting and infected WT and AIM2 KO mice with B. abortus virulent strain and monitored their susceptibility to infection. Analysis of bacterial burdens showed that AIM2 KO mice harbored significantly greater numbers of Brucella in the spleen than did WT mice at 1, 3, and 6 wk postinfection.

Bacterial ligands must secure entry into the cytoplasm to activate inflammasomes; however, the mechanisms by which concealed ligands are liberated in the cytoplasm have remained unclear. Several studies have shown that activation of AIM2 inflammasome by intracellular bacteria requires bacteriolysis and subsequent release of bacterial chromosomal DNA into the cytosol (48, 49). However, whether the bacteriolysis is accidental or is an active GBP-directed process has to be clarified. Recently, others (50) have shown that IFN-inducible protein IRGB10 is essential for activation of the DNA-sensing AIM2 inflammasome by Francisella novicida. IRGB10 directly targeted cytoplasmic bacteria through a mechanism requiring GBPs. Localization of IRGB10 to the bacterial cell membrane compromised bacterial structure integrity and mediated cytosolic release of ligands for recognition by inflammasome sensors. We also observed a decreased expression of IRGB10 in Brucella-infected macrophages of STING KO; however, whether this molecule plays a role in Brucella bacteriolysis remains unclear (data not shown).

Putting these results together in a pathway, it is possible that Brucella triggers initial direct STING signaling, type I IFN, and GBP activation via bacterial c-di-GMP. Our findings suggest that GBPs associate with BCVs and, by an as-yet-undefined mechanism, induce lysis of the BCV or maybe direct bacteriolysis, which result in bacterial products such as DNA being released into the cytosol to be recognized by cytosolic sensors such as AIM2. After DNA is released into the cytosol, the STING signal also could be further amplified by cGAS sensing of genomic DNA generating cGAMP (Fig. 10). However, further experiments are necessary to support this hypothesis and define how this DNA signaling hierarchy is governed. Additional questions remain to be answered such as how GBP targeting is regulated and how GBPs act mechanistically to expose Brucella ligands to cytosolic recognition pathways.

In conclusion, our results provide insights into the mechanism by which bacterial-associated ligands are liberated in the cytoplasm to activate inflammasomes and establish a cGAS-independent mechanism of STING-mediated protection against an intracellular bacterial infection.

We thank Dr. Xi Chen (Division of Biostatistics, Department of Public Health Sciences, Sylvester Comprehensive Cancer Center) and Dr. Tianli Xia (Department of Cell Biology, University of Miami, FL) for performing the bioinformatic analysis of the microarray data and RNA preparation.

The authors have no financial conflicts of interest.