TRIM56-mediated production of type I interferon inhibits intracellular replication of Rickettsia rickettsii

ABSTRACT Rickettsia rickettsii (R. rickettsii), the causative agent of Rocky Mountain spotted fever (RMSF), is the most pathogenic member among Rickettsia spp. Previous studies have shown that tripartite motif-containing 56 (TRIM56) E3 ligase-induced ubiquitination of STING is important for cytosolic DNA sensing and type I interferon production to induce anti-DNA viral immunity, but whether it affects intracellular replication of R. rickettsii remains uncharacterized. Here, we investigated the effect of TRIM56 on HeLa and THP-1 cells infected with R. rickettsii. We found that the expression of TRIM56 was upregulated in the R. rickettsii-infected cells, and the overexpression of TRIM56 inhibited the intracellular replication of R. rickettsii, while R. rickettsii replication was enhanced in the TRIM56-silenced host cells with the reduced phosphorylation of IRF3 and STING and the increased production of interferon-β. In addition, the mutation of the TRIM56 E3 ligase catalytic site impairs the inhibitory function against R. rickettsii in HeLa cells. Altogether, our study discovers that TRIM56 is a host restriction factor of R. rickettsii by regulating the cGAS-STING-mediated signaling pathway. This study gives new evidence for the role of TRIM56 in the innate immune response against intracellular bacterial infection and provides new therapeutic targets for RMSF. IMPORTANCE Given that Rickettsia rickettsii (R. rickettsii) is the most pathogenic member within the Rickettsia genus and serves as the causative agent of Rocky Mountain spotted fever, there is a growing need to explore host targets. In this study, we examined the impact of host TRIM56 on R. rickettsii infection in HeLa and THP-1 cells. We observed a significant upregulation of TRIM56 expression in R. rickettsii-infected cells. Remarkably, the overexpression of TRIM56 inhibited the intracellular replication of R. rickettsii, while silencing TRIM56 enhanced bacterial replication accompanied by reduced phosphorylation of IRF3 and STING, along with increased interferon-β production. Notably, the mutation of the TRIM56’s E3 ligase catalytic site did not impede R. rickettsii replication in HeLa cells. Collectively, our findings provide novel insights into the role of TRIM56 as a host restriction factor against R. rickettsii through the modulation of the cGAS-STING signaling pathway.

largely unknown, which brings barriers to the development of more effective prevention and control measures against RMSF.
Innate immunity is the first line of defense to fight against various pathogen infections.Type I interferons (IFN-I) constitute a critical component of innate immun ity and have a nearly universal anti-virus role (4) but have little restricted effect on facultative bacterial pathogens (5).The effect of IFN-I on obligate cytosolic bacterial pathogens, including Rickettsia species, has been reported in a few studies.Studies have reported that the host cells, such as human THP-1 macrophages, murine bone marrow-derived macrophages (BMDMs), and human microvascular endothelial cells (HMECs), secrete IFN-I during infection with Rickettsia sp.(6)(7)(8)(9).Human THP-1 macro phages infected with a mildly pathogenic agent such as Rickettsia parkeri, Rickettsia africae, or Rickettsia massiliae produce different levels of interferon beta (IFN-β) to trigger different proteome signatures and differentially impact critical components of innate immune responses to interfere the replication of the SFG rickettsial agent (8).Also, R. parkeri-infected BMDMs produce IFN-β mediated by the DNA sensor cGAS, inducing the expression of interferon regulatory factor IRF5, which upregulates the expression of the genes encoding guanylate binding proteins and inducible nitric oxide synthase to inhibit the replication of R. parkeri (9).Moreover, under infection with Rickettsia conorii, a highly pathogenic agent, HMECs secrete IFN-β to activate signal transducer and activator of transcription protein 1 by phosphorylation in an autocrine/paracrine manner, promoting the expression of transcription factors interferon regulatory factor 7 (IRF7) and IRF9 and inhibiting the expression of suppressor of cytokine signaling protein SOCS1 and UBP43, which finally lead to the significant suppression of the intracellular replication of R. conorii (6,7).However, the effect of IFN-I on the host cells infected with R. rickettsii, a highly pathogenic agent, has not been well known.
The host TRIM56 protein is reported to contribute to the IFN-I signal pathway and is a restriction factor of several RNA viruses (influenza virus, yellow fever virus, dengue virus, and bovine viral diarrhea virus) (10)(11)(12) and DNA viruses (Newcastle disease virus and herpes simplex virus) (13,14) both in an E3 ligase-dependent and -independent manner.Furthermore, Salmonella typhimurium SopA HECT-type E3 ligase targets TRIM56 to stimulate RIG-I and MDA5 innate immune receptors, which subsequently modulates inflammatory responses (15).However, the role of TRIM56 in anti-bacterial infection remains vastly unexplored.
Here, we investigated the role of TRIM56 in the host innate immune response against R. rickettsii infection.Our results demonstrated that TRIM56 suppressed the intracellular replication of R. rickettsii in HeLa and THP-1 cells by inducing the production of IFN-β.This anti-microbial effect mediated by TRIM56 is dependent on the cysteine residues of the RING domain.Our findings not only gain insights into the mechanism of host innate immune response against R. rickettsii but also provide the potential therapeutic targets for RMSF.

R. rickettsii infection upregulates the expression of TRIM56 in host cells
To explore the potential role of TRIM56, we first examined whether R. rickettsii infection alters TRIM56 expression in HeLa and THP-1 cells.HeLa and THP-1 were infected with R. rickettsii, respectively; the protein samples of HeLa and THP-1 cells were collected for analyses at 0, 1, 2, and 4 days post-infection (dpi).The TRIM56 mRNA level (Fig. S1) and protein levels were significantly increased following R. rickettsii infection in quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting analysis in HeLa cells (Fig. 1A and B; Fig. S2A) and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells (Fig. 1C and D; Fig. S2B) compared to uninfected control.Cell viability assessment revealed that around 30% of HeLa cells (Fig. 1E) and THP-1 cells (Fig. 1F) underwent cell death on the 4 dpi using the Cell Counting Kit-8 (CCK-8) assay, whereas cell viability remained above 90% during the initial 3 days.These results demonstrated that TRIM56 exhibited upregulation upon R. rickettsii infection in HeLa and THP-1 cells

The alteration of TRIM56 expression level affects the intracellular replication of R. rickettsii
To investigate the impact of TRIM56 on R. rickettsii replication, HeLa cells were trans fected with the small interfering RNA (siRNA) of TRIM56 (siTRIM56) and overexpres sion plasmid pcDNA3.1-hTRIM56-HA(pcTRIM56), while the nonsense siRNA (siNC) and plasmid vector pcDNA3.1-HA(vector) were set as controls.After R. rickettsii infection, total DNA was extracted, and the genomic equivalent (GE) of R. rickettsii DNA was quantitated using quantitative polymerase chain reaction (qPCR).The transfection of siTRIM56 led to a reduction in TRIM56 expression and an increased intracellular burden of R. rickettsii at 4 dpi (Fig. 2A; Fig. S3A).Conversely, the transfection of pcTRIM56 resulted in an enhanced TRIM56 expression and decreased bacterial loads at 4 dpi (Fig. 2B; Fig. S3B).To gain further insight into the anti-R.rickettsii role of TRIM56, the trim56 gene was knocked out in HeLa and THP-1 cells.In order to understand the course of R. rickettsii infection regulated by TRIM56, wild-type (WT) cells, trim56 −/− cells, and TRIM56 overexpression cells were infected with R. rickettsii.The infected cells were collected at 0, 1, 2, 3, and 4 dpi for analyses of bacterial burden.Notably, trim56 −/− HeLa and THP-1 cells exhibited a complete lack of TRIM56 expression validated by Western blotting, accompanied by sustained high rickettsial load levels (Fig. 2C through F; Fig. S3C and  D).TRIM56 overexpression cells displayed a sustained low level of rickettsial load (Fig. rickettsii using qPCR, while protein lysates from the cells were subjected to Western blotting analysis to assess TRIM56 protein expression, and densitometry was (Continued on next page) 2G), highlighting the positive role of TRIM56 in inhibiting intracellular replication of R. rickettsii.Furthermore, to elucidate the function of the RING domain characterized by E3 ligase activity of TRIM56, the TRIM56 point mutation plasmids containing cysteine residues at 21 and 24 to N-terminal of the RING domain (C21A and C24A) were transfec ted to trim56 −/− HeLa cells, but these mutations failed to suppress the replication of R. rickettsii in trim56 −/− HeLa cells at 4 dpi (Fig. 2H), suggesting that the inhibitory effect of TRIM56 on R. rickettsii replication was involved in 21 and 24 (C21 and C24) of the E3 catalytic site within the RING domain.Collectively, these findings provided evidence that TRIM56 exerted an inhibitory effect on R. rickettsii intracellular replication in a manner of C21 and C24 E3 catalytic site-dependent way.

TRIM56 deficiency inhibits IFN-β transcription and secretion after R. rickettsii infection
Previous studies displayed that IFN-β caused a robust, dose-dependent restriction of R. parkeri growth in mouse bone marrow-derived macrophages (9).Studies also have shown that TRIM56 is involved in innate anti-viral immunity response, including the initiation of IFN response (16).To investigate the impact of TRIM56 on R. rickettsii-induced IFN-β response, WT and trim56 −/− HeLa cells, as well as THP-1 cells, were infected with R. rickettsii, and mRNA was collected to analyze the transcriptional level of IFN-β.As depicted in Fig. S4, the mRNA levels of IFN-β significantly increased following infection, with the WT cells exhibiting a significantly higher induction compared to trim56 −/− cells.
To further analyze the role of TRIM56 in the regulation of the IFN-I pathway during R. rickettsii infection, WT and trim56 −/− HeLa cells were transfected with pGL3.0-IFN-β-lucplasmid with pcTRIM56, pcTRIM56 (C21A), pcTRIM56 (C24A), or vector, followed by R. rickettsii infection.As shown in Fig. 3A, the IFN-β promoter activity was decreased in trim56 −/− HeLa cells compared with the WT HeLa cells after transfection.In previous results, we found that the overexpression of TRIM56 inhibited the replication of R. rickettsii in HeLa cells.The transfection of the TRIM56 full-length plasmid group induced higher IFN-β expression levels, whereas the cells transfected with TRIM56 E3 ligase catalytic site mutation plasmids showed no significant changes in IFN-β expression.Therefore, we speculated that TRIM56 was able to affect the secretion of IFN-β after R. rickettsii infection.To verify whether TRIM56 deficiency affected IFN-β secretion, the supernatant of PMA-differentiated WT and trim56 −/− THP-1 cells infected by R. rickettsii was detected by the enzyme-linked immunosorbent assay (ELISA) at the 0, 0.5, 1, 2, and 3 dpi (Fig. 3B).As a result, WT THP-1 cells significantly increased IFN-β secretion, while trim56 −/− THP-1 cells did not during R. rickettsii infection.Meanwhile, TNF-α expression was not affected by trim56 deletion (Fig. 3C).Next, to analyze the role of IFN-β during R. rickettsii infection, we treated the PMA-differentiated WT and trim56 −/− THP-1 cells with IFN-β (200 ng/mL) for 48 hours.Compared to the untreated groups, R. rickettsii replication was significantly decreased in IFN-β-treated WT and trim56 −/− THP-1 cells (Fig. 3D).Collectively, the above results suggested that TRIM56 deficiency specificity inhibited the transcription and secretion of IFN-β, thereby promoting the intracellular replication of R. rickettsii.presented as the fold change between the ratios of TRIM56/GAPDH (n = 3, mean ± SD). (C-F) trim56 −/− THP-1 cells (C, D) and trim56 −/− HeLa cells (E, F) were infected with R. rickettsii at an MOI of 1 for 0, 1, 2, 3, and 4 days.The cells were washed and collected for DNA extraction to quantify the GE of R. rickettsii using qPCR, while protein lysates from the cells were subjected to Western blotting analysis to assess TRIM56 protein expression, and densitometry was presented as the fold change between the ratios of TRIM56/GAPDH (n = 3, mean ± SD). (G) HeLa cells were transfected with vector and pcTRIM56, followed by R. rickettsii infection at an MOI of 1 for 0, 1, 2, 3, and 4 days.The cells were washed and collected for DNA extraction to quantify the GE of R. rickettsii using qPCR (n = 3, mean ± SD). (H) trim56 −/− HeLa cells were transfected with pcTRIM56 (WT), pcTRIM56 (C21A), pcTRIM56 (C24A), and vector, followed by R. rickettsii infection at an MOI of 1 for 4 days.The cells were washed and collected for DNA extraction to quantify the GE of R. rickettsii using qPCR (n = 3, mean ± SD).Data presented were representative of at least three independent experiments.ns P ≥ 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

TRIM56 positively regulates the cGAS-STING signaling pathway through ubiquitination of STING during R. rickettsii infection
TRIM56 has been previously shown to directly modulate STING activity to induce type I IFN production in response to double-stranded DNA stimulation (13).To examine the activation of cGAS-STING signaling, PMA-differentiated WT and trim56 −/− THP-1 cells were infected with R. rickettsii for 0, 0.5, 2, 4, 6, and 12 hours, respectively, followed by Western blotting analysis.Compared with WT THP-1, the phosphorylation of IRF3 (p-IRF3) (Fig. 4A and B; Fig. S5A) and STING (p-STING) (Fig. 4C and D; Fig. S5B) in trim56 −/− THP-1 cells was significantly inhibited as evaluated by densitometry analysis.To further gain insights into how TRIM56 regulated the activation of STING during R. rickettsii infection, we investigated the potential of TRIM56-mediated ubiquitination of STING by co-immunoprecipitation (co-IP).The WT and trim56 −/− HeLa cells were transfected with STING-FLAG or Ub-HA, followed by R. rickettsii infection at an MOI of 1 for 2 days, and then, the cells were treated with MG132 and subjected to immunoprecipitation with an anti-FLAG antibody, followed by immunoblotting with an anti-HA antibody.We found that after knocking out TRIM56, the ubiquitination of STING was significantly ns P ≥ 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.Statistical analyses in (B-D) were performed using two-way analysis of variance (ANOVA) with multiple comparisons, compared to WT. reduced (Fig. 4E and F; Fig. S5C).These results demonstrated that TRIM56 E3 ligase positively regulated the cGAS-STING signaling pathway through the ubiquitination of the STING, finally leading to the influence production of IFN-β to suppress the intracellular replication of R. rickettsii.

DISCUSSION
RMSF, a severe life-threatening tick-borne zoonotic disease with high fatality rates in people, is caused by R. rickettsii, the most pathogenic species of genus Rickettsia.If untreated, it can quickly progress into a life-threatening illness in people, with high fatality rates (17)(18)(19).Therefore, the search for effective anti-bacterial effectors is of great significance for the prevention and treatment of R. rickettsii infection.The activation of IFN-β signaling constitutes an important component of host defense mechanisms, yet the status of this pathway during Rickettsia infection of macrophage cells, the were infected by R. rickettsii, and then, the infected cells were collected at 0, 0.5, 2, 4, 6, and 12 hours post-infection for Western blotting analysis, followed by probing with anti-p-IRF3, anti-IRF3, and anti-GAPDH, respectively.(B) Densitometry of (A) was presented as a fold change between the ratios of p-IRF3/IRF3 (n = 3, mean ± SD). (C-D) PMA-differentiated WT and trim56 -/− THP-1 cells were infected by R. rickettsii, and then, the infected cells were collected for Western blotting analysis, followed by probing with anti-p-STING, anti-STING, and GAPDH, respectively (C).Densitometry of (C) was presented as a fold change between the ratios of p-STING/STING (n = 3, mean ± SD) (D).(E-F) WT and trim56 -/-HeLa cells transfected with STING-FLAG and HA-Ub plasmids were infected with R. rickettsii at an MOI of 1 for 2 days, and then, the protein complexes were extracted from the cell lysates with anti-FLAG M2 beads.The immunoprecipitation with FLAG lysates (E) and whole-cell lysates (F) was probed with FLAG and HA antibodies.Data presented were representative of at least three independent experiments.ns P ≥ 0.05; ** P < 0.01; **** P < 0.0001.immune cells preferably targeted by pathogenic rickettsiae during human spotted fever syndromes, has so far remained an insufficient area of scientific inquiry (20,21).Here, we report the TRIM56-mediated down-regulation of the intracellular replication of R. rickettsii with upregulation of IFN-β secretion by modulating cGAS-STING signaling pathways in HeLa and THP-1 cells, suggesting that TRIM56 also participated in the immune response against intracellular bacteria such as R. rickettsii.This is in agreement with published evidence documenting the production of endogenous IFN-β by cultured human THP-1 macrophages, BMDMs, and HMECs in response to infection with mildly pathogenic typhus group species (6)(7)(8)(9).Previous studies have also shown that Rickettsia species have undergone extensive genome reduction and are dependent on host processes for replication.IFN-β alters the cytosol to an uninhabitable environment for obligate microorganisms; this may occur through a combination of IFN-stimulated genes and alterations to metabolism (9).The present study unequivocally demonstrates that spotted fever group species R. rickettsii infection stimulates the expression and secretion of IFN-β in THP-1 cells compared to trim56 −/− cells, indicating that R. rickettsii-induced IFN-β expression likely occurs via TRIM56-medi ated IFN-β secretion.
Upon infection, the presence of intracellular Rickettsiae is sensed by an as-yet unknown host surveillance system leading to the initiation of an IFN-β expression response; several intracellular signaling pathways are known to induce IFN-β expression during microbial infections (22)(23)(24).TLRs sense microbial DNA and RNA to activate transcription factors IRF3, resulting in the expression of IFN-β.Furthermore, RIG-1 and MDA5 in the cytosol detect double-stranded RNA from RNA viruses, triggering the induction of IFN-β.Additionally, the cGAS-STING signaling axis detects pathogenic DNA to trigger an innate immune reaction involving a strong IFN-I response against microbial infections.The previous study demonstrates that the typhus group species R. parkeriinduced IFN-I production of macrophages depends on cGAS, but the key host molecules mediating the signaling axis of the Rickettsia-induced cGAS-STING pathway remain to be elucidated (9).In the cGAS-STING signaling axis, IRF3 phosphorylation is essential for IFN-β production to initiate immune responses (25)(26)(27)(28).Previous studies have shown that the induction of IFN-β in HUVECs infected with Chlamydia pneumoniae is reliant on IRF3 (29).In this study, the expression of the phosphorylation of IRF3 was significantly increased in THP-1 cells compared to trim56 −/− THP-1 cells upon R. rickettsii infection, suggesting that TRIM56 may facilitate the initiation of IRF3 phosphorylation in response to R. rickettsii infection.
The ubiquitination of STING, an important upstream regulator of IRF3 phosphoryla tion, is crucial on the cGAS-STING signaling axis (14,(30)(31)(32).According to reported studies, TRIM56 is capable of the ubiquitination of STING under the stimulation of virus infection (13,33,34).However, whether TRIM56 participates in the ubiquitination of STING under obligate intracellular bacterial infection is unclear.It has been reported that the facultative anaerobe Salmonella typhimurium effector SopA targets TRIM56 to inhibit its E3 ligase activity by occluding the E2-interacting surface, culminating in the suppression of RING ubiquitination to facilitate intracellular amplification of bacteria (15,35).In this study, the ubiquitination of STING was reduced in trim56 −/− cells followed by R. rickettsii infection, demonstrating that the ubiquitination of STING in response to obligate intracellular bacteria R. rickettsii infection was in a TRIM56-dependent way.
Our study adds TRIM56 to the list of anti-pathogen TRIMs and provides novel insights for a better understanding of the anti-pathogen mechanisms of TRIM56, specifically against Rickettsia.Here, we also demonstrated that the depleting TRIM56 expression significantly reduced IFN-β mRNA expression during R. rickettsii infection, suggesting that TRIM56 might be a potential therapeutic target for infection with R. rickettsii.However, this study was limited to in vitro cell culture assays, and the in vivo role of TRIM56 remains unclear.In the future, animal-based experiments will be conducted to explore the in vivo role of TRIM56.
R. rickettsii was propagated in Vero cells for the preparation of stocks.R. rickettsii (Sheila Smith strain) was grown in Vero cells and isolated by isopycnic density gradient centrifugation in the BSL-3 laboratory as previously described (17,36).Briefly, confluent monolayers of Vero cells grown in DMEM supplemented with 2% fetal bovine serum and 2 mM L-glutamine were infected with R. rickettsii at an MOI of 1 and then incubated in a 33°C incubator set at 5% CO 2 until 50% of the monolayer was disrupted due to infection.The number of R. rickettsii organisms and viable rickettsial organisms in suspension was detected by qPCR (37) and plaque assay (38), respectively.

R. rickettsii purification
Four to five days post-infection, the culture supernatant of Vero cells infected with R. rickettsii was discarded and replaced with PBS (Gibco, United States, catalog no.C10010500BT) in a culture bottle.The cells were then scraped using a cell scraper (Corning, United States, catalog no.3010) and transferred into 50-mL centrifuge tubes, which were placed on ice.Subsequently, 8 mL of Vero cell suspension was aliquoted into 15-mL centrifuge tubes (Corning, United States, catalog no.430790) and kept on ice.The UP-250 ultrasonic cell mill probe (Xinzhi, Ningbo, China) was used with ultrasonic intensity set at 30%, oscillating for 2 seconds followed by a 2-second pause, repeating this cycle for a duration of 1 minute (a total of 12 tubes of 15 mL).Low-speed centrifuga tion was performed at 4°C and 3,000 rpm for 10 minutes using a HITACHI himac CR 21GII centrifuge.The supernatant was collected from three centrifuge tubes in the low-speed centrifugation and then was added into one 50-mL centrifuge tube for the high-speed centrifugation at 12,000 rpm for 10 minutes at 4°C.The pellet was then resuspended with surose-phosphate-glutamate (SPG) and stored at −80°C.

Plasmid and transfection
TRIM56 expression plasmid was constructed with routine molecular cloning techniques.The full-length human TRIM56 gene was synthesized by GeneScript (Nanjing, China) and cloned into pcDNA3.1-C-HAbetween the BamHI and XhoI restriction sites to create the mammalian expression construct pcDNA3.1-hTRIM56-HA.The point mutation plasmids of TRIM56 C21A and C24A were generated through homologous recombination.HeLa cells were seeded in a 12-well plate at a density of 1 × 10 5 cells per well with 1 mL of complete growth medium; then, the recombinant plasmid was transiently transfected with Lipofectamine 3000 (Invitrogen, United States, catalog no.100022052) according to the manufacturer's instructions.The transfected cells were infected with R. rickettsii at an MOI of 1 for 3 hours in a 5% CO 2 incubator at 33°C.Then, each cell well was washed three times with PBS and replaced with a fresh medium.Finally, the cells in wells were collected at different times post-infection, while whole DNA and protein samples were extracted from the collected cells for qPCR and Western blot analysis, respectively.

RNA interference experiment
TRIM56 small interfering RNA (siTRIM56: 5′-GCAGCAGAAUAGUGUGGUATT-3′) and ControlSiRNA (siNC: 5′-UUCUUCGAACGUGUCACGUTT-3′) were synthesized by Gene Pharma (Suzhou, China).HeLa cells were inoculated in 12-well plates at 1 × 10 5 cells per well in a 1 mL medium for 1 day.Then, the HeLa cells were transfected by siTRIM56 or siNC with RNAiMAX (Invitrogen, United States, catalog no.100022052) at a final concentration of 20 nM according to the manufacturer's instructions.The transfected cells were infected with R. rickettsii at an MOI of 1 for 3 hours in a 33°C 5% CO 2 incubator; then, each cell well was washed three times with PBS and replaced with a fresh medium.The infected cells were collected at different times post-infection, while DNA and protein samples were extracted from the infected cells for qPCR and Western blotting analysis.

Western blotting analysis
The cells were harvested and washed three times with PBS and then lysed in cell lysis buffer for Western and IP buffer (Beyotime, China, catalog no.P0013) with phenylme thanesulfonyl fluoride (PMSF, Beyotime, China, catalog no.ST506-2).The mixture was centrifuged at 12,000 g for 10 minutes, and the supernatant was collected.Samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, United States, catalog no.IPVH00010).After blocking with 5% skim milk, the membranes were incubated with the indicated primary antibodies, followed by appropriate secondary antibodies.Blots were developed with an enhanced chemiluminescence kit (Gene protein link technology, China, catalog no.P06M31X).The primary antibodies used are as follows: rabbit polyclonal antibodies against TRIM56 purchased from Abcam (Cambridge, United Kingdom, catalog no.10583), and rabbit polyclonal antibodies against IRF3 (catalog no.4302S), p-IRF3 (catalog no.4947S), STING (catalog no.13647S), p-STING (catalog no.50907S), and α-tubulin (catalog no.2148S) were purchased from Cell Signaling Technology (United States).Mouse polyclonal antibodies against GAPDH were purchased from Proteintech (United States, catalog no.60004-1-Ig); goat anti-rabbit (catalog no.P03S02S) and goat anti-mouse (catalog no.P03S01S) secondary antibodies were purchased from Gene protein link technology (China).

RNA extraction and real-time PCR
Total RNA was isolated from cells by the PureLink RNA kit (Invitrogen, United States, catalog no.1218302), and quantitative PCR reaction was monitored with the One-Step TB Green PrimeScript PLUS RT-PCR Kit (Takara, Japan, catalog no.RR096A).All the primers used in this study are listed in Table 1.

IFN-β luciferase reporter assay
The IFN-β luciferase reporter construct was purchased from Transvector (Chengdu, China).WT and trim56 -/-HeLa cells were seeded in a 12-well plate at a density of 1 × 10 5 cells per well with 1 mL of complete growth medium.The next day, cells were transfected with IFN-β-Luc reporter plasmid, vector, pcTRIM56, pcTRIM56 (C21A), or pcTRIM56 (C24A) at a final concentration of 20 nM for 4 hours, followed by R. rickettsii infection at an MOI of 1 for 3 hours; then, the cells were lysed with the Dual-Luciferase Reporter Gene Assay Kit (Beyotime, China, catalog no.RG027) according to the manufacturer's instructions, and luminescence was measured using the SpectraMax i3x microplate reader (Molecular Devices, United States).

IFN-β treatment
WT and trim56 −/− THP-1 cells were seeded at a density of 1 × 10 5 cells per well in 24-well plates in RPMI 1640 with 10% FBS supplemented with 162 nM PMA overnight.The next day, the media were exchanged for media without PMA supplemented.The cells were infected with R. rickettsii at an MOI of 1 for 3 hours at 33°C in a 5% CO 2 incubator, then washed three times with PBS and replaced with a fresh medium containing 200 ng/mL IFN-β (MCE, catalog no.HY-P7024) for 48 hours.The cells were washed and collected for DNA extraction to quantify the GEs of R. rickettsii using qPCR.

Enzyme-linked immunosorbent assay
Culture supernatants of uninfected and Rickettsia-infected THP-1 macrophages at the respective time points post-infection were collected, and IFN-β and TNF-α levels were measured with the human IFN-β ELISA Kit (Solarbio, China, catalog no.SEKH-0410) and human TNF-α ELISA Kit (Solarbio, China, catalog no.SEKH-0047), following the manufac turer's instructions (8).Protein concentrations were determined based on the optical density at 450 nm using a BioTek Epoch2 microplate reader and compared to standard curves for purified IFN-β.

Immunoprecipitation
Immunoprecipitation was performed following established protocols, as described previously (39).HeLa cells were seeded in a 12-well plate at a density of 1 × 10 5 cells per well with 1 mL of complete growth medium, then co-transfected with pCMV-Flag-STING (Sino Biological, China, catalog no.HG29810-NF) and pCMV-HA-Ub (Ke Lei Biotechnol ogy, China, catalog no.kl-zl-0513) for 12 hours followed by R. rickettsii infection at an MOI of 1 for 2 days, and 100 nM MG132 were added to cells for 8 hours before cells were lysed in IP buffer (Beyotime, China, catalog no.P0013).After pre-clearing with protein A/G agarose beads for 1 hour at 4°C, whole-cell lysates were used for immunoprecipitation with the indicated antibodies.A 50% slurry of FLAGM2 beads (Sigma, United States, catalog no.M8823) was added to 1 mL of cell lysates and incubated at 4°C for 6 hours.Immunoprecipitates were extensively washed five times with lysis buffer and eluted with SDS loading buffer by boiling for 5 minutes.The following immunoblot steps were consistent with Western blotting analysis.

Statistical analysis
Statistical analyses and calculations were performed with GraphPad Prism 8 software.Student's t-tests or two-way ANOVA was used to determine the difference, and P-values less than 0.05 were considered statistically significant.

FIG 1 FIG 2
FIG 1 Upregulated expression of TRIM56 during R. rickettsii infection.HeLa cells were infected with R. rickettsii at an MOI of 1 or uninfected (A).Densitometry of HeLa cells was presented as fold change between the ratios of TRIM56/GAPDH (n = 3, mean ± SD) (B).PMA-differentiated THP-1 cells were infected with R. rickettsii at an multiplicity of infection (MOI) of 1 or uninfected (C).Densitometry of PMA-differentiated THP-1 cells was presented as a fold change between the ratios of TRIM56/GAPDH (n = 3, mean ± SD) (D).The cell viability of R. rickettsii-infected HeLa cells (E) and PMA-differentiated THP-1 cells (F) was measured by CCK-8.The infected cells were collected at 0, 1, 2, and 4 dpi to determine the protein levels of TRIM56 by Western blotting analysis, and GAPDH served as the loading control.The data presented were representative of at least three independent experiments.ns P ≥ 0.05; **** P < 0.0001.