B. anthracis vaccine Pasteur II strain was kindly provided by the Institute of Lanzhou Biological Products in China.

Overnight cultures of strains tested were incubated in LB medium and grown to mid-log phase (OD₆₀₀ = 0.6) at 37°C with shaking. The cells were harvested by centrifugation, and total RNA was purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. The primers used for each target are listed in Table 1. cDNA was synthesized from 1 μg RNA using Superscript III reverse transcriptase (Invitrogen) and random hexamers following the manufacturer's protocol. The cDNA was diluted 5-fold and was amplified by qPCR. The gene expression levels as indicated by CT values were compared among the strains tested. 16S rRNA was used as a reference gene for normalization. The qPCR was repeated three times.

Strains were cultured overnight, and samples of the supernatant were collected and filtered through 0.22 μm-pore-size syringe filters. An aliquot of 5 ml of the filtered supernatant was mixed with an equal volume of 95% ethanol and incubated at 4°C overnight, followed by centrifugation. The precipitate was dried at 22–25°C and resuspended in 50 μl PBS. Following the addition of the loading buffer, the sample was heated to 100°C for 5 min. Twenty microliter of each sample (total protein content 5 μg) was loaded onto a 12% SDS-PAGE gel. The samples were then transferred onto a nitrocellulose membrane (Bio-Rad). The membranes were blocked for 1 h with TBS-T (20 mm Tris base, 137 mm NaCl, 0.1% Tween20) containing 5% dry milk at room temperature. The membranes were then incubated with either an anti-protective antigen (PA) antibody or anti-LF antibody (Thermo Scientific), and then diluted 1:2000 in TBS-T-5% milk overnight at 4°C. Membranes were washed in TBS-T and incubated with HRP-conjugated goat anti-mouse IgG secondary antibody diluted 1:4,000 in TSB-T-5% milk for 1 h at room temperature. After the same washing procedure, proteins were detected using the ECL Western Blot Substrate (Pierce) according to the manufacturer's instructions.

To explore whether protective antigen (PA) is able to directly regulate the transcription of acpA gene, we performed a gel shift assay. A DNA fragment of the upstream region of acpA was amplified by PCR using biotin-labeled primers: acpApromo_F and acpApromo_R (Table 1). The amplified DNA fragments were purified using the QIAquick PCR Purification kit (Qiagen) according to the manufacturer's instructions. DNA binding and electrophoresis were performed as described (Yang et al., 2015). Briefly, commercial-grade protective antigen protein (Listbiological Laboratories, Inc.) was added to a 20 μl mixture containing 0.2 pmol biotin-labeled DNA, 1 mg of poly(dI-dC), 25 mM NaH₂PO₄ (pH 8.0), 50 mM NaCl, 2 mM MgCl₂, 1 mM dithiothreitol (DTT), 10% glycerol, and 0.1 mM EDTA. The concentrations of PA protein used were 0.4, 1, 2, 4, and 8 μM (corresponding to 0.2, 0.5, 1, 2, and 4 μg, respectively). Unlabeled DNA fragments of the promoter region were added into the reaction at a 120-fold concentration over the labeled probe to inhibit non-specific binding. Bovine serum albumin (BSA) was added to prevent non-specific protein binding. The binding reaction was initiated by the addition of PA, and the reaction mixture was incubated at room temperature for 25 min. Samples were then loaded onto a 6% native polyacrylamide gel (acrylamide-bisacrylamide [29:1] in 0.5 × Tris-borate-EDTA [TBE] buffer). Electrophoresis was performed for 3 h at 4°C with 7 V cm⁻¹, and the gels were electroblotted onto nylon membranes in 0.5 × TBE at 300 mA for 90 min at 4°C. After cross-linking the DNA-protein hybrids with UV radiation, the membranes were hybridized with stabilized streptavidin-HRP conjugate (Thermo Scientific) followed by incubation with DAB substrate at room temperature for 20–30 min to optimize signal intensity.

The B. anthracis isolates were incubated in a LB medium with 0.9% NaHCO₃ under 5% CO₂ for 8 h. The bacterial cells were stained with India ink and were observed under a microscope. The Sterne strain without capsule formation was used as negative control. The bacterial capsule is visible in India ink because it excludes the ink particles. Each experiment was repeated at least three times.

Six to eight week old BALB/C male mice (18–20 g) were used for the assessment of pathogenicity of the Pasteur II pag knockout mutant. Briefly, mice were randomly divided into two groups (n = 6) and injected with 10⁴ CFU ml⁻¹ Pasteur II and PasteurII-pagKO, respectively, via subcutaneous injection to the inner thigh. The mice were observed continuously for 60 min, and afterwards once every 12 h. All experiments involving animals were performed in accordance with the protocols approved by the Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, China. Significance in animal survival was determined by Fisher exact test.

The expression levels of the capsule genes on pXO2 affect the expression of toxin genes, including cya, lef, and pag on pXO1 (Barkus and Leppla, 1989; Koehler et al., 1994; Sirard et al., 1994), indicating the interplay between the two plasmids. During our spontaneous mutagenesis studies, we found that the pag gene on pXO1 might affect the expression of the capsule genes on pXO2. To determine the role of pag in capsule production, we deleted the pag gene promoter region in the pXO1 plasmid (Figure 1A). Compared to the Pasteur II control (Figure 1B), the pag promoter knockout mutant, PasteurII-pagpromoterKO (generated with PApr-175SacF and PA-RSacII), produced dramatically less capsule (Figure 1C), which is similar to the capsule negative control Sterne strain (Figure 1G). To verify if the capsule deficiency was caused by the deletion of the pag promoter, we subcloned fragments of the pag gene and promoter into the pag promoter knockout strain for functional complementation assays (Figure 1A). Our results show that only the strains complemented with fragments encoding the entire pag gene restored capsule synthesis (Figure 1D), suggesting that pag is involved in capsule formation.

To confirm the role of pag gene in capsule formation, we deleted the entire pag gene. Similar to the capsule negative control Sterne strain (Figure 1G), the pag-deletion strain displayed capsule deficiency (Figure 1E). The morphology of PasteurII-pagKO deletion strain showed hair-like, dry, smooth, and non-sticky colonies, which was significantly different from the morphology of the Pasteur II control (data not shown). To further confirm the effect of pag on capsule formation, we conducted the complementation experiment by introducing pFF40 carrying the pag gene into the pag deletion strain. The complementary strain completely recovered the capsule formation, with thicker capsule morphology and increased capsule substance as compared to the Pasteur II strain (Figure 1F).

To elucidate whether the capsule defect phenotype of pag null knockout mutants is a result of the lack of PA production, we performed Western blot assays. The results showed that the expression of pag was not detected in either the pag promoter or the entire pag gene null mutants (lane 4 and lane 6 in Figure 2A), whereas the pag null mutation did not affect LF production (lane 4 and lane 6 in Figure 2B). This indicates the specific effect of the pag promoter or pag null mutation on PA production.

Taken together, the above results demonstrated that the PA protein is involved in capsule formation.

To elucidate the potential mechanism of PA's involvement in capsule formation, we examined the impact of the pag deletion on the transcription of selected virulence genes located on pXO1 and pXO2. The total RNA was extracted from PasteurII-pag KO and the original Pasteur II control, and was analyzed by qPCR. In the pag deletion strain, no pag expression was detected, as had been expected (Table 2). However, the expression of virulence genes gerXB and atxA, which are located in plasmid pXO1, showed a 2-fold increase and 2-fold decrease, respectively, in PasteurII-pag KO compared with that in Pasteur II control. In addition, the expression levels of genes responsible for capsule synthesis, including capA, capB, capC, and capD, decreased 4-fold in the PasteurII-pag KO strain compared with those in Pasteur II control, whereas the expression level of both acpA and acpB decreased 8-fold (Table 2). Surprisingly, the pag deletion also altered the transcription of the spoA, abrB, and rpoB genes located in chromosomal DNA (Table 2).

Our qPCR results suggested that PA is probably involved in the capsule formation by mediating the transcription of acpA and acpB. To test it, we constructed a constitutional acpA or acpB expression strain in the pag promoter and pag gene knockout mutant, respectively. Compared to the Pasteur II control (Figure 3) and the pag null mutants (Figures 1C,E), the ability of capsule formation was fully restored in the pag promoter, the entire pag gene knockout mutant carrying on the constitutive acpA expression system (Figures 3B,C), or carrying on the constitutive acpB expression system (Figures 3D,E). To explore the possibility of whether PA directly affects the acpA transcription by binding to the promoter region of acpA, we conducted a gel mobility shift assay using purified recombinant PA, and labeled the acpA gene promoter region. No shift of the DNA/protein complex band was observed (data not shown), indicating that the PA protein did not directly mediate the acpA transcription.

The role of PA in pathogenicity is controversial in different mouse models of infection (Welkos et al., 1993; Heninger et al., 2006; Moayeri et al., 2015). In this study, we examined the effect of pag on the pathogenicity of B. anthracis Pasteur II strain in the BALB/c mice model of subcutaneous infection. All BALB/c mice infected with PasteurII-pagKO survived for at least 96 h, whereas 50% (3 of 6) of the mice infected with the Pasteur II control strain died by 72 h, and 100% of them died by 96 h after infection. The mortality of mice did not increase when infected with more than 2-fold higher amounts of the pag knockout mutant than wild type control in subsequent experiments. The results indicated that the pag gene is an important virulence factor for B. anthracis Pasteur II strain in subcutaneous infection (Figure 4).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.