Ehrlichia chaffeensis Arkansas strain was cultured in human acute leukemia THP-1 cell line in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% fetal bovine serum (FBS; Tianhang, Zhejiang, China) at 37°C in 5% CO₂ and 95% air, as described previously (Cheng et al., 2008). Escherichia coli strains DH5α and BL21 (DE3) for DNA cloning and protein expression were cultured at 37°C in Luria–Bertani (LB) broth supplemented with appropriate antibiotics (50 μg/ml of kanamycin, 100 μg/ml of ampicillin, or 34 μg/ml of chloramphenicol) when necessary (Cheng et al., 2011). Pseudomonas aeruginosa strains were cultured at 37°C in LB broth supplemented with 150 μg/ml of carbenicillin or 50 μg/ml of gentamicin when necessary (Weng et al., 2016).

The plasmids used in this study are listed in Supplementary Table S1. For DNA manipulation, standard protocols or manufacturer instructions of commercial products were followed. The primers used for gene cloning, EMSA, and qRT-PCR are listed in Supplementary Table S1.

Ehrlichia chaffeensis was isolated from infected THP-1 cells as described previously (Teymournejad and Rikihisa, 2020). Briefly, E. chaffeensis-infected THP-1 cells (~2 × 10⁷ cells, >95% infected) were harvested at 600 × g at room temperature for 5 min. The pellet was suspended in fresh culture medium and passed through a 23-gauge needle with a syringe on ice for 20 times to crush the host cell membrane. To remove unbroken cells and cell debris, the mixture was centrifuged at 1,000 × g at 4°C for 5 min. The supernatant was collected by additional centrifugation at 10,000 × g at 4°C for 10 min. The bacterial pellet was suspended in fresh culture medium for synchronous culture or in 0.3 M sucrose for PNA transfection.

The DNA fragments encoding full-length CtrA and Tr1 were cloned into pET-33b(+) for recombinant CtrA (rCtrA) and recombinant Tr1 (rTr1), pET-41a(+) for GST-rCtrA as described previously (Cheng et al., 2006). Escherichia coli BL21 (DE3) cells were transformed with plasmids to express the recombinant proteins. The strains were induced to express rCtrA with 1 mM isopropyl-thio-β-D-galactoside (IPTG; Solarbio, Beijing, China) at 37°C for 4 h, or to express rTr1 with 1 mM IPTG at 20°C for 5 h, or to express GST and GST-rCtrA with 0.1 mM IPTG at 37°C for 4 h. rCtrA and rTr1 were purified from E. coli inclusion bodies using 6 M urea, and GST and GST-rCtrA were purified from E. coli soluble fraction (Cheng et al., 2006). The purified proteins were dialyzed against stocking buffer [10 mM Tris-HCl (Genview, Beijing, China), pH 7.5, 1 mM dithiothreitol (DTT; Solarbio)] for further experiments.

Antibodies against E. chaffeensis CtrA and Tr1 were prepared as described previously (Cheng et al., 2006). Purified rCtrA and rTr1 were cut from the SDS-PAGE gel, then sent to ABclonal Biotechnology Co., Ltd. (Wuhan, China) for rabbit polyclonal antibody preparation. Purified rCtrA or rTr1 was injected into Japanese white rabbits four times every 2 weeks. Twelve days after the fourth injection, 20 ml of total blood was collected from the rabbits to prepare the antiserum.

Peptide nucleic acid transfection to knockdown CtrA in E. chaffeensis was performed as described previously (Yan et al., 2021). An antisense PNA oligomer targeting 31–46 bp following the start codon of ctrA (CtrA PNA) 3′-CGTACACGTTTCCGTC-5′ and a control PNA (CTL PNA) 3′-CACATATCTCGG-5′ were synthesized by KareBay™ Biochem, Inc. (Ningbo, China). Three micrograms of CtrA PNA or CTL PNA dissolved in nuclease-free water was mixed with 100 μl of host cell-free E. chaffeensis in 0.3 M sucrose, then incubated on ice for 15 min. Electroporation was conducted at 2,000 V, 25 μF, and 400 Ω with a 10-ms pulse using a Gene Pulser Xcell™ electroporation system (Bio-Rad, Hercules, CA, United States) in a 2-mm electroporation cuvette (Bio-Rad). Then the PNA-transfected E. chaffeensis was transferred to a T25 flask to infect 5 × 10⁵ THP-1 cells and incubated at 37°C for 2 h with gentle shaking every 15 min to facilitate bacterial internalization. To detect the effect of CtrA PNA, infected cells were harvested at 36 h p.i. The expression level of ctrA and the protein level of CtrA were examined by qRT-PCR and Western blotting, respectively.

The susceptibility of P. aeruginosa to H₂O₂ was determined as described previously (Weng et al., 2016). Overnight-cultured P. aeruginosa strains were diluted in LB broth to an OD₆₀₀ of 0.03 and cultured at 37°C. At an OD₆₀₀ of 1.0, bacteria from 1 ml of culture were collected and washed three times with 1× PBS. The pellet was resuspended in 1× PBS and treated with H₂O₂ at a final concentration of 50 mM at 37°C for 15 min. The number of live bacteria was determined by gradient dilution and plate counting.

Ehrlichia chaffeensis was treated with H₂O₂ as described previously (Yen et al., 2020). THP-1 cells were synchronously infected with host cell-free E. chaffeensis, CTL PNA-, or CtrA PNA-transfected E. chaffeensis. At 36 h p.i., 20 μl of 10 mM H₂O₂ diluted in culture medium was added into 2 ml of the synchronous culture, reaching a final concentration of 100 μM. After 2 h of treatment at 37°C, the expression levels of gshA and gshB were determined with qRT-PCR.

Total RNA was extracted from each sample and reverse transcribed to cDNA as described previously (Duan et al., 2021). The amounts of E. chaffeensis 16S rRNA, ctrA, gshA, gshB, p28, and human GAPDH were determined with qRT-PCR using specific primers (Supplementary Table S1) and the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, MA, United States). The expression levels of the relative genes were normalized against that of E. chaffeensis 16S rRNA. Bacterial growth was determined as the amount of bacterial 16S rRNA normalized against that of human GAPDH mRNA.

Swimming and swarming motilities were performed as described previously (Zhang et al., 2019). Swimming media consisting of 0.3% agar was supplemented with 10 g/L of tryptone and 5 g/L of NaCl (pH 7.2). Swarming media consisting of 0.35% agar was supplemented with 62 nM K₃PO₄, 2 nM MgSO₄, 10 μM FeSO₄, 0.4% glucose, and 0.1% casein hydrolysate. Pseudomonas aeruginosa strains were cultured overnight at 37°C and then diluted to an OD₆₀₀ of 0.1. Two microliters of the diluted bacterial solution was center spotted onto the surface of the corresponding agar plates. The swimming plates were incubated at 30°C for 16 h, while the swarming plates were incubated at 37°C for 14 h. Bacterial motilities were imaged using a ChemiDoc™ XRS+ camera (Bio-Rad) and assessed by measuring the diameter of the widest point of spread on each plate. All experiments were repeated five times for data analysis.

Electrophoretic mobility shift assay was performed as described previously (Cheng et al., 2011). Briefly, the promoter regions of gshA (391 bp), gshB (460 bp), and p28 (260 bp) were amplified with PCR using the specific primers (Supplementary Table S1). The purified GST-rCtrA (2.5 μM) was incubated with 50 ng of DNA probes in a 20-μl reaction mixture containing 10 mM Tris-HCl (pH 7.5), 1 mM DTT, and 1% glycine (Solarbio) on ice for 30 min. Samples were loaded onto 8% native polyacrylamide gel in 1× TBE buffer, which had been prerun for 1 h at 100 V, and electrophoresed at 10 mA on ice for 1.5 h. The gel was stained in 1× TBE containing 0.5 μg/ml ethidium bromide at room temperature for 10 min. Bands were visualized using a molecular imager ChemiDoc™ XRS+ (Bio-Rad).

Enhanced green fluorescent protein fusions were constructed as described previously (Duan et al., 2021). Briefly, the promoter region of gshA, gshB, or p28 was amplified and inserted upstream of the promoter-less egfp gene in the pQE60 vector. Escherichia coli BL21 (DE3) strain containing pACYCDuet-1 harboring ctrA (pACYCDuet-1-rCtrA) or the empty pACYCDuet-1 vector was transformed with the pQE60–promoter–EGFP fusion constructs. After induction of rCtrA expression with 0.05 mM IPTG at 37°C for 3 h, bacterial samples were collected and subjected to Western blotting to measure the amounts of EGFP, RpoA, and rCtrA in each sample.

To detect the CtrA protein levels after PNA transfection, CTL PNA- or CtrA PNA-transfected E. chaffeensis-infected THP-1 cells were harvested by centrifugation at 500 × g for 5 min at 36 h p.i. The pellet was suspended in 1× PBS and immediately sonicated. The samples were then subjected to 12% SDS-PAGE, transferred to a PVDF membrane, and incubated with anti-CtrA or anti-Tr1 rabbit antiserum, respectively. After being washed, the membranes were incubated with secondary HRP-conjugated goat anti-rabbit IgG (Promega, WI, United States, W401B). The specific bands were detected with an Immobilon Western kit (Millipore, MA, United States) and a molecular imager ChemiDoc™ XRS+. The relative amount of CtrA or Tr1 in CtrA PNA-transfected E. chaffeensis was normalized against that in CTL PNA-transfected E. chaffeensis, respectively.

To detect the amount of EGFP in the reporter assay, an equivalent number of E. coli cells was collected. The protein levels of EGFP, RpoA, and rCtrA were determined using a mouse monoclonal anti-GFP antibody (GeneTex Inc., North America, GTX628528), mouse monoclonal anti-E. coli RNA polymerase α antibody (BioLegend, San Diego, CA, United States, 663104), or specific rabbit polyclonal anti-CtrA antiserum, respectively. The relative amounts of EGFP or RpoA in strains expressing rCtrA were normalized against those in strains containing pACYCDuet-1 vector.

All experiments were repeated at least three times. Statistical analyses were performed using GraphPad Prism 7.0. Statistical significance of a two-group comparison was assessed using Student’s t-test (two-tailed). A value of p < 0.05 was considered significant.

The production of ROS by host cells is one of the major mechanisms of host defense against bacterial infections (Ziltener et al., 2016). We first investigated whether GshA and GshB in E. chaffeensis are required for bacteria to respond to oxidative stress. At 36 h p.i., E. chaffeensis-infected THP-1 cells were treated with 100 μM H₂O₂ at 37°C for 2 h. We found that the expression of gshA and gshB was significantly upregulated after H₂O₂ treatment (Figure 1), suggesting that GshA and GshB might be involved in E. chaffeensis response to oxidative stress.

We then investigated the function of E. chaffeensis GshA and GshB. No E. chaffeensis deletion mutant of GshA or GshB is currently available, because classical bacteriology techniques, such as targeted mutagenesis, are not readily applicable for obligatory intracellular bacteria (McClure et al., 2017). To overcome this limitation, we used P. aeruginosa as a surrogate system to study the functions of E. chaffeensis GshA and GshB. In P. aeruginosa, GshA and GshB are critical for bacteria to resist oxidative stress (Van Laar et al., 2018; Zhang et al., 2019). The enzyme domain of E. chaffeensis GshA or GshB shows 55.0% or 64.7% identity to that of P. aeruginosa GshA or GshB, respectively (Supplementary Figure S1). We then expressed gshA or gshB from E. chaffeensis or P. aeruginosa in a gshA::Tn mutant or a gshB::Tn mutant of the P. aeruginosa reference strain PA14 (Liberati et al., 2006). The growth rates of the wild-type PA14, the GshA mutant, the GshB mutant, and the complemented mutants showed no difference (Supplementary Figures S2A,B). The bacteria were grown to the exponential phase and treated with 50 mM H₂O₂ at 37°C for 15 min. The survival rate of the gshA::Tn or the gshB::Tn mutant strain was significantly lower than that of the wild-type PA14 (Figures 2A,B), which was restored by the expression of gshA from E. chaffeensis or P. aeruginosa (Figure 2A), or by the expression of gshB from E. chaffeensis or P. aeruginosa (Figure 2B), respectively. These results indicate that E. chaffeensis GshA and GshB are functional and confer the ability of bacterial defense against oxidative stress, which aids E. chaffeensis in establishing infection in hosts.

Deletion of GshA or GshB affects P. aeruginosa swimming and swarming motilities due to the disruption in the redox status (Van Laar et al., 2018; Zhang et al., 2019). We then examined whether E. chaffeensis GshA or GshB can restore the swimming and swarming motilities of the P. aeruginosa gshA::Tn or gshB::Tn mutant, respectively. The swimming and swarming motilities of the gshA::Tn or the gshB::Tn mutant strain were significantly reduced compared with that of the wild-type PA14, which were restored by the expression of gshA from E. chaffeensis or P. aeruginosa (Figure 2C and Supplementary Figure S3A), or by the expression of gshB from E. chaffeensis or P. aeruginosa (Figure 2D and Supplementary Figure S3B), respectively. These results further confirm that E. chaffeensis GshA and GshB are functional.

As GshA and GshB are functional in E. chaffeensis, we next investigated the mechanisms by which their expression is regulated. Ehrlichia chaffeensis CtrA upregulates the expression of genes that confer physicochemical stress resistance to bacteria (Cheng et al., 2011). We found one 8-mer binding motif in the gshA promoter region (−217 to −210 calculated from the translational start site) and one 9-mer binding motif containing a 1-bp mismatch in the gshB promoter region (−296 to −281 calculated from the translational start site). CtrA is expressed at the late stage of E. chaffeensis intracellular growth (Cheng et al., 2011). Using quantitative RT-PCR (qRT-PCR), we examined the expression patterns of gshA and gshB in synchronously cultured E. chaffeensis in THP-1 cells. After normalization against bacterial 16S rRNA, the expression of gshA and gshB was also upregulated at the late stage of E. chaffeensis intracellular growth (Figure 3), indicating that the expression of gshA and gshB might be regulated by CtrA.

PNA is a DNA mimic that has been shown to bind single- and double-stranded DNA and RNA with high affinity and specificity (Pelc et al., 2015), and inhibits transcription from double-stranded DNA (Nielsen et al., 1991) and translation from RNA (Good and Nielsen, 1999). Therefore, we designed CtrA PNA that specifically binds near the translation start site of ctrA (Figure 4A). Transfection of host cell-free E. chaffeensis with CtrA PNA significantly reduced ctrA mRNA level at 36 h p.i. (Figure 4B). The CtrA PNA specificity was confirmed by the results that CtrA PNA transfection significantly reduced the CtrA protein level but had no effects on the protein level of another transcription regulator, Tr1 (Figure 4B). The transfection with CtrA PNA significantly reduced the expression of gshA and gshB in E. chaffeensis, while it had no effects on the expression of p28, a gene regulated by Tr1 (Figure 4C). These results indicate that CtrA is involved in the regulation of gshA and gshB expression.

To investigate whether CtrA directly regulates the expression of gshA and gshB, we performed EMSA using GST-rCtrA (Cheng et al., 2011). The purified GST-rCtrA showed a single band on the SDS-PAGE gel (Supplementary Figure S4). The DNA probes derived from the promoter regions of gshA and gshB were shifted upon the incubation with GST-rCtrA (Figure 5A). The binding specificity was confirmed by the results that no shifted band was detected when the DNA probes were incubated with purified GST protein or when a DNA probe derived from the p28 promoter region, which does not contain CtrA 8-mer or 9-mer binding motifs allowing 1-bp mismatch, was incubated with GST-rCtrA (Figure 5A). These results indicate that CtrA directly binds to the promoters of gshA and gshB in E. chaffeensis.

We then examined whether CtrA activates the expression of gshA and gshB. The promoter region of gshA, gshB, or p28 was inserted upstream to the promoter-less egfp gene in the pQE60 plasmid to generate gshA-EGFP, gshB-EGFP, or p28-EGFP fusion constructs. Escherichia coli BL21 (DE3) strain containing pACYCDuet-1 vector harboring E. chaffeensis ctrA gene (pACYCDuet-1-rCtrA) or pACYCDuet-1 vector only (negative control) was transformed with the EGFP fusion constructs, respectively. The rCtrA expression induced by IPTG resulted in a significantly higher expression of EGFP in bacteria harboring gshA-EGFP or gshB-EGFP compared with the vector control, while it had no effects in bacteria harboring p28-EGFP (Figure 5B and Supplementary Figure S5). These results indicate that CtrA activates the expression of gshA and gshB in E. chaffeensis.

We then investigated whether the upregulation of gshA and gshB upon oxidative stress is activated by CtrA. Host cell-free E. chaffeensis was transfected with CTL PNA or CtrA PNA, then used to infect THP-1 cells. At 36 h p.i. the infected THP-1 cells were treated with 100 μM H₂O₂ at 37°C for 2 h. H₂O₂ treatment significantly induced the expression of gshA and gshB in E. chaffeensis transfected with CTL PNA, while CtrA PNA transfection blocked the upregulation of gshA and gshB induced by H₂O₂ treatment (Figure 6). As negative controls, the p28 expression did not change under all conditions (Figure 6). These results indicate that CtrA activates the expression of gshA and gshB upon oxidative stress.