Animal experiments were approved by Animal Ethics Committee of Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (CAAS) and carried out in strict accordance with the recommendations of the Animal Ethics Procedures and Guidelines of the People's Republic of China. All efforts were made to minimize animal suffering.

The bacterial strains and plasmids used for this study are described in Table 1. The A. pleuropneumoniae strains were cultured in a brain heart infusion (BHI, Difco Laboratories, Detroit, MI, USA) medium supplemented with 10 μg/ml nicotinamide adenine dinucleotide (NAD) (Sigma-Aldrich, U.S.). For culture of A. pleuropneumoniae transconjugants (single crossovers), BHI medium was supplemented with 10 μg/ml of NAD and 7 μg/ml of chloramphenicol. E. coli ATCC 25922 strain and S. enterica ATCC 51741 strain (American Type Culture Collection, ATCC) were cultured in a Luria-Bertani (LB, Difco Laboratories, Detroit, MI, USA) medium. E. coli β2155 was grown in LB medium supplemented with 1 mM diaminopimelic acid (DAP) (Sigma-Aldrich, U.S.). All strains were routinely grown at 37°C.

A microdilution broth method was performed to determine the minimal inhibitory concentration (MIC) of antimicrobial peptide PR-39 according to the broth micro dilution guideline of the Clinical and Laboratory Standards Institute (CLSI, 2013). PR-39 was purchased from AnaSpec (San Jose, CA). Standardized bacterial suspensions of log-phase cultures of E. coli ATCC 25922, S. enterica ATCC 51741, A. pleuropneumoniae strains ATCC 27090, S-8 and MD12 were prepared and diluted to a concentration of 1 × 10⁶ CFU/ml. MIC determinations were performed using commercially sterile 96-well microtiter plates (Costar 3599, U.S.A.). The MIC value was determined as the lowest concentration of PR-39 that prevented visible growth. Then 20 μl of each bacteria-peptide suspension in the 96-well microtiter plates was plated onto LB or BHI agar plates and incubated for 20 h at 37°C. The MBC value was determined as the lowest concentration of PR-39 that showing no visible growth on the plates (Hu et al., 2016).

The A. pleuropneumoniae wild-type strain MD12 was grown in 5 ml of BHI medium for 15 h, and then diluted to an optical density at 600 nm (OD₆₀₀) of 0.1. Fresh cultures in 5 ml of BHI medium were supplemented with PR-39 (concentration range 0–0.2 μM) and incubated while shaking at 37°C. Growth was monitored by measuring the OD₆₀₀ values at an interval of 1 h using the Eppendorf BioPhotometer (Eppendorf, Germany).

For RNA isolation, A. pleuropneumoniae MD12 strain was grown to mid-logarithmic phase in 3 ml of BHI medium supplemented with PR-39 (concentration range 0–0.2 μM). The cultures were harvested by centrifugation at 10,000 g at 4°C. Total RNA was extracted using RNeasy kit (Qiagen) and cDNA was synthesized using the PrimeScript RT reagent kit (TaKaRa, Japan) according to the manufacturer's instructions. The primers used for analysis of sapA expression are listed in Table S1. The cDNA samples were amplified using SYBR Green I (TakaRa). Quantitative real-time polymerase chain reactions (qRT-PCR) were performed in a MicroAmp Optical 96-well reaction plate using a Stratagene Mx3000P system (Agilent Technologies, Germany). Amplification efficiency was evaluated using a standard curve generated by qRT-PCR using the cDNA dilution series with three replicates. The stability of the six housekeeping genes recF, glyA, rho, tpiA, pykA (Nielsen and Boye, 2005) and syp (Lone et al., 2009) was examined using the program geNorm (Vandesompele et al., 2002). The geometric mean of the best-scoring reference genes glyA, tpiA, and syp was used to normalize the target gene expression levels. The qRT-PCR experiments were performed in triplicate with three independent biological replicates. Relative expression levels were analyzed by a threshold cycle (ΔΔCt) method to calculate the fold change in gene expression (Pfaffl, 2001).

The primers used for the construction of the deletion mutant ΔsapA are listed in Table S1. Primers AUF/AUR, and ADF/ADR were used to amplify the two segments flanking the sapA gene. Using single-overlap extension PCR (SOE PCR), the fragment with a 570 bp internal in-frame deletion in the sapA gene (from nt 24 to 593) was generated, and cloned into the conjugative vector pEMOC2 (Baltes et al., 2003) to produce the plasmid pEMΔsapA. Using E. coli β2155 and a single-step transconjugation system (Dehio and Meyer, 1997; Oswald et al., 1999), plasmid pEMΔsapA was used to introduce the sapA mutation into the wild-type strain MD12. After two homologous recombination steps, the A. pleuropneumoniae ΔsapA mutant was verified by sequencing and PCR analyses using AJDF/AJDR primers.

The 2,471 bp PCR product including the entire sapA open reading frame (ORF) and 675 bp of the upstream region containing the native promoter was amplified with the primers AHBF/AHBR (Table S1). The PCR reaction was performed under the following conditions: 95°C for 3 min, 30 cycles with 94°C for 30 s, 52 °C for 30 s and 72°C for 2 min, the final extension at 72°C for 8 min. The PCR product was digested with SalI/SacI and ligated to SalI/SacI-digested pGZRS-19 plasmid (West et al., 1995), yielding plasmid pGZRS-sapA. The recombined plasmid pGZRS-sapA was confirmed by DNA sequencing (Comate Bioscience Co., Ltd.) and electroporated into the ΔsapA mutant for trans complementation. The electroporation conditions were set to 2,500 V, 200 Ω, and 25μF. Transformants were selected on BHI agar containing 20 μg/ml of ampicillin. The complemented mutant strain, verified by colony PCR and DNA sequencing, was designated PΔsapA.

Bactericidal assays were performed as described previously (Mason et al., 2005). The A. pleuropneumoniae strains MD12, ΔsapA, and PΔsapA were grown in BHI medium to OD₆₀₀ 0.8. Cells of each strain from the broth cultures were harvested and diluted in PBS (pH 7.4) to a concentration of 10⁶ CFU/ml. The wells of a sterile, polystyrene 96-well microtiter plate (Costar 3599, U.S.A.) were filled with 90 μl of PBS. PR-39 was serially diluted in the wells and each well retained 90 μl of the appropriate concentration (0.5–4 μM) of PR-39. Ten microliters of the bacterial suspension were added to each well, and the plate was incubated for 0.5–3 h at 37°C. Bacteria incubated with PBS served as controls. Serial dilutions of the bacteria were plated on BHI agar. The bactericidal effect was expressed as the percentage of surviving cells, using the bacterial counts obtained with bacteria incubated in PBS as 100%.

SDS-EDTA sensitivity assay was performed as described previously (Carpenter et al., 2014). A. pleuropneumoniae strains MD12, ΔsapA, and PΔsapA were incubated in BHI medium at 37°C with shaking at 180 rpm to OD₆₀₀ 1.0. Each strain was serially diluted with PBS, and 2 μl of these dilutions were spotted in triplicate onto fresh BHI agar plates containing 0.1% SDS and 0.5 mM EDTA. All the plates were incubated overnight at 37°C.

The 1-N-phenylnaphthylamine (NPN) uptake assay was performed as described previously (Martinez De Tejada and Moriyon, 1993). A. pleuropneumoniae strains MD12, ΔsapA, and PΔsapA were grown to OD₆₀₀ 0.6 and harvested by centrifugation at 2,500 g for 15 min. Pellets were washed three times and resuspended in 5 mM HEPES buffer (pH 7.2) containing 10 μM NPN (Sigma–Aldrich, USA). NPN uptake into the A. pleuropneumoniae ΔvacJ mutant was used as a positive control, as vacJ encodes VacJ lipoprotein and the membrane permeability of this mutant was increased as described previously (Xie et al., 2016). Fluorescence was measured using the EnVision Multilabel Reader (PerkinElmer, UK), with emission at 420 nm and excitation at 350 nm.

The A. pleuropneumoniae strains MD12, ΔsapA and PΔsapA were cultivated in BHI medium at 37°C to mid-logarithmic growth phase. Cells of each strain from the broth cultures (10⁶ CFU/ml) were incubated with 4 μM PR-39 for 1 h, and harvested by centrifugation. The cells were washed three times with PBS, and fixed overnight using 2.5% glutaraldehyde at 4°C. Dehydration was performed in upgraded ethanol (washed once with each of 50, 70, 85, 95%, and three times with 100%). Then the samples were dried using a critical point drying method and sputter-coated with gold. The cell morphology of all samples was visualized using a scanning electron microscope (JSM-7500F, JEOL, Japan).

The BALB/c mouse model has been acknowledged as an appropriate one to assess A. pleuropneumoniae infection (Chiang et al., 2009; Seo et al., 2013). Specific-pathogen-free, 6-week-old female BALB/c mice (Beijing Vital River Laboratory Animal Co., Ltd.) were purchased from the VitalRiver Laboratories (VRL, Beijing, China). A. pleuropneumoniae strains MD12 and ΔsapA were cultured in BHI medium at 37°C, and harvested during the mid-exponential phase and washed three times with sterile PBS. A total of 50 mice were randomly divided into 5 groups (n = 10/group). Group 1 and group 2 were respectively inoculated intraperitoneally with 100 μl of PBS containing 10⁸ and 10⁷ CFU of MD12. Group 3 and group 4 were respectively inoculated intraperitoneally with 100 μl of PBS containing 10⁸ and 10⁷ CFU of ΔsapA. Non-infected mice in the control group were inoculated with 100 μl of sterile PBS (pH 7.4). The health status and the weight of the mice were monitored twice daily for a 14-day period and humane endpoints used to determine if the mice met criteria to be euthanized (Nemzek et al., 2004). These criteria included weight loss >10–15%, lethargy, inability to stand, anorexia or flocked together for more than 6 h. Mice meeting criteria were euthanized by cervical dislocation under isoflurane anesthesia.

A total of 10 specific-pathogen-free, 6 week-old female BALB/c mice were randomly divided into 2 groups (n = 5), and each group was intraperitoneally administered with 5.0 × 10⁶ CFU of the MD12 strain or the ΔsapA mutant. Three days post-infection, mice from each group were humanely euthanized and the organs of lung, liver, and kidney were removed aseptically. Samples were weighed, and homogenized using a tissue homogenizer (100 mg weight/ml of PBS). Viable counts in serial dilutions of homogenates were determined following culture on BHI agar plates for 24 h at 37°C.

Statistical analyses were performed using GraphPad Prism version 5.01 (GraphPad Software Inc., U.S.A.). The data are expressed as the means ± standard deviation. The statistical analysis of the data was performed using one-way ANOVA, two-way ANOVA, or the Student's t-test. P-values less than 0.05 were considered statistically significant.

To explore the antibacterial activity of PR-39, the MICs of PR-39 for A. pleuropneumoniae strains ATCC 27090, S-8, MD12, E. coli ATCC 25922 and S. enterica ATCC 51741 were measured (Table S2). For E. coli and S. enterica, the MICs of PR-39 ranged from 0.5 to 1 μM, the MBCs of PR-39 were 1 μM. However, PR-39 had higher MICs for A. pleuropneumoniae strains than E. coli and S. enterica, ranging from 4 to 8 μM. Similarly, the MBCs of PR-39 for A. pleuropneumoniae strains were 8 μM, much higher than those of E. coli and S. enterica. These results showed that A. pleuropneumoniae exhibited a certain resistance to PR-39 compared to E. coli and S. enterica.

The expression of the sapA gene was analyzed using qRT-PCR in A. pleuropneumoniae MD12 when exposed to PR-39. Three housekeeping genes glyA, tpiA, and syp were selected to normalize sapA gene expression levels, and PCR efficiency for each gene was not less than 1.92. When the MD12 strain was exposed to sublethal concentrations of PR39, growth curves were similar to that of untreated bacteria (Figure 1A), but the transcription levels of sapA were higher than that of untreated bacteria (Figure 1B). In the presence of increasing concentrations of PR-39, the expression of sapA was upregulated in a dose- dependent manner, suggesting sapA may contribute to a resistance mechanism in A. pleuropneumoniae MD12 against PR-39.

Analysis of the A. pleuropneumoniae L20 genome sequence revealed the presence of an intact sap operon (Figure 2A). This operon consists of four genes: sapA (APL_RS04170), sapB (APL_RS04165), sapC (APL_RS04160), sapD (APL_RS04155). However, sapF (APL_RS06520) is not linked to the sapABCD locus.

To investigate the function of the SapA protein, an in-frame-deletion mutant of sapA in A. pleuropneumoniae was constructed using double-crossover homologous recombination and confirmed by PCR and DNA sequencing (Figures 2A,B, Supplementary Materials). PCR with primers AJDF/AJDR was used to amplify the 925 bp amplicon from the wild-type MD12 strain, and the 355 bp amplicon from the sapA deletion mutant ΔsapA (Figure 2B). The ΔsapA mutant contains a 570 bp in-frame deletion in the sapA gene. The results of qRT-PCR showed that the transcription levels of the downstream genes sapB, sapC, and sapD were unaffected, confirming that the mutation in ΔsapA was nonpolar (Figure S1). The complemented mutant strain PΔsapA was generated using the plasmid pGZRS-sapA, with transformants selected on plates containing ampicillin, and confirmed by PCR (Figure 2C).

To determine whether the SapA protein has a role in the survival of A. pleuropneumoniae upon exposure to the antimicrobial peptide PR-39, the MD12, ΔsapA, and PΔsapA strains were tested in a bactericidal assay. Cells of each strain were incubated with specific concentrations of PR-39 for 3 h. The results showed that the mutant devoid of sapA was significantly more sensitive to PR-39 over a concentration range of 0.5–4 μM than was the isogenic wild type strain (Figure 3A). In addition, following the elongation of treatment time, the sensitivity of ΔsapA to PR-39 was increased (Figure 3B). Trans-complementation with sapA expressed on pGZRS-19 partially restored resistance to PR-39. These findings indicate that the A. pleuropneumoniae SapA protein is required for the bacterium's resistance to the PR-39.

To exclude the possibility of impaired outer membrane integrity due to the deletion of the sapA gene, the sensitivity of the MD12, ΔsapA, and PΔsapA strains to SDS-EDTA was analyzed. As shown in Figure S2A, all these strains did not exhibit sensitivity to SDS-EDTA. In addition, the outer membrane integrity of each strain was further evaluated using the fluorescent probe NPN, which exhibits fluorescence weakly in aqueous but strongly in hydrophobic environments (Lee et al., 2015). In Figure S2B, no significant difference in uptake of NPN was observed between MD12, ΔsapA, and PΔsapA, while NPN fluorescence was significantly higher in the ΔvacJ mutant whose membrane permeability was increased as described previously (Xie et al., 2016). These data indicated that the sapA gene deletion did not cause alterations in the outer membrane integrity in A. pleuropneumoniae.

To further confirm the increased sensitivity of the ΔsapA strain to PR-39, the morphology of the MD12, ΔsapA, and PΔsapA strains treated with PR-39 was assessed using scanning electron microscopy. After PR-39 treatment, a significant morphological variation was observed among the MD12, ΔsapA, and PΔsapA strains (Figure 4). MD12 displayed a smooth surface, which is typical of this A. pleuropneumoniae strain (Figure 4). However, when exposed to PR39, cells of the ΔsapA mutant had an irregular and crinkled appearance and a sunken shape compared to that of the wild-type MD12 and complemented PΔsapA strains (Figure 4). These data indicated that the A. pleuropneumoniae SapA protein functions, at least to some extent, to protect this pathogen from the lethal effects of PR-39.

To address whether sapA deletion affected the virulence of A. pleuropneumoniae, BALB/c mice were inoculated intraperitoneally with wild type strain MD12 and the ΔsapA mutant at various doses. The MD12 strain gave rise to a higher mortality rate than ΔsapA (Figure 5), which suggested that the deletion of sapA attenuates the virulence of A. pleuropneumoniae.

The capacity of the MD12 and ΔsapA strains to colonize mice was then tested. The A. pleuropneumoniae load in tissues of systemically infected mice was determined by culturing the lungs, livers, and kidneys homogenates 3 days post-infection. As shown in Figure 6, the viable counts in lung were significantly decreased in the ΔsapA mutant-infected mice compared with the WT-infected mice (P < 0.01). Similarly, significant differences (P < 0.05) in bacterial loads were also found between the MD12-inoculated and ΔsapA-inoculated mice in livers and kidneys (Figure 6). Taken together, the results showed that the ΔsapA mutant of A. pleuropneumoniae displayed a reduced ability to colonize BALB/c mice.

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.