NADH-Dependent Flavin Oxidoreductase of Mycoplasma Hyopneumoniae Functions as a Potential Novel Virulence Factor and not Only as a Metabolic Enzyme


 Background: Mycoplasma hyopneumoniae (Mhp) is the main pathogen of enzootic pneumonia, pigs infected with Mhp demonstrate mainly poor growth and a reduced feed conversion rate, a disease that has a significant impact on the pig industry and pock production worldwide. The pathogenesis, especially possible virulence factors of Mhp, has still not been fully clarified.Results: The transcriptome and proteomic analysis of Mhp strains differed in virulence based on reported literature, and RNA transcription expression levels between high- and low-virulence strains initially indicated that nicotinamide adenine dinucleotide (NADH)-dependent flavin oxidoreductase (NFOR) was related to Mhp pathogenicity. Prokaryotic expression and purification of the NFOR protein of Mhp were performed, a rabbit-origin polyclonal antibody of NFOR was prepared, and multiple sequence alignment and evolutionary analysis of Mhp NFOR were performed. For the first time, it was found that the NFOR protein was conserved among all Mhp strains, and NFOR was localized on the cell surface and could adhere to immortalized porcine bronchial epithelial cells (hTERT-PBECs). Adhesion to hTERT-PBECs could be specifically inhibited by an NFOR polyclonal antibody, and the adhesion rates of both high- and low-virulence strains, 168 and 168L, significantly decreased by more than 40%. Moreover, Mhp NFOR could not only recognize and interact with host fibronectin and plasminogen but could also induce cellular oxidative stress and apoptosis in hTERT-PBECs. Lactate dehydrogenase release of Mhp NFOR in hTERT-PBECs was significantly positively correlated with the virulence of Mhp.Conclusions: Overall, in addition to being a metabolic enzyme related to oxidative stress, NFOR may also function as a potential novel virulence factor of Mhp, thus contributing to the pathogenesis process of Mhp, providing new ideas and theoretical support for studying the pathogenic mechanisms of other mycoplasmas.


Background
As the simplest self-replicating organism, Mycoplasma has the smallest genome. It is speculated that they evolved from the degeneration of gram-positive bacteria and are most closely related to certain clostridia in phylogeny [1,2]. Due to its limited biosynthesis and metabolism capabilities, Mycoplasma relies on infected host cells to provide nutrients to meet the necessary needs of its parasitic life [1], and they have also developed mechanisms to invade and further survive in host cells. The highly plastic variable surface protein group is responsible for the rapid changes of the main surface protein antigens and the invasion of nonphagocytic host cells and the subsequent regulation of the host immune system [3][4][5][6][7]. These mechanisms contribute to the establishment of chronic infection and further to the persistence of Mycoplasma in the host.
Many species of mycoplasmas are pathogens that cause various diseases in livestock production and eventually lead to heavy economic losses [8,9]. Among them, Mycoplasma hyopneumoniae (Mhp), the pathogen of porcine endemic pneumonia, is a highly contagious and globally distributed porcine respiratory disease. It usually destroys the epithelium along with the respiratory tract, making pigs vulnerable to secondary infections by other bacteria and viruses and ultimately causing signi cant economic losses to the global pig industry [9]. However, a basis for the immune pathogenesis of Mhp has not yet been fully clari ed. Some scholars have considered that the onset of porcine enzootic pneumonia depends on Mhp virulence factors, which enable the pathogen to evade the host defense mechanism, and the production of molecules that participate in processes including cell-host adhesion, response to environmental stress of the host, and immune regulation [10]. Therefore, uncovering novel virulence factors would be of great importance in illuminating the mechanism of Mhp pathogenesis.
Despite the presence of surface proteins, a variety of metabolic enzymes have been identi ed and described as critical pathogenicity determinants not limited to their roles in basic metabolism [11,12]. The utilization of available substrates and the metabolic potential and growth rate of bacteria all play indispensable roles related to their pathogenicity [8]. A previous transcriptomics study demonstrated that nicotinamide adenine dinucleotide (NADH)-dependent avin oxidoreductase (NFOR) was overrepresented in Mhp pathogenic strain 7448 but not in M. occulare [13]. In addition, previous comparative proteomics reports demonstrated that there are signi cant differences in the expression of NFOR between high-and low-virulence Mhp strains [14,15]. NFOR is an oxidoreductase, the largest class of enzymes, and its function is to catalyze the oxidation of NADH to NAD + by reducing molecular O2 to H2O or H2O2 at the same time. Some organisms, such as Nomuraea rileyi, contain these kinds of enzymes, which play important roles in regulating cellular redox and osmotic pressure balance to maintain normal cell growth and development [16]. Therefore, we hypothesized that Mhp NFOR, in addition to its effect on metabolic processes, may have a certain relationship with Mhp virulence.
As a new candidate virulence factor discovered in this study, NFOR was located at the surface of Mhp, as determined by electron microscopy observation and ow cytometry analysis. NFOR could adhere to immortalized porcine bronchial epithelial cells (hTERT-PBECs) and recognize host bronectin and plasminogen, with higher a nity for bronectin. It could also induce host cytotoxicity, oxidative stress damage, and apoptosis. Our ndings support the notion that NFOR may be a potential novel virulence factor of Mhp, which will provide new ideas and theoretical support for studying the pathogenic mechanisms of Mhp and other mycoplasmas.

Mhp strains and growth conditions
All ve Mhp strains were thawed from frozen Mhp bacterial stocks and subcultured for three generations before use for subsequent analysis. The strains were cultured in modi ed Friis medium, designated KM2 cell-free medium, which contained 20% (v/v) pig serum (sterilized by irradiation from a clean snatchfarrowed porcine colostrum-deprived piglet and produced in our lab) cultivated in a humidi ed incubator at 37°C [17]. Mhp strain 168 was isolated and cultured from a pig exhibiting representative characteristics of mycoplasma pneumonia of swine (MPS) in China[18]. This eld strain was gradually attenuated through continuous passage to the 380 th generation, resulting in the low-virulence strain 168L [19]; the Mhp 168L strain used in this study was passage 353. Strain JS is a virulent strain that can induce typical characteristics of MPS with a lung lesion score of approximately 15, as mentioned previously [17].
Strain LH is a virulent clinical strain that was isolated in our lab. Strain J (ATCC 25934) was passaged once from the ATCC stock to yield frozen stocks. The titers of Mhp strains were quanti ed using the 50% color change unit (CCU 50 ) assay [20], which was modi ed from the CCU assay [21] and tested by quantitative PCR.

RNA transcriptional analysis
The ve Mhp strains were cultured in the abovementioned culture conditions at 37°C for 48 h.
Then, total RNA was extracted using a Total RNA Extraction Kit (Cat No. R6834, Omega Biotek, Guangzhou, China). HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) (Cat No. R223-01, Vazyme, Nanjing, China) was used to reverse transcribe at least 1 µg of total RNA in a 10-µL reaction volume before performing qRT-PCR on an ABI 7500 Real Time PCR System with the help of a HiScript® II One Step qRT-PCR SYBR® Green Kit (Cat No. Q221-01, Vazyme, Nanjing, China). qRT-PCR was run using cDNA for the NFOR gene under speci c conditions. The P46 gene of Mhp was selected as the internal control. The PCR primers used in the quantitative assays are listed in Table 1. The fold changes in mRNA expression of the NFOR genes of various Mhp strains differing in virulence were determined using the 2 -ΔΔCT method as described previously [22].

Multiple sequence alignment
Seventeen amino acid sequences of the NFOR protein from Mhp were retrieved from the National Center for Biotechnology Information and UniProt protein databases, and their homologies were analyzed. All sequences were aligned with the CLUSTAL W program. Phylogenetic inference according to the neighborjoining criterion was performed through Molecular Evolutionary Genetics Analysis version 10 (MEGA 10). A total of 2000 nonparametric bootstrap analyses were used to test the robustness of the hypothesis.

Protein expression and preparation of polyclonal antibody
The Mhp NFOR gene (MHP168_RS01740) was synthesized by GenScript Biotech Corp. (Nanjing, China) and was expressed in the BL21(DE3) E. coli strain through the pET32a vector, puri ed using High A nity Ni-Charged Resin FF a nity columns (Cat No. L00666, GenScript, Nanjing, China), and identi ed by Western blot analysis. Puri ed proteins were determined with the BCA Protein Assay Kit (Cat No. P0012S, Beyotime, Shanghai, China), and the protein concentration was calculated before storage at -70°C.
Polyclonal antibodies against Mhp NFOR were obtained by subcutaneously immunizing 1-month-old New Zealand white rabbits with 1.2 mg of recombinant protein (rNFOR) emulsi ed in Freund's complete adjuvant for the rst immunization (Cat No. F5881, Sigma-Aldrich, St Louis, MO, USA). Each rabbit was then immunized with 1.2 mg of rNFOR emulsi ed in Freund's incomplete adjuvant (Cat No. F5506, Sigma-Aldrich, St Louis, MO, USA) twice at 2-week intervals. Booster immunization was performed once a week after three immunizations before sera were collected.

Enzymatic activity assays
The enzymatic activity of puri ed rNFOR was determined by calculating the oxidation of NADH to NAD + at 25°C. In brief, 5 μg/mL rNFOR, 0.1 M potassium phosphate buffer (pH 7.5, containing 1 mM dithiothreitol), 10 μM avin mononucleotide (FMN) (Cat No. F107158, Aladdin, Shanghai, China) and 0.5 mM NADH (Cat No. N106933, Aladdin, Shanghai, China) were used in this study. The reaction system was 2 mL, and the puri ed rNFOR was preincubated with FMN for 5 min before NADH was incubated.
The optical density (OD) was measured at 340 nm (OD340). The speci c activity was calculated by the following equation: Surface-exposed NFOR detection by ow cytometry Flow cytometry analysis was used to test whether NFOR is located on the surface of Mhp strains and to probe NFOR surface distributions between the high-virulence strain 168 and the low-virulence strain 168L.
Fluorescein isothiocyanate (FITC)-conjugated anti-IgG (Cat No. BA1105, Boster, Wuhan, China) was then used to stain the above Mhp strains, and a BD Accuri C6 ow cytometer was used to measure the uorescence intensity. Surface-exposed NFOR detection by immunoelectron microscopy Mhp strains were cultured and grown to mid-log phase at 10 000 × g by centrifugation at 10°C for 20 min before each Mhp bacterium was harvested. A total of 1×10 8 CCU of bacterial suspension was washed three times and nally resuspended in a volume of 50 µL of 0.1 M phosphate-buffered saline (PBS, pH 7.4). Immunoelectron microscopy was performed according to previous studies [25,26] with some modi cations. Brie y, 5 µL of the sample was added to a 400 mesh formvar-coated nickel grid and allowed to stand for 5 min. Then, the grid was xed with 2% paraformaldehyde in PBS for 5 min at room temperature (RT) followed by blocking with 1% negative rabbit serum and blocking buffer (1% (w/v) BSA in PBS) for 1 h. The samples were then incubated with anti-rNFOR antibody or preimmune serum (negative control) at a 1:10 dilution in blocking buffer for another 1 h (PBS as a blank control). After washing ve times in blocking buffer, the samples were incubated with a secondary gold-conjugated antibody (goat anti-rabbit IgG, 10 nm-gold particles, Cat No. GA1014, Boster, Wuhan, China) at a dilution of 1:20 for 1 more hour. The samples were washed 5 times with PBS for 5 min each time before being xed in 2% paraformaldehyde in PBS for 5 min. Then, the grids were washed eight times in distilled water and stained with 1% phosphotungstic acid (pH 6.5) for 15 s. After the samples were dried by an infrared lamp, they were observed under a Tecnai high-eld transmission electron microscope.
Indirect immuno uorescence assay (IFA) Immortalized porcine bronchial epithelial cells (hTERT-PBECs) were established and cultured to a density of 80% in 24-well cell plates with Dulbecco's modi ed Eagle's medium:nutrient mixture F-12 (DMEM/F12) medium plus 2% (v/v) fetal bovine serum (Gibco, Grand Island, NY, USA) supplemented with growth factors (Cat No. CC-4175, Lonza, Basel, Switzerland), as we previously made and reported [22]. Cells were washed three times with cold PBS before being xed with 4% paraformaldehyde for 10 min at RT. Subsequently, 0.2% Triton X-100 was used at RT for 3 min, followed by blocking for 2 h using 3% (w/v) BSA in PBS. Cells were incubated with 100 μg of puri ed rNFOR for 1 h at 37°C in a cell incubator before they were washed three times with PBS and incubated with anti-rNFOR antibody at a 1:250 dilution for another 2 h at 37°C. After three washes with PBS, the cells were then incubated with a 1:100 dilution of tetraethyl rhodamine isothiocyanate (TRITC)-conjugated anti-IgG (Cat No. SA00007-2, Proteintech, Rosemont, IL, USA) for 1 h in a 37°C incubator. 6-Diamidino-2-phenylindole (DAPI, Cat No. D8417, Sigma-Aldrich, St Louis, MO, USA) was used for nuclear staining before the cells were observed using a uorescence microscope (Zeiss, Tokyo, Japan). Instead of rNFOR, BSA was selected as a negative control.

Antibody-mediated adhesion inhibition
Mhp strains (high-virulence 168 and low-virulence 168L, the titers of which were 1 × 10 7 CCU/mL) were collected by centrifugation at 10 000 × g for 20 min at 10°C and resuspended in 500 µL of PBS after washing three times with PBS. The samples were preincubated with polyclonal antibody against rNFOR or preimmune serum at a 1:20 dilution for 30 min in a 37°C incubator. Mhp bacteria were suspended in DMEM/F12 before being added to con uent hTERT-PBECs seeded in 24-well cell plates. Plates were then centrifuged at 1 000 × g for 10 min before being placed at 4°C for 2 h. After washing three times with PBS, hTERT-PBECs were collected after digestion with 0.125% trypsin (twice diluted with Hanks medium with 0.25% trypsin, Cat No. 25200072, Gibco, Grand Island, NY, USA), and the cells were centrifuged at 1 300 rpm for 10 min after adding DMEM/F12 containing 10% FBS to stop cell digestion. Following Mhp bacterial genome extraction, quantitative real-time PCR was then performed as previously reported [27]; the real-time PCR primers are shown in Table 1. Experiments were performed in triplicate, and data were analyzed using SPSS 20.0. Mhp titers were quanti ed using the CCU 50 assay mentioned above.
Surface plasmon resonance (SPR) analysis SPR analysis was performed according to our previous study [26] using a Biacore X100 Plus instrument (GE Healthcare, Boston, MA, USA). Fibronectin and plasminogen were diluted to 50 µg/mL before they were linked covalently to the CM5 sensor chip as a ligand using an amine coupling kit (Biacore AB, Cytiva, Guangzhou, China). The immobilization of soluble bronectin and plasminogen produced approximately 2000 resonance units (RUs). The binding kinetics were measured by increasing the concentration (0-4000 nmol/L) of the analyte (Mhp NFOR) in running buffer (HBS-EP), which consisted of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% (v/v) surfactant P20 (Biacore AB, Cytiva, Guangzhou, China), with a ow rate of 30 μL/min, passing through immobilized Mhp NFOR at 20°C for 3 min. The dissociation phase was monitored for 1000 seconds by allowing buffer to ow through the chip. Biacore X100 control software was used to manually analyze the binding kinetics.

Far-Western blot (Far-WB) analysis
Fifteen micrograms of rNFOR was resolved by 10% SDS-PAGE and then transferred to a PVDF membrane (Cat No. IPFL00010, Millipore, Darmstadt, Germany). After three washes with PBS, the membrane was blocked with 5% skimmed milk in TBST (TBS containing 0.5% Tween 20), which served as the blocking buffer, before being placed in a 37°C incubator for 2 h with gentle shaking. Then, the membrane was incubated with 15 μg/mL bronectin (Cat No. F1056, Sigma-Aldrich, Darmstadt, Germany) or plasminogen (Cat No. SRP6518, Sigma-Aldrich, Darmstadt, Germany) at 37°C for another 2 h. After another three washes with TBST, membranes were subsequently incubated with anti-bronectin antibody (Cat No. ab299, Abcam, Cambridge, UK) at a 1:1000 dilution or anti-plasminogen antibody (Cat No. ABP55618, Annkine, Wuhan, China) at a 1:400 dilution in blocking buffer for 2 h in a 37°C incubator. The membranes were then incubated with the secondary antibody (HRP-conjugated goat antirabbit IgG) (Cat No. BA1055, Boster, Wuhan, China) at a 1:2000 dilution for 2 h in a 37°C incubator after three washes with TBST. Finally, the membranes were developed with Electro-Chemi-Luminescence (ECL) substrate using a ChemiDoc XRS+ system (Bio-Rad). Instead of rNFOR, BSA was used as a negative control.
Quanti cation of lactate dehydrogenase (LDH) release hTERT-PBECs were seeded in 24-well cell plates one night before cell growth reached 80% con uence. First, the cells were incubated with puri ed rNFOR protein at different concentrations (5 μg, 10 μg, 15 μg, 20 μg). Six hours later, culture supernatants were collected, and LDH activity was measured with a CytoTox 96 ® Non-Radioactive Cytotoxicity Assay (Cat No. G1780, Promega, Madison, WI, USA) following the manufacturer's instructions. The corrected values in the formula below were used to calculate the percentage of cytotoxicity: percent cytotoxicity = 100 × experimental LDH release (OD 490 )/maximum LDH release (OD 490 ). PBS was used instead of rNFOR as a negative control, and three Mhp strains (strains JS and J and 168L, 1 × 10 8 CCU/mL) that differed in virulence were used as positive controls.
Reactive oxygen species (ROS) detection hTERT-PBECs were seeded in 24-well plates one day before cell growth reached 80% con uence. Cells were incubated with 20 μg of puri ed rNFOR in a 37°C incubator for 6 h. ROS detection was measured using a ROS-Glo™ H 2 O 2 Assay (Cat No. G8820, Promega, Madison, WI, USA), and relative luminescence units (RLU) were recorded using a plate reader. PBS was used as a negative control, and positive controls were the RLUs from three Mhp strains (strain JS, J and 168 L, 1 × 10 8 CCU/mL).
Apoptosis assay hTERT-PBECs were seeded one night before at 2×10 5 cells/well in culture medium of a total volume of 500 µL in 24-well cell plates. hTERT-PBECs were then incubated with 20 μg of puri ed rNFOR at 37°C for 12 h, with cells grown from culture medium DMEM/F12 plus 2% FBS and epithelial growth factors to maintain the DMEM/F12 without adding the above reagents as a negative control, and Mhp strains that differed in virulence (high-virulence strain JS and low-virulence strain 168L, 1 × 10 8 CCU/mL) were used as positive controls. To explore whether antiserum to rNFOR could block and reduce the apoptosis induced by Mhp in hTERT-PBECs, we preincubated Mhp strains (JS and 168L, 1 × 10 8 CCU/mL) with rabbit polyclonal antibody raised against rNFOR at a 1:20 dilution at 37°C for half an hour before they were applied to hTERT-PBECs seeded in 24-well plates in a 37°C incubator for 12 h. The apoptosis rate was detected and calculated using a dual apoptosis detection kit (Cat No. A211, Vazyme, Nanjing, China) with Annexin V-FITC/PI.

Statistical analysis
Data for the adhesion rate from quantitative real-time PCR analysis and the apoptosis rate were analyzed by GraphPad Prism 6 software and FlowJo software v7.6. Relative NFOR mRNA expression levels between the moderate-virulence strain J or the low-virulence strain 168L and the high-virulence Mhp strains 168, JS, and LH were assessed via multiple comparisons of analysis of variance (ANOVA). The adhesion rates between anti-NFOR serum or negative serum and groups of the high-virulence strain 168 or between low-virulence strain 168L and anti-NFOR serum or negative serum were compared by the multiple t test. P < 0.05 was considered a signi cant difference, and P < 0.01 was considered an extremely signi cant difference.

Signi cant differences in NFOR transcription levels between high-and low-virulence Mhp strains
The relative quantitative RT-PCR results showed that there indeed existed signi cant differences in NFOR transcription levels among the ve Mhp strains that differed in virulence. Figure 1 shows the mRNA expression levels of the NFOR gene in ve Mhp strains, with the Mhp strains 168L (Fig. 1A) and J (Fig. 1B) used as controls. The mRNA expression levels of the NFOR gene were signi cantly upregulated in the high-virulence Mhp strains 168, LH and JS compared to the low-virulence Mhp strain 168L. The mRNA expression levels of the NFOR gene in Mhp strain J remained unchanged, as the expression levels were far less than a 2-fold change (Fig. 1A), which is usually regarded as the criterion for distinguishing whether expression is signi cantly different [28]. Similarly, the mRNA expression levels of the NFOR gene were signi cantly upregulated in the high-virulence Mhp strains 168, LH and JS compared to the moderate-virulence Mhp strain J (Fig. 1B). In contrast, the mRNA expression levels of the NFOR gene in Mhp strain 168L were decreased compared with those of strain J, but the difference was not signi cant.
Bioinformatics analysis, protein expression and enzymatic activity of Mhp NFOR Seventeen protein sequences of NFOR from Mhp were retrieved from the NCBI and UniProt databases, and the homologies between them were analyzed. As shown in Fig. 2A, all protein sequences were aligned with the CLUSTAL W program (Fig. 2A). Molecular Evolutionary Genetics Analysis version 10 (MEGA 10) was used to make phylogenetic inferences based on the neighbor-joining criterion. The robustness of the hypothesis was tested through 2000 nonparametric bootstrap analyses (Fig. 2B). It was found that all Mhp NFOR proteins had only minimal differences in amino acid positions from the results of multiple sequence alignments. Similarly, evolutionary analysis revealed that the overall homology of NFOR among all Mhp strains reached approximately 96.92%, suggesting that NFOR is a relatively highly conserved protein and has little effect among different Mhp strains.
The full-length coding sequence (CDS) of Mhp 168 strain NFOR is 1173 bp, with the predicted protein containing 390 amino acids. On the basis of codon optimization, a prokaryotic expression vector pET-32a-NFOR was constructed, and it was expressed in Escherichia coli BL21 (DE3) to study the enzyme activity of rNFOR after puri cation. The enzymatic speci c activity of the rNFOR protein was determined to be 29.12 IU/mg.

NADH-dependent avin oxidoreductase is located at the surface of Mhp cells
To con rm whether NFOR is located on the surface of Mhp, two tests were performed. Flow cytometry analysis revealed that the outer membrane-localized NFOR can approach the NFOR-speci c antibody through the surface in both Mhp strains 168 and 168L, indicating that the NFOR antigen is present on the cell surface of the Mhp strain. There was no signi cant difference in the mean uorescence intensity (MFI) between Mhp strain 168L incubated with preimmune serum and 168L treated with antirecombinant protein NFOR (anti-rNFOR) serum (Fig. 3A), while the MFI of strain 168 treated with preimmune serum was approximately 4-fold lower than that of Mhp 168 treated with anti-rNFOR serum (Fig. 3B). In addition, Mhp cells bind antibodies against the Mhp NFOR protein surrounding their surface, as revealed by immune electron microscopy. The antibodies were localized and restricted to the peripheral area of Mhp cells in both the Mhp strain JS and strain 168, while in the preimmune serumtreated negative group, gold particles were not visible (Fig. 3C). rNFOR adheres to hTERT-PBECs, and treatment with anti-rNFOR serum inhibits adherence to Mhp An indirect immuno uorescence assay (IFA) was used to determine whether Mhp NFOR could speci cally adhere to the surface of hTERT-PBECs. The results revealed obvious orange-red punctate uorescence on the surface of immortalized hTERT-PBECs incubated with rNFOR, whereas in the negative control group, no speci c uorescence was found around cell nuclei stained with DAPI (Fig. 4A), suggesting that rNFOR could speci cally bind to hTERT-PBECs.
To further assess the role of NFOR in Mhp adhesion to hTERT-PBECs, an antibody inhibition assay was conducted. Anti-rNFOR serum decreased the adherence of Mhp (both strains 168 and 168L) to hTERT-PBECs compared to that of the control group, which was treated with negative serum. Compared with the adhesion of Mhp in the absence of anti-rNFOR polyclonal antibody, the adhesion level was shown as the adhesion rate. Mhp strains incubated with anti-rNFOR antibody led to 48.7% (strain 168) and 51.75% (strain 168L) (p < 0.001) reductions in the adhesion rate of Mhp to hTERT-PBECs (Fig.  4B). The results further indicated that NFOR of Mhp plays an irreplaceable role in the adherence of Mhp to host cells.

Identi cation of rNFOR binding ligands
To determine whether the components of hTERT-PBECs interact with NFOR, surface plasmon resonance (SPR) and far-Western blot analysis were used to examine the interactions between NFOR of Mhp and bronectin and plasminogen. Mhp NFOR could bind to bronectin and plasminogen in a dose-dependent manner, with equilibrium dissociation constant (KD) values of 233.3 nmol and 394.8 nmol, respectively (Fig. 5A), indicating that Mhp NFOR could speci cally bind to both bronectin and plasminogen and had a relatively high a nity for bronectin.
Expression of puri ed recombinant protein rNFOR in the pET-32a expression vector is shown in panel "a" of Fig. 5B, with a recombinant protein rNFOR size of 44 kDa. The expression of rNFOR was detected using a polyclonal antibody in a Western blotting experiment. As shown in panel "b" of Fig. 5B, a clear band appeared at position 44 kDa regardless of whether it was from the whole bacterial protein or the puri ed protein rNFOR. As shown in Fig. 5C, before the nal reaction with bronectin or plasminogen, the corresponding band was observed in the reaction of rNFOR with anti-rNFOR antibody (positive control), but no speci c reaction was observed in the negative control (which used BSA was instead of anti-rNFOR antibody). The results showed that Mhp NFOR has a strong a nity for plasminogen and bronectin and that the binding ability of NFOR and brinogen is better than that of the Mhp and plasminogen. rNFOR induced cytotoxicity, oxidative stress damage and apoptosis of hTERT-PBECs The cytotoxicity of rNFOR on hTERT-PBECs was determined by the CytoTox 96 ® Non-Radioactive Cytotoxicity test, and it was found that the viability of cells was signi cantly reduced by rNFOR in a dosedependent manner (Fig. 6A). In addition, rNFOR induced oxidative stress damage in hTERT-PBECs (Fig.   6B). The results also demonstrated that the cytotoxicity and host cell cellular oxidative stress of Mhp against hTERT-PBECs were signi cantly correlated with the differing virulence of pathogenic Mhp strains ( Fig. 6A and 6B).
The ow cytometry results further indicated that rNFOR could induce apoptosis in hTERT-PBECs (Fig. 7A), and the late-stage apoptosis (upper right quadrant) rate was upregulated by 82.4% compared with the negative control group (4.9%). The late-stage apoptosis induced by Mhp strain JS or 168L in hTERT-PTECs was signi cantly decreased when rNFOR was blocked with an anti-rNFOR antibody. The late-stage apoptotic cell percentage decreased by 41.9% and 35.8%, respectively (Fig. 7B and 7C).

Discussion
To date, all mycoplasmas cultured and identi ed are known to be host parasites, the hosts of which include humans or animals, with a higher degree of speci city to hosts and tissues [29]. The main habitats of mycoplasmas are respiratory and genitourinary tracts, serous membranes and epithelial surfaces of mammary glands of certain animal species. Mhp is a host-speci c pathogen that infects only pigs. Currently, many virulence factors of Mhp have been revealed, and their roles include adhesion, invasion, and intracellular proliferation; however, the molecular mechanisms of infection and pathogenesis have not been fully elucidated [9]. In recent years, some studies have indicated that several metabolic enzymes play an important role in host-pathogen interactions during Mycoplasma infections[8, 23,26]. "Omics" studies have shown that there are signi cant differences in the expression of NFOR between high-virulence Mhp strain 7448 and moderate-virulence strain J and nonpathogenic M.
occulare through transcriptomics comparison [13] or between high-and low-virulence Mhp strains through proteomics comparison [14,15]; however, a previous study classi ed NFOR as a "nonvirulent" protein [14] in the supplementary le, but with no veri cation.
However, here, the mRNA expression levels of the NFOR gene in pathogenic high-virulence Mhp strains 168, LH and JS were signi cantly upregulated compared with either moderate-virulence strain J or lowvirulence strain 168L. This result preliminarily con rmed that NFOR was correlated with the virulence of Mhp. Further investigation showed that the NFOR antigen was located on the cell surface of Mhp by both MFI analysis through ow cytometry and immunoelectron microscopy observation. Compared with the nonpathogenic 168L strain, the uorescence intensity of the pathogenic high-virulence strain 168 was increased by approximately 4-fold, further indicating that NFOR may be responsible for the virulence of Mhp.
The adhesion of all microorganisms, including mycoplasma, to their host cells is a key step for their colonization and subsequent infection of the host. Adhesion ability is an important factor that re ects bacterial virulence [30]. Mhp is mainly found on the mucosal surface along the entire swine respiratory tract, including the trachea, bronchi and bronchioles, inducing ciliostasis and loss of cilia [31]. The rst stage of pathogenesis is the adhesion of Mhp to cilia in epithelial cells of the respiratory mucosa by means of adhesins. Here, we found that NFOR could adhere to the immortalized porcine bronchial epithelial cells (hTERT-PBECs) established in our previous study [22]. In addition, preneutralizing Mhp with polyclonal antiserum to rNFOR obviously decreased the adherence of Mhp to host cells.
Fibronectin has been studied more extensively, and a popular extracellular matrix protein has been identi ed that forms a molecular bridge between pathogens and host cell receptors [32,33]. According to our previous studies [23,26], several bronectin-binding bacterial proteins have been found to mediate the adhesion of bacteria to host cells and subsequent invasion by binding to bronectin. Therefore, bronectin binding protein plays a critical role in the pathogenic process of bacteria, including mycoplasmas. In addition to bronectin, a variety of bacteria can sequester the host zymogen plasminogen to the cell surface. Once localized to the bacterial surface, plasminogen can act as a cofactor for adhesion or, after being activated as plasmin, can provide an effective source of proteolytic activity. The recruitment of plasminogen to the surface of bacterial cells is directly mediated by specialized cell surface receptors or cytoplasmic and glycolytic pathway proteins located on the surface of bacterial cells or is indirectly mediated through interactions with host plasma proteins such as brinogen [34]. In this study, we found that rNFOR could speci cally bind to bronectin and plasminogen and had a higher a nity for bronectin. It is possible that bronectin might function as the main receptor of NFOR and that plasminogen acts as a cofactor to mediate the adhesion process of Mhp. Further characterization of the interaction between bronectin and plasminogen would be useful and helpful in improving the understanding of the adhesion-related factors of Mhp.
Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that converts lactate to pyruvate. LDH release assays of damaged cells are a method to detect cell viability from the perspective of cell membrane integrity [35]. Our results showed that NFOR could induce cytotoxicity in hETRT-PBECs in a dose-dependent pattern, and the LDH release of NFOR in host cells was positively correlated with various Mhp strains that differed in virulence. ROS are constantly generated during numerous cellular processes, one of the main sources of which is aerobic respiration, but most are often offset by antioxidant proteins. In addition, a large amount of ROS is produced from bacterial or viral infections, in ammatory reactions, ionizing radiation, and many chemical drugs [36][37][38]. In these processes, if the induced ROS is more than the upper limit offset by antioxidant proteins, so-called "oxidative stress" will be generated. Unlike cell necrosis, apoptosis is not a passive process but a phenomenon that induces autologous injury through a series of signal activation, protein expression and regulation processes [39]. A recent study demonstrated that ROS deposition is thought to be a direct cause of apoptosis because ROS induce strong cytotoxicity to host cells. Thus, when the intracellular ROS content shows a large increase, it will stimulate oxidative stress and induce apoptosis [40][41][42].
In this study, we found that rNFOR could induce oxidative stress damage in immortalized porcine bronchial epithelial cells (hTERT-PBECs) in Mhp strains that differed in virulence. Moreover, the H 2 O 2 release induced against cells by the low-virulence Mhp strain 168L was signi cantly lower than that induced by one high-virulence strain and one moderate-virulence strain (JS and J). Flow cytometry results further indicated that rNFOR could also induce apoptosis in hTERT-PBECs, and the late-stage apoptosis rate was upregulated by 82.4% compared with the negative control group (4.9%) after changing the medium to maintenance medium without FBS and growth factors after 12 h. The late-stage apoptosis induced by either Mhp from the high-virulence strain JS or the low-virulence strain 168L in hTERT-PBECs was signi cantly decreased when rNFOR was blocked with anti-rNFOR polyclonal antibody, with latestage apoptotic cell percentages decreasing by 41.9% and 35.8%, respectively. In conclusion, these results suggested that NFOR, functioning as more than only a metabolic enzyme, may act as a potential new virulence factor of Mhp, which will provide certain theoretical support and new ideas for the research and development of live-attenuated or subunit vaccines against Mhp.

Conclusions
In this study, in addition to being a metabolic enzyme related to oxidative stress, NFOR may be a potential novel virulence factor of Mhp, thus contributing to the pathogenesis process of Mhp, providing new ideas and theoretical support for studying the pathogenic mechanisms of other mycoplasmas.

Consent for publication
Not applicable.

Data availability
All data and materials are included in the manuscript and will be available from the corresponding author on reasonable request. XX carried out most of the experiments described in the manuscript and wrote the article; FH, RC, JJW and JL helped preparing the recombinant protein and preparing the rabbit hyperimmune sera; YNW cultured Mhp strains; HYW and ZZZ helped doing relevant in vivo cell cytotoxicity, cell apoptosis experiment; YB performed real-time PCR experiment; and QYX and GQS helped to revise the manuscript; ZXF conceived the study and contributed in its design and coordination. All authors read and approved the nal manuscript.   Detection of surface-exposed NFOR by ow cytometry and immunoelectron microscopy techniques.