Coxiella burnetii Employs the Dot/Icm Type IV Secretion System to Modulate Host NF-κB/RelA Activation

Coxiella burnetii is the causative agent of Q fever and an obligate intracellular pathogen in nature that survives and grows in a parasitophorous vacuole (PV) within eukaryotic host cells. C. burnetii promotes intracellular survival by subverting apoptotic and pro-inflammatory signaling pathways that are typically regulated by nuclear transcription factor-κB (NF-κB). We and others have demonstrated that C. burnetii NMII proteins inhibit expression of pro-inflammatory cytokines and induce expression of anti-apoptotic genes during infection. Here, we demonstrate that C. burnetii promotes intracellular survival by modulating NF-κB subunit p65 (RelA) phosphorylation, and thus activation, in a Type Four B Secretion System (T4BSS)-dependent manner. Immunoblot analysis of RelA phosphorylated at serine-536 demonstrated that C. burnetii increases NF-κB activation via the canonical pathway. However, RelA phosphorylation levels were even higher in infected cells where bacterial protein or mRNA synthesis was inhibited. Importantly, we demonstrate that inhibition of RelA phosphorylation impairs PV formation and C. burnetii growth. We found that a T4BSS-defective mutant (CbΔdotA) elicited phosphorylated RelA levels similar to those of wild type C. burnetii infection treated with Chloramphenicol. Moreover, cells infected with CbΔdotA or wild type C. burnetii treated with Chloramphenicol showed similar levels of GFP-RelA nuclear localization, and significantly increased localization compared to wild type C. burnetii infection. These data indicate that without de novo protein synthesis and a functional T4BSS, C. burnetii is unable to modulate NF-κB activation, which is crucial for optimal intracellular growth.

Intracellular bacterial pathogens including C. burnetii manipulate eukaryotic cell functions by secreting bacterial proteins, or effectors, that interact with host cell factors to promote intracellular survival (Voth and Heinzen, 2009;Voth et al., , 2011Toman et al., 2012;van Schaik et al., 2013;Newton et al., 2014). C. burnetii produces a T4BSS, and bacterially-derived virulence determinants are delivered to the host cytosol via this machinery throughout infection (Voth and Heinzen, 2009;Chen et al., 2010;Toman et al., 2012;van Schaik et al., 2013). Employing bioinformatics, bacterial two-hybrid approaches, yeast screens, and genetic screens, approximately 120 putative C. burnetii T4BSS effectors have been identified Chen et al., 2010;van Schaik et al., 2013;Newton et al., 2014). However, it remains unknown whether the C. burnetii T4BSS modulates NF-κB signaling. Many immune responses seen in in vitro and in vivo C. burnetii studies have been attributed to LPS and intrinsic properties of the bacterium (Honstettre et al., 2004;Zamboni et al., 2004;Shannon et al., 2005a,b;Andoh et al., 2007;Zhang et al., 2007;Lu et al., 2008). These approaches have not addressed the possibility that C. burnetii actively modulates the NF-κB-mediated immune response at the cellular level using the T4BSS. We hypothesized that C. burnetii modulates host NF-κB signaling via the T4BSS to promote intracellular survival. In this study, we analyzed C. burnetii-mediated temporal modulation of NF-κB activation/signaling throughout the infectious cycle. We also identified the NF-κB signaling pathway that is activated/modulated during C. burnetii infection. In addition, we analyzed the effect of inhibiting NF-κB activation on C. burnetii growth and development. Finally, using a T4BSS-defective mutant and transient inhibition of bacterial protein synthesis, we investigated whether C. burnetii protein(s) activate NF-κB in a T4BSS-dependent manner.

MATERIALS AND METHODS
Growth of C. burnetii, Tissue Culture, and Infection C. burnetii Nine mile phase II strain stocks were cultivated in African green monkey kidney Vero cells (CCL-81; ATCC, Manassas, VA) and purified as previously described (Morgan et al., 2010). The C. burnetii T4BSS dotA mutant strain (Beare et al., 2012) and C. burnetii T4BSS dotA mutant complemented strain were generously provided by Dr. Bob Heinzen (NIAID Rocky Mountain Laboratories, Montana) and grown as previously described (Beare et al., 2012). Non-adherent human monocytic leukemia derived THP-1 cells (TIB-202; ATCC) were grown in 75-cm 2 tissue culture flasks in RPMI 1640 medium (Gibco, Carlsbad, CA) supplemented with 1 mM sodium pyruvate, and 10% fetal bovine serum (FBS) at 37 • C in 5% CO2 (Mahapatra et al., 2010). Hela 229 epithelial cells (CCL-1.2; ATCC) were grown in RPMI 1640 supplemented with 10% FBS and gentamicin (Invitrogen) at 37 • C in 5% CO 2 and 95% humidified air. Infections of THP-1 cells with C. burnetii NMII were initiated in 24-well tissue culture plates at a multiplicity of infection (MOI) of 25. Bacteria were added to 2 × 10 6 THP-1 cells per well and incubated at 37 • C for 4 h to allow close host cell-bacteria contact. Fresh media was then added to each well for a final concentration of 10 6 cells/ml. This time point represents T = 0 for infectious studies.

NF-κB Modulation Assay
To assay C. burnetii modulation of NF-κB activation, uninfected or C. burnetii-infected THP-1 cells were incubated for 72 hpi. Cells were then incubated in media with (+) or without (−) bacteriostatic levels (10 µg/ml) of Cm for an additional 24 h (Mahapatra et al., 2010). Total protein lysates were collected from cell pellets using laemmli sample buffer (Bio-Rad, Hercules, CA) containing protease and phosphatase inhibitor cocktails (Sigma). An NF-κB activation control was generated by treating uninfected THP-1 cells with 50 ng/ml of recombinant human TNF-α (R&D systems) for 8 h prior to total cellular protein collection . For temporal analysis, protein lysates from paired infected and uninfected THP-1 cells were collected at 0, 24, 48, 72, 96, and 120 hpi, with one experimental set transiently treated with 10 µg/ml of Cm for the final 24 h while the other set was mock-treated. Cell culture media was exchanged daily using centrifugation to harvest the cells and removal of the spent media followed by suspension of the cells in fresh media ± Cm. Infected and uninfected cells were handled identically and a minimum of three experiments (N = 3) were carried out for each time point and condition. Temporal analysis of infected and uninfected differentiated THP-1 cells treated with Rifampin to inhibit mRNA synthesis was performed by treating cells with phorbol 12-myristate 13-acetate (PMA; 200 nM; EMD Biosciences, San Diego, CA) overnight. Cells were infected with C. burnetii, and Rifampin (10 µg/ml) was added (+Rif) or not (−Rif) at the time of infection. Total protein lysates were collected at 2, 24, 48, 72, and 96 hpi for immunoblot analysis as described below.

RelA Inhibition
An inhibitory peptide (IP)-DRQIKIWFQNRRMKWKKNGLL SGDEDFSS (Novus Biologicals)-that competitively inhibits phosphorylation of RelA (S529/S536) and a custom synthesized control peptide (CP)-RMDRKWKQIFQNKIWRKSSDELLN DFGGD (Thermofisher scientific)-were used to determine if inhibition of NF-κB activation alters C. burnetii survival/growth in host cells. Initially, the IP's ability to suppress NF-κB activation in uninfected THP-1 cells was determined. Briefly, 150 µm IP or CP was added to 10 6 THP-1 cells and incubated at 37 • C, 5% CO 2 for 4 h. Recombinant human TNF-α (200 ng/ml; R&D systems) was then added to induce NF-κB activation. Controls included untreated THP-1 cells, TNF-α only treated cells, IP only treated cells, and CP only treated cells. After an additional 12 h incubation, cells were pelleted and total protein lysates collected. After confirmation via immunoblotting that the IP does inhibit NF-κB activation, it was used to determine the requirement of NF-κB activation for C. burnetii survival/growth. Briefly, 10 6 THP-1 cells treated with either 10 µg/ml of Cm (Sigma), 200 ng/ml of TNF-α (R&D systems), or IP (150 µm) were inoculated with purified C. burnetii NMII at an MOI of 25. Prior to inoculation, cells were treated with IP for 4 h or TNFα for 1 h. To ensure consistent cellular response, fresh TNF-α was added every 12 h and IP every 24 h, respectively. At 72 hpi, cells were harvested for (i) total protein lysates, (ii) IFA after cytospin as previously described (Mahapatra et al., 2010) to detect PV formation, or (iii) C. burnetii infectious unit enumeration by sonication lysis of infected cells, serial dilution, and infection of HeLa cell monolayers followed by Fluorescent Forming Unit (FFU) calculation as described (Coleman et al., 2004;Omsland et al., 2009). Briefly, IFA was used to visualize infected cells using a Nikon Eclipse TE 2000-S fluorescence microscope. Fifteen randomly chosen fields of view (FOV) visualized at 20x magnification were counted per well to determine the average number of PVs per FOV. For FFU/ml determination, the FOV average times the area FOV/well and inoculum volume (100 ul) were multiplied by the dilution factor to determine infectious particles from each sample treatment.

Immunoblot Blot Analysis
Cell lysates were separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane (Pierce, Rockford, IL). The membranes were blocked for 1 h at room temperature with 5% nonfat milk in Tris-buffered saline (150 mM NaCl, 100 mM Tris-HCl, pH 7.6) containing 0.1% Tween-20 (TBST) . Following blocking, membranes were incubated overnight at 4 • C with 5% nonfat milk in TBST having primary antibodies for the various target proteins (see following). Detection of NF-κB was performed using a rabbit monoclonal anti-human primary antibody specific to the phosphorylated Serine 536 form of RelA (Cell Signaling Technology, Danvers, MA). Activation of the non-canonical NF-κB pathway was assayed using rabbit anti-human polyclonal antibody against p100 (the precursor), and p52 (active form of NF-kappaB2, Cell Signaling Technology, Danvers, MA). Determination of C. burnetii density within infected THP-1 cell lysates was performed using rabbit polyclonal antibody against the C. burnetii 27-kDa outer membrane protein Com1 as previously described (Morgan et al., 2010). Mouse monoclonal antibodies directed against human β-actin (Sigma, Saint Louis, MO) were employed as a loading control. After primary antibody incubation, the membranes were washed 3X with TBST then incubated with either anti-rabbit or anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (KPL, Gaithersburg, MD) for 1 h at room temperature, washed 3X in TBST and detected by chemiluminescense following the manufacturer's directions (ECL SuperSignal West Pico Chemiluminescent Substrate, Pierce, Rockford, IL). Visualization and digital imaging of immunoblots was performed on a FluorChem HD2 Imaging System (Alpha Innotech Corporation, Leandro, CA).

Densitometry
The signal density of the detected bands in experimental samples were analyzed by ImageJ (version 1.46 h) as described previously (Schneider et al., 2012). Briefly, relative phosphorylated RelA band intensity was normalized to β-actin and quantified with respect to uninfected THP-1 cells ( Figure 1A) and C. burnetii infected THP-1 cells at 24 hpi (Figure 2A). To examine the role of non-canonical NF-κB signaling pathway, relative NF-κB p100 and NF-κB p52 band intensity was normalized to β-actin and quantified with respect to C. burnetii infected THP-1 cells at 24 hpi (Figure 3). In Figure 4D, relative C. burnetii Com1 band intensity was normalized to β-actin and quantified with respect to C. burnetii infected THP-1 cells at 72 hpi. For Figure 5A, relative phosphorylated RelA band intensity was normalized to β-actin and quantified with respect to uninfected THP-1 cells.

Immunofluorescent Microscopy
An indirect immunofluorescence assay (IFA) was used to enumerate PV and infectious C. burnetii. For immuno-staining, methanol-fixed C. burnetii-infected cells were incubated 1 h with polyclonal Guinea pig anti-C. burnetii primary sera diluted in PBS, 2% BSA. After 3 PBS washes, samples were incubated 1 h with Alexa488-conjugated goat anti-Guinea pig secondary IgG (Molecular probes, Thermo Fisher) also diluted in PBS, 2% BSA. DAPI (4' ,6-diamidino-2-phenylindole) was used in the secondary incubation to stain total DNA. Labeled cells were visualized using a Nikon Eclipse TE 2000-S fluorescence microscope with a Nikon DS FI1 camera and NIS-ELEMENTS F 3.00 software (Mahapatra et al., 2010). A minimum of 15 fields per sample were counted using the 20x objective. All experiments were performed with 3 biological samples and statistical analyses were performed using a paired Student's t-test.

Transfections and GFP-RelA/DAPI Co-Iocalization Assay
Hela cells were transiently transfected with the GFP-RelA plasmid (Addgene) using X-tremeGENE 9 DNA Transfection Reagent (Roche Life Sciences), as described by the manufacturer. Transfected cells were grown overnight and then inoculated with either C. burnetii NMII, C. burnetii ∆dotA (Beare et al., 2012), or a C. burnetii dotA-complemented strain (Beare et al., 2012). Separate cells were also infected with C. burnetii NMII concurrent with the addition of Cm (Mahapatra et al., 2010). C. burnetii ∆dotA strain and dotA-complemented strains were generously provided by Dr. Robert A. Heinzen (Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT) (Beare et al., 2012). All strains were used at an MOI of 25. At 24 and 48 hpi, cells were methanol-fixed and IFA performed as above using Guinea pig anti-C. burnetii primary antibody and Alexa555-conjugated goat anti-Guinea pig secondary IgG (Molecular probes, Thermo Fisher). DAPI was used to stain total DNA. Subcellular colocalization of GFP-RelA and DAPI in transfected cells was visualized as above using a 60x oil objective. A minimum of 100 transfected cells were scored per sample. Data shown are the means of three independent experiments and statistical analyses were performed using a paired Student's t-test.

NF-κB Activation is Modulated by C. burnetii Proteins during Infection
RelA is one of the most extensively studied NF-κB complex subunits (Sakurai et al., 2003;Bonizzi and Karin, 2004;Perkins, 2007). Similar to cRel and RelB, it contains a 300-amino acid region with homology to the Rel proto-oncogene (RH domain)  and a transactivation domain (Sakurai et al., 2003;Bonizzi and Karin, 2004;Perkins, 2007). The RH domain harbors motifs for nuclear localization and binding to specific DNA sequences, while the transactivation domain contains phosphorylation sites that remain bound to the inhibitor IκB while in the cytoplasm (Sakurai et al., 2003;Bonizzi and Karin, 2004;Perkins, 2007). Phosphorylation of serine 536 (S536) in the transactivation domain is required for optimal activation (Sakurai et al., 2003;Bonizzi and Karin, 2004;Viatour et al., 2005;Perkins, 2007). To determine if NF-κB is modulated by C. burnetii, NF-κB activation was assayed in THP-1 cells via detection of RelA phosphorylation during infection and treatment with Cm (Sakurai et al., 2003). Figures 1A,B reveal that NF-κB is activated during infection as evidenced by increased levels of phosphorylated RelA, and that C. burnetii protein synthesis is required to modulate the level of activation. Compared to uninfected cells, RelA phosphorylated protein levels increased ∼10-fold in C. burnetii-infected cells. However, phosphorylated RelA levels were ∼20-fold higher in infected cells treated with Cm. These results indicate that C. burnetii proteins modulate NF-κB signaling during infection.

C. burnetii Modulates NF-κB Activation Temporally during Infection
To assess the dynamics of NF-κB activation throughout infection, we examined infected THP-1 cells from 24-144 hpi. Cells were either mock-treated or transiently treated with Cm. We hypothesized that NF-κB activation would respond directly to de novo bacterial protein synthesis depending on the stage of infection (early [24-48 hpi], mid [48-96 hpi], or late [96-144 hpi]). To test this hypothesis, we first investigated the total levels of RelA. As shown in Figure 2A, RelA levels at each time post-infection were consistent throughout infection, indicating C. burnetii does not modulate the relative expression of RelA. We next assessed RelA phosphorylation in the presence and absence of transient Cm treatment. Figures 2A,B show that in the absence of transient Cm treatment, phosphorylated RelA levels remain low at 24 hpi (early infection). However, when compared to 24 hpi, RelA phosphorylation levels significantly increase at 48 hpi (early to mid-infection) and remain elevated through 96 hpi (mid infection). Notably, transient application of Cm at 0, 24, and 48 hpi resulted in even higher levels of phosphorylated RelA at 24, 48, and 72 hpi, respectively, suggesting that C. burnetii proteins dampen NF-κB activation during infection. Interestingly, application of Cm at 72 hpi did not alter RelA phosphorylation levels at 96 hpi relative to infected cells in the absence of Cm. During late infection times (96-144 h), RelA phosphorylation was reduced and de novo C. burnetii protein synthesis did not alter NF-κB activation. Combined, these results indicate that infection of THP-1 cells by C. burnetii involves temporal modulation of NF-κB activation via RelA phosphorylation. While C. burnetii infection triggers NF-κB activation, de novo C. burnetii proteins significantly suppress NF-κB activation during early and middle stages of Bottom panel-β-actin loading control. Sample treatments (±) are indicated above immunoblot panels and correlate to the lanes below. (B) Inhibition of RelA phosphorylation in THP-1 cells impairs C. burnetii PV formation. 15 fields of view at 20x magnification (>100 cells/field of view) from three independent samples were analyzed for PV enumeration. Error bars show ±S.E.M. Statistical differences relative to I−Cm were calculated using a t-test for paired samples (*signifies P < 0.05). (C) Infectious progeny enumeration in Hela cells from three independent samples as measured by IFA and Fluorescent Forming Units (FFUs). Error bars show ± SEM. Statistical differences relative to I−Cm were calculated using a t-test for paired samples (*signifies P < 0.05). (D) Representative immunoblot showing Com1 levels in C. burnetii-infected THP-1 cells either untreated or treated with Cm, TNF-α, or RelA IP and harvested at 72 hpi. Top panel-β-actin control. Bottom panel-Com1 detected with a rabbit polyclonal antibody. (E) Differences in C. burnetii Com1 levels relative to normalized β-actin from three independent samples were analyzed. All samples were analyzed at 72 hpi. C. burnetii growth was examined in THP-1 cells either untreated (I−Cm), or treated with Cm (I+Cm), TNF-α, or RelA IP. Error bars represent ± SEM. Statistically significant differences relative to I-Cm are represented as *P ≤ 0.05, (Student's t-test). intracellular growth. To confirm these findings, we examined the effect of treatment with the mRNA synthesis inhibitor rifampin on temporal NF-κB activation during infection. Additionally, to determine if these observations were infection model-specific, we assessed infection of PMA-differentiated THP-1 macrophagelike cells. Cells were treated with rifampin at the time of infection, and RelA phosphorylation assessed at 2, 24, 48, 72, and 96 hpi. Figure 2C shows that RelA phosphorylation levels in differentiated THP-1 cells infected with C. burnetii increased relative to uninfected cells. Furthermore, inhibition of bacterial mRNA synthesis resulted in even higher levels of RelA phosphorylation that were evident through 96 hpi similar to Figure 2A. These observations support our findings that indicate C. burnetii proteins temporally modulate NF-κB activation during infection.

C. burnetii Does Not Modulate NF-κB Activation via the Non-canonical Pathway
The results above demonstrate the involvement of RelA in NF-κB activation. NF-κB transcription factors are typically activated by either the canonical or non-canonical signaling pathway (Beinke and Ley, 2004;Bonizzi and Karin, 2004;Park et al., 2005;Viatour et al., 2005;Perkins, 2007). The canonical pathway transmits signals via RelA activation, while the non-canonical pathway functions through NF-κB p52 activation (Beinke and Ley, 2004;Bonizzi and Karin, 2004;Park et al., 2005;Viatour et al., 2005;Perkins, 2007). Formation of active p52 occurs via proteolytic processing of the p100 precursor (Beinke and Ley, 2004;Bonizzi and Karin, 2004;Park et al., 2005;Viatour et al., 2005;Perkins, 2007). Therefore, to determine if the non-canonical pathway is activated during C. burnetii infection of THP-1 cells, we used immunoblot analysis to detect NF-κB p100/p52. Figure 3 clearly demonstrates that C. burnetii does not modulate host cell NF-κB p100 and p52 levels over the course of infection (24-144 hpi) in the presence or absence of bacterial protein synthesis. When compared to C. burnetii-infected cells at 24 hpi, NF-κB p100 levels remain constant throughout infection, and addition of Cm does not significantly impact p100 expression ( Figure 3B). In contrast, p52 levels are barely detectable and do not significantly change in the presence or absence of Cm ( Figure 3C). Together, these data reveal that NF-κB activation in infected THP-1 cells does not involve non-canonical NF-κB signaling.

Inhibition of NF-κB Activation Impairs C. burnetii Development
To determine if C. burnetii-mediated NF-κB activation is essential for intracellular survival and growth, we treated THP-1 cells with an inhibitory peptide (IP) that competitively inhibits RelA (S529/S536) phosphorylation to suppress NF-κB activation. Inhibition of RelA phosphorylation was confirmed by immunoblotting using TNF-α to trigger RelA phosphorylation ( Figure 4A). Figure 4A demonstrates the effectiveness of TNFα to induce RelA phosphorylation and the IP's ability to inhibit RelA phosphorylation. To measure C. burnetii development following induction (TNFα) or inhibition (IP) of NF-κB activation, we analyzed (i), the number of treated THP-1 cells with PV relative to infected, untreated cells, (ii) the number of infectious progeny produced in Hela cells by treated THP-1 cell lysates relative to infected, untreated THP-1 cell lysates, and (iii) levels of the major outer membrane protein Com1 in treated and untreated THP-1 cells as a measure of C. burnetii total protein. Figures 4B-D show that PV formation, infectious progeny, and C. burnetii Com1 levels were reduced by approximately 20-30% in THP-1 cells pretreated with TNFα. Of particular interest, Figure 4B demonstrates that, compared to C. burnetii infected cells, PV are reduced by approximately 60% in IP-treated cells. Figures 4C-E confirm and support these observations, demonstrating that the number of infectious C. burnetii produced (Figure 4C) within IP-treated cells as well as the amount of Com1 detected in these cells (Figures 4D,E) was reduced by approximately 60%. Collectively, these results demonstrate that C. burnetii development is significantly impaired when NF-κB activation is inhibited. Additionally, unlike Cm treatment, C. burnetii is able to overcome high levels of activated NF-κB induced by TNF-α application. However, their growth is marginally inhibited.

C. burnetii Requires a Functional Dot/Icm T4BSS to Modulate NF-κB Signaling
Pathogenic microorganisms employ unique strategies to interfere with NF-κB signaling (Rahman and McFadden, 2011). Bacteria modulate NF-κB signaling (activation or suppression) depending on requirements of their intracellular lifestyle and niche (Rahman and McFadden, 2011). C. burnetii's close relative, L. pneumophila, utilizes over 10 Dot/Icm effectors to induce a biphasic pattern of NF-κB activation (Rahman and McFadden, 2011;Shin, 2012). To determine if the C. burnetii T4BSS plays a role in modulating NF-κB, we analyzed RelA phosphorylation in THP-1 cells infected with either wild type C. burnetii (± Cm), a C. burnetii T4BSS ∆dotA mutant, or a dotA-complemented strain. Figures 5A,B reveal that, compared to cells infected with wild type C. burnetii (I−Cm), levels of phosphorylated RelA were higher in cells infected with C. burnetii (I+Cm) or the ∆dotA mutant. However, levels of phosphorylated RelA were similar in cells infected with either wild type C. burnetii (−Cm) or the ∆dotA complemented strain. Together, these results suggest that C. burnetii protein synthesis and a functional T4BSS are required for NF-κB modulation. To confirm this observation and determine if this event extends to RelA nuclear translocation, we transiently transfected Hela cells with GFP-RelA and examined nuclear translocation when infecting cells with either wild type C. burnetii (± Cm), the ∆dotA mutant, or the ∆dotA-complemented strain. Migration of GFP-RelA into the nucleus indicates NF-κB activation. Using immunofluorescence microscopy we visualized RelA colocalization with DAPI (DNA stain). Figure S1 and   Figure 5C show that at 24 and 48 hpi, GFP-RelA localized to the nucleus regardless of the C. burnetii strain used to infect the cells. However, quantification of the amount of GFP-RelA in the nucleus of infected cells revealed that wild type C. burnetii (+Cm) and the ∆dotA mutant had approximately 50% more cells with nuclear-localized GFP-RelA ( Figure 5D). These data clearly demonstrate that NF-κB activation levels are significantly higher in infected cells treated with Cm and cells infected with the ∆dotA mutant, suggesting that T4BSS effectors play a crucial role in modulating NF-κB signaling.

DISCUSSION
Manipulation of host NF-κB signaling via secreted effector activity is a strategy used by many microbial pathogens to thwart innate and adaptive immune responses (Rahman and McFadden, 2011). NF-κB activation typically induces the expression of hundreds of genes (Beinke and Ley, 2004;Bonizzi and Karin, 2004;Park et al., 2005;Viatour et al., 2005;McFadden, 2006, 2011;Perkins, 2007). Genes targeted by NF-κB include those encoding pro-inflammatory cytokines, chemokines, and adhesion molecules that regulate recruitment and trafficking of immune cells to the site of infection (Beinke and Ley, 2004;Bonizzi and Karin, 2004;Park et al., 2005;Viatour et al., 2005;Perkins, 2007). NF-κB activation also induces the transcription of genes such as defensins that have direct microbicidal activity, and enzymes that generate reactive intermediates (Beinke and Ley, 2004). NF-κB acts as a major molecular link between the launch of innate and adaptive immunity by facilitating T cell activation via induction of MHC proteins and CD80/86 in antigen-presenting cells (Beinke and Ley, 2004). B cell differentiation is also usually stimulated by NF-κB activation (Beinke and Ley, 2004). Additionally, NF-κB plays a critical role in expression of anti-apoptotic proteins (e.g., c-IAP-1/2, AI/Bfl-1, Bcl-2, and Bcl-X L ) (Beinke and Ley, 2004;Rahman and McFadden, 2011). Regulation of the cell-cycle protein cyclin D1, which increases cellular survival and proliferation, is also dependent on NF-κB activation (Beinke and Ley, 2004;Rahman and McFadden, 2011). Thus, master regulators such as NF-κB are prime targets for pathogenic microorganisms that promote survival by "regulating the regulator" to meet the requirements of their intracellular life cycle. In the current study, we demonstrate that C. burnetii modulates host NF-κB during infection in a T4BSS-dependent manner.
We discovered that C. burnetii modulates NF-κB activation through a process that requires de novo bacterial protein and mRNA synthesis (Figures 1, 2). Our findings clearly indicate that C. burnetii promotes NF-κB activation via RelA phosphorylation in a temporal manner, and bacterial protein and mRNA synthesis inhibitors alter activation. These findings indicate that C. burnetii maintains a balance between activation and suppression of NF-κB signaling during infection. Depending on the stage of infection, bacteria typically either activate or suppress NF-κB signaling (Rahman and McFadden, 2011). Studies on C. burnetii's close phylogenetic relative reveal that L. pneumophila induces a biphasic pattern of NF-κB activation in human epithelial cells (Bartfeld et al., 2009;Rahman and McFadden, 2011;Shin, 2012). Short term activation at early time of infection (<8 hpi) is followed by decreased activation, which is then followed by long term induction of NF-κB later in infection (Bartfeld et al., 2009). Unlike L. pneumophila, our data suggest that C. burnetii activates NF-κB at a low level for at least the first 5 days of a ∼6 day infection cycle (Figure 2A). Importantly, activation is suppressed relative to Cm-and rifampin-treated infections, indicating the response of the host cell is robust NF-κB activation in the absence of ongoing C. burnetii macromolecular synthesis. Activation and suppression of NF-κB signaling during early to mid-infection likely promotes cell survival and prevents a robust immune response.
NF-κB can be activated by canonical and non-canonical pathways. Our findings indicate that C. burnetii does not modulate NF-κB via the non-canonical pathway (Figure 3). This finding is crucial to narrow C. burnetii modulation of NF-κB activation to canonical pathway molecular components upstream and downstream of RelA. Canonical NF-κB pathway-associated molecular components, such as host inducer ligands, receptors, adaptor molecules, IκB proteins, and kinases, may play a crucial role in C. burnetii-mediated activation or suppression of NF-κB signaling and are commonly targeted by intracellular bacterial pathogens (Rahman and McFadden, 2011). Examples include Shigella flexneri and Yersinia spp. that use the type III secretion system effectors OspG and YopP/J, respectively, to prevent IκB degradation, maintaining NF-κB inactive in the cytosol (Bhavsar et al., 2007). Conversely, activation of canonical NF-κB signaling protects several intracellular pathogens including Mycobacterium tuberculosis (Dhiman et al., 2007), Bartonella henselae (Kempf et al., 2005), Chlamydia pneumonia (Wahl et al., 2001), Rickettsia rickettsii (Clifton et al., 1998), and L. pneumophila (Abu-Zant et al., 2007) from cell death. Our results clearly indicate that inhibition of RelA phosphorylation restricts PV formation and reduces infectious progeny production (Figure 4). It is tempting to speculate that in the absence of some level of NF-κB activation, THP-1 cells prevent C. burnetii development by promoting pro-apoptotic mechanisms. It is likely that NF-κB-mediated anti-apoptotic genes such as c-iap2 and a1/bfl-1, which are up-regulated in C. burnetii-infected cells , would not be expressed when RelA phosphorylation is abrogated. Together, these findings suggest that two opposing effects of NF-κB activation occur in C. burnetii-infected cells: (1) some level of NF-κB activation is required to suppress pro-apoptotic pathways, which is beneficial for the pathogen, while (2) robust NF-κB activation would induce the expression of pro-inflammatory cytokines, ultimately leading to pathogen clearance from the host.
Finally, to identify C. burnetii factors that modulate NF-κB signaling during infection, we investigated whether the C. burnetii T4BSS was involved. In a series of experiments (Figures 5A-D and Figures S1A,B) we demonstrate that bacteria lacking a functional T4BSS do not modulate NF-κB activation during infection. This suggests that C. burnetii uses T4BSS effectors to either directly or indirectly modulate host NF-κB activity. Multiple studies indicate that C. burnetii T4BSS effectors include proteins with eukaryotic-like domains, including ankyrin repeat domains, tetratricopeptide repeats, coiled-coil domains, leucine-rich repeats, GTPase domains, ubiquitination-related motifs, and multiple kinases and phosphatases (Pan et al., 2008;Chen et al., 2010;Toman et al., 2012;van Schaik et al., 2013). Importantly, a significant number of C. burnetii T4BSS effectors are translocated into the host cell cytoplasm when expressed in a surrogate L. pneumophila system (Pan et al., 2008;Chen et al., 2010;Toman et al., 2012;van Schaik et al., 2013). Distinct T4BSS effectors associate with the PV membrane, microtubules, mitochondria, and the nucleus (Pan et al., 2008;Chen et al., 2010;Toman et al., 2012;Larson et al., 2013Larson et al., , 2015Weber et al., 2013;van Schaik et al., 2013;Weber et al., 2016). However, the specific function of the vast majority of confirmed and putative C. burnetii T4BSS effectors remains unknown. Interestingly, C. burnetii's close phylogenetic relative, L. pneumophila, regulates host NF-κB activation using more than 10 Dot/Icm-dependent effectors (Rahman and McFadden, 2011;Shin, 2012). As NF-κB is one of the principal regulators of the host immune response, identifying and characterizing C. burnetii T4BSS effectors that modulate this pathway and thoroughly characterizing the mechanism of NF-κB modulation will significantly contribute to our understanding of C. burnetii pathogenic mechanisms.