NADH oxidase of Mycoplasma hyopneumoniae functions as a potential mediator of virulence
BMC Veterinary Research volume 18, Article number: 126 (2022)
Mycoplasma hyopneumoniae (M. hyopneumoniae) is the etiological agent of enzootic pneumonia, a highly infectious swine respiratory disease that distributed worldwide. The pathogenesis and virulence factors of M. hyopneumoniae are not fully clarified. As an important virulence factor of bacteria, nicotinamide adenine dinucleotide (NADH) oxidase (NOX) participates in host-pathogen interaction, however, the function of NOX involved in the pathogenesis of M. hyopneumoniae is not clear.
In this study, significant differences in NOX transcription expression levels among different strains of M. hyopneumoniae differed in virulence were identified, suggesting that NOX may be correlated with M. hyopneumoniae virulence. The nox gene of M. hyopneumoniae was cloned and expressed in Escherichia coli, and polyclonal antibodies against recombinant NOX (rNOX) were prepared. We confirmed the enzymatic activity of rNOX based on its capacity to oxidize NADH to NAD+. Flow cytometry analysis demonstrated the surface localization of NOX, and subcellular localization analysis further demonstrated that NOX exists in both the cytoplasm and cell membrane. rNOX was depicted to mediate adhesion to immortalized porcine bronchial epithelial cells (hTERT-PBECs). Pre-neutralizing M. hyopneumoniae with anti-rNOX antibody resulted in a more than 55% reduction in the adhesion rate of high- and low-virulence M. hyopneumoniae strains to hTERT-PBECs. Moreover, a significant difference appeared in the decline in CCU50 titer between virulent (168) and virulence-attenuated (168L) strains. NOX not only recognized and interacted with host fibronectin but also induced cellular oxidative stress and apoptosis in hTERT-PBECs. The release of lactate dehydrogenase by NOX in hTERT-PBECs was positively correlated with the virulence of M. hyopneumoniae strains.
NOX is considered to be a potential virulence factor of M. hyopneumoniae and may play a significant role in mediating its pathogenesis.
Porcine respiratory diseases, which are characterized by high morbidity and mortality, medications cost, feed efficiency and growth rate decreasing , and lower quality of pig carcasses, led to a considerable impact on the pig industry . Enzootic pneumonia (EP) is a highly infectious and prevalent, global, chronic, swine respiratory disease that is caused by Mycoplasma hyopneumoniae (M. hyopneumoniae) infection, which is characterized by nonproductive, dry coughing, severe respiratory distress, growth retardation, and inefficient feed conversion . M. hyopneumoniae is considered to be one of the primary pathogens involved in porcine respiratory disease complex (PRDC) [1, 3]. Infected pigs often develop secondary infections involving a range of other pathogens, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus (PCV), and Pasteurella multocida . This comorbidity phenomenon hinders proper diagnosis of specific diseases due to complicated symptomatic manifestations, which leads to an increased mortality rate in infected pigs. Thus, the mechanisms underlying M. hyopneumoniae pathogenesis are not fully understood. Some reports have suggested that the onset of EP depends on virulence factors of M. hyopneumoniae that enable the pathogen to override host defense mechanisms by inducing a biochemical host response that promotes cell adhesion and immunomodulation . Therefore, the identification and functional analysis of novel virulence-related genes would be helpful in building a more complete understanding of M. hyopneumoniae pathogenesis.
Several studies have also demonstrated that mycoplasma metabolic enzymes play more than a basic role by possibly serving as an important determinant of pathogenicity [6,7,8,9]. Among them, nicotinamide adenine dinucleotide (NADH) oxidases (NOX) of Mycoplasma bovis have shown possible participation in virulence . Moreover, comparative proteomics findings have demonstrated significant variations in the expression of NOX among various M. hyopneumoniae strains that differed in degree of virulence [10, 11]. NOX belongs to the largest group of enzyme oxidoreductases, which function in catalyzing the reduction of reactive superoxide species into H2O2 or H2O by simultaneously oxidizing NADH to NAD+. Various organisms contain these enzymes, which play important roles in regulating cellular redox and osmotic pressure balance to maintain normal cell growth and development. In addition to its effect on metabolic processes, NOX also plays a vital role in bacterial oxidative stress response, bacterial membrane formation, and virulence regulation . More importantly, NOX of M. hyopneumoniae has already been reported to reside on the cell surface and is suspected to bind to fibronectin, heparin, actin, and plasminogen [9, 13]. Proteins located in multiple subcellular locations with different functions are called “moonlighting” proteins . Several dozen of these proteins have been identified as ubiquitous intracellular enzymes or intracellular/ surface moonlighting proteins (ISMPs), which function in essential cellular processes . Thus, NOX of M. hyopneumoniae may be linked to its virulence.
As a novel virulence factor candidate identified in this study, NOX was found in both the cytoplasm and cell membrane of M. hyopneumoniae. NOX specifically adhered to immortalized porcine bronchial epithelial cells (hTERT-PBECs) and recognized host fibronectin. NOX not only shows its enzyme characteristics but also induces obvious cytotoxicity, oxidative stress damage, and apoptosis in hTERT-PBECs. Our findings support the notion that NOX may be a potential novel virulence factor of M. hyopneumoniae, and these findings will provide new ideas and theoretical support for studying the pathogenic mechanisms of M. hyopneumoniae and other mycoplasmas.
Significant differences in the transcription level of nox genes between high and low virulence strains of M. hyopneumoniae
To investigate mRNA expression differences of the nox gene between M. hyopneumoniae (Mhp) strains of differing virulence, relative quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed. As shown in Fig. 1, the mRNA expression levels of the nox gene were significantly upregulated in the highly virulent strains Mhp 168, LH and JS compared to the virulence-attenuated strain Mhp 168L (p < 0.001). In addition, the mRNA expression levels of the nox gene in low virulent strain J were higher than that in the virulence-attenuated strain Mhp 168L, but with no significant difference (p > 0.05).
Bioinformatic analysis, protein expression, and enzymatic activity of M. hyopneumoniae NOX
Eighteen amino acid sequences of NOX from M. hyopneumoniae were retrieved from the NCBI and UniProt Protein databases (Genbank accession numbers were shown in supplementary file), and the homologies among them were analyzed. All sequences were aligned using the CLUSTAL W program, as shown in supplementary file Fig. S1A. Molecular Evolutionary Genetics Analysis version 10 (MAGE10) was used for phylogenetic inference according to the neighbor-joining criterion. The robustness of the hypothesis was evaluated with 1000 nonparametric bootstrap analyses (Fig. S1B). The nox gene of M. hyopneumoniae contains an open reading frame of 1383 base pairs (bp) encoding 460 amino acids. The results of comparative sequence analysis indicated that the amino acid sequences of NOX among all M. hyopneumoniae strains shared more than 99.34% homology with each other. It also showed that NOX is a highly conserved protein, and there were few differences among various M. hyopneumoniae strains. Based on codon optimization, the prokaryotic expression plasmid pET-21a-nox was constructed and expressed in Escherichia coli BL21 (DE3). Expression of purified recombinant NOX protein (rNOX) with a size of 50.6 KDa was shown in panel “a” of Fig. S2. As shown in panel “b” of Fig. S2, the Western blot analysis results showed that the prepared polyclonal antibody could specifically recognize the recombinant protein rNOX. Then, the enzymatic activity of rNOX was determined after purification and renaturation. The enzymatic specific activity of the rNOX protein was determined to be 25.23 IU/mg.
NOX located both on the surface and in the cytoplasm of M. hyopneumoniae cells
To investigate whether NADH oxidase is present on the surface of M. hyopneumoniae, flow cytometry analysis was performed. The results showed that the outer membrane-localized NOX was surface-accessible to NOX-specific antibodies in Mhp strains 168 and 168L, initially suggesting that NOX antigen was present on the bacterial cell surface of M. hyopneumoniae strains. There was no significant difference in the mean fluorescence intensity (MFI) between the M. hyopneumoniae strain 168L treated with anti-rNOX serum and strain 168L incubated with pre-immune serum (Fig. 2A), whereas the MFI of strain 168 treated with anti-rNOX serum was approximately 10-fold higher than that of strain 168 treated with pre-immune serum (Fig. 2B), suggesting that NOX may correlate with the virulence of M. hyopneumoniae strains.
The whole-cell, membrane, and cytoplasmic fractions of M. hyopneumoniae were prepared for Western blot to determine the subcellular localization of NOX using rabbit anti-rNOX serum. The results showed that NOX protein was distributed in the M. hyopneumoniae cytoplasm and cell membrane, NOX was more distributed in the cytoplasm (Fig. 3). Moreover, the results also showed that the content of NOX in the membrane of virulent strain 168 was higher than that in virulence-attenuated strain 168L.
rNOX binds hTERT-PBECs and anti-rNOX serum inhibits M. hyopneumoniae adherence to hTERT-PBECs.
Indirect immunofluorescence assays (IFAs) were used to determine whether M. hyopneumoniae NOX could specifically adhere to the surface of hTERT-PBECs. The IFA results revealed significant punctate fluorescence on the cell surface of hTERT-PBECs incubated with rNOX; however, no specific punctate fluorescence was found around 6-diamidino-2-phenylindole (DAPI)-stained cell nuclei in negative controls (Fig. 4). Moreover, the results show that there was dose-dependent binding of rNOX to hTERT-PBECs, and the upper limit of concentration to saturate the host cell surface was 200 μg. However, no adhesion was observed when the lower limit concentration of rNOX was below 15 μg. Thus, it appears that rNOX may bind specifically to hTERT-PBEC membranes.
To further evaluate the role of NOX in M. hyopneumoniae adhesion to hTERT-PBECs, an antibody inhibition assay was performed. Compared with the control group treated with negative serum, the anti-rNOX serum could reduce the adherence of M. hyopneumoniae (both strains 168 and 168L) to hTERT-PBECs. The adherence degree was shown as the adhesion rate compared with the adherence of M. hyopneumoniae in the absence of antibody. Incubation with anti-rNOX antibody resulted in 52.73% (strain 168) and 57.47% (strain 168L) (p < 0.001) reductions in the adhesion rate of M. hyopneumoniae to hTERT-PBECs (Fig. 5A). In addition, pre-neutralizing M. hyopneumoniae with anti-rNOX antibody resulted in a 2.67 (strain 168; p < 0.001) and 1.25 (strain 168L; p < 0.01) reduction in the 50% color changing unit (CCU50) of M. hyopneumoniae, and there was a significant difference in CCU50 titer decline between virulent strain 168 and virulence-attenuated strain 168L (p < 0.001) (Fig. 5B). This result further confirmed that M. hyopneumoniae NOX may function as a potential adhesion factor, playing an essential role in the adherence of M. hyopneumoniae to host cells.
Identification of rNOX binding ligands
To determine whether hTERT-PBEC components interact with NOX we conducted ELISA and Far-Western blot analyses to examine the interaction between M. hyopneumoniae NOX and fibronectin. ELISA results showed that rNOX had a stronger binding ability to fibronectin than to phosphate buffered saline (PBS) and the unrelated protein (heat shock protein Hsp60 of Aeromonas hydrophila, rGroEL) (Fig. 6A). The ELISA results also indicated that rGroEL has no binding ability to fibronectin compared to that of the PBS control. As shown in panel “b” of Fig. 6B, a corresponding band was observed in the reactions of rNOX to the anti-rNOX antibody (positive control) before finally reacting to fibronectin, while no specific reaction was observed in the negative control (bovine serum albumin, BSA). All these results showed that M. hyopneumoniae NOX could specifically bind to and exhibit high affinity for fibronectin.
rNOX induced cytotoxicity, oxidative-stress damage, and apoptosis of hTERT-PBECs
As shown in Fig. 7A, rNOX induced 59.73%, 60.82%, 79.99%, 91.71% cytotoxicity at concentrations of 5 μg, 10 μg, 15 μg and 20 μg, respectively. At the same time, the cytotoxicity of strain infection controls of M. hyopneumoniae infection was 37.86% (168L), 56.45% (J), 69.10% (168) and 69.35% (JS). The cells treated with lipopolysaccharides (LPS) and dimethyl sulfoxide (DMSO) (positive controls) induced cytotoxicity at 156.43% and 183.14%, respectively.
rNOX could induce oxidative stress damage in hTERT-PBECs, as measured by the average luminescence expressed in relative luminescence units (RLUs). As shown in Fig. 7B, 20 μg rNOX induced hTERT-PBECs to produce 13735 RLU of reactive oxygen species (ROS), which was higher than that of the negative control. At the same time, there were 79386 RLU and 31405 RLU production of ROS in the positive control (cells treated with LPS and DMSO), respectively. Moreover, rNOX could not induce oxidative stress damage in hTERT-PBECs treated with low concentrations of rNOX (< 20 μg, data not shown). Up to 20 μg rNOX or more could induce oxidative stress damage in hTERT-PBECs. The ROS release from hTERT-PBECs induced by the M. hyopneumoniae strain was 27045 RLU (168L), 271068 RLU (J), 313941 RLU (168), and 535685 RLU (JS), indicating that virulence-attenuated M. hyopneumoniae strain 168L-induced ROS release against cells was significantly lower than the three virulent strains (JS, 168 and J).
Moreover, flow cytometry results indicated that rNOX could also induce apoptosis in hTERT-PBECs, and the late-stage apoptosis rate was upregulated by 87.4% compared with the negative control group. The late-stage apoptosis induced by M. hyopneumoniae strains 168 or 168L in hTERT-PTECs was significantly decreased when rNOX was blocked with an anti-rNOX antibody. The late-stage apoptotic cell percentage (upper right quadrant) decreased by 58.7% and 46.2%, respectively (Fig. 8).
Mycoplasmas are the simplest self-replicating organisms and are thought to have evolved from gram-positive bacteria through reductive evolution . As a result of their limited biosynthetic and metabolic capabilities, mycoplasmas rely on infected host cells for nutrition and possibly other metabolic needs . Bacterial pathogens, including M. hyopneumoniae , form a biofilm by adhering to the proximal, solid-phase vector, which enhances their transmission. Adhesion ability is a crucial factor that appears to correlate with bacterial virulence. Fimbriae and cell wall components are the usual constituents involved in adhesion. However, because the dramatic genomic downsizing of mycoplasmas has resulted in the loss of the cell wall, surface lipoproteins play a vital role in their adhesion to, and invasion of hosts as well as their ability to evade host immune responses [13, 17, 18]. Apart from surface lipoproteins, metabolic enzymes are also known to play a role in host-pathogen interactions . Furthermore, beyond the role of oxidoreductase in metabolism, it appears that NOX may also play a role in mediating the virulence of bacterial pathogens [7, 12].
M. hyopneumoniae is known as a host-specific pathogen that infects only pigs including domestic pigs and wild boars . Many factors in the virulence of M. hyopneumoniae have been shown to play roles in adhesion, cellular invasion, and intracellular proliferation. However, the molecular mechanisms of infection and pathogenesis of M. hyopneumoniae have remained unclear [2, 5, 19]. Previous comparative proteomics studies have demonstrated that NOX was overexpressed in the high virulent 7448 strain compared to nonpathogenic M. flocculare . Here, in this study, mRNA expression levels of the nox gene in the pathogenic 168, LH, and JS strains were significantly upregulated compared to those in the virulence-attenuated strain 168L. This result preliminarily suggests that NOX may have a correlation with the virulence of M. hyopneumoniae. Further analyses using flow cytometry showed that NOX is present on the surface of M. hyopneumoniae, which is consistent with a previous report . Subcellular localization analysis further indicated that NOX is distributed in both the M. hyopneumoniae cytoplasm and the cell membrane and, compared with the low virulence strain 168L, fluorescence intensity of the pathogenic strain 168 was greater by a factor of 10. The results of subcellular localization analysis also showed that the content of NOX in the membrane of virulent strain 168 is higher than that in virulence-attenuated strain 168L. All these results further indicated that NOX may be correlated with virulence of M. hyopneumoniae strains.
As is the case with most ISMPs, further study is needed to understand NOX production, attachment to cell surfaces, and performance in extracellular functions. Various hypotheses have been suggested regarding these processes . For example, some ISMPs are produced and found to be associated with cell surfaces. An increase in extracellular pH has been shown to cause some Lactobacillus crispatus ISMPs to be released from cell surfaces . In most cases, the components of cell surfaces (e.g.: proteins, or lipids, etc.) that bind ISMPs remain unknown. However, recent studies have shown that extracellular enolase is bound to a rhamnose residue in the cell membrane of mycoplasmas , while enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can covalently bind to lipoteichoic acid on L. crispatus , and NADH-dependent flavin oxidoreductase (NFOR) of M. hyopneumoniae has been shown to interact with host fibronectin and plasminogen . Although it is still unclear how NOX acts as an enzyme in the cell matrix, stimulating adhesion on cell surfaces, the investigations described here provide findings that can help to guide further study.
The adhesion of microorganisms, including mycoplasmas, to their host cells is a crucial step in their colonization and subsequent infection of a host. M. hyopneumoniae is commonly found on the mucosal surfaces of swine trachea, bronchi and bronchioles, where it can induce ciliostasis and loss of cilia . The first stage of pathogenesis is adhesion to cilia in epithelial cells of the respiratory mucosa by means of adhesins. In this study, we found that NOX can adhere to hTERT-PBECs which were established in our previous work . In addition, M. hyopneumoniae pre-neutralized with polyclonal antiserum to rNOX significantly reduced the adherence of M. hyopneumoniae to host cells. Fibronectin has been widely studied and determined to be a prevalent extracellular matrix protein that can form a molecular bridge between a pathogen and host cellular receptors . Several fibronectin-binding bacterial proteins were found to mediate adhesion and subsequent invasion of bacteria into host cells through binding to fibronectin . Therefore, fibronectin-binding proteins play a key role in the pathogenic process of bacteria. NOX of M. hyopneumoniae has already been shown to reside on the cell surface and is reported to bind to fibronectin, heparin, actin and plasminogen [9, 13]. Similarly, in this study, we confirmed that rNOX has a high affinity for, and can bind specifically to fibronectin. It may be possible that fibronectin functions as a primary receptor of NOX, thus mediating the adhesion process of M. hyopneumoniae.
Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that converts lactate to pyruvate. Measurement of LDH release by damaged cells offers a method for detecting cell viability from the perspective of cell membrane integrity . We found that NOX can induce dose-dependent host cell cytotoxicity, and LDH release of NOX in host cells is positively correlated with the virulence of various M. hyopneumoniae strains. ROS are generated during many cellular processes, for example aerobic respiration, but most ROS are neutralized by antioxidants. Bacterial or viral infections, inflammatory reactions, ionizing radiation, and many chemical drugs also produce a considerable amount of ROS [28, 29]. In these processes, if the level of ROS exceeds the neutralizing limit of antioxidant proteins, so-called “oxidative stress” will result. Apoptosis refers to the autonomous and orderly death of cells controlled by genes to maintain the stability of the overall intracellular and extracellular environment. Unlike cell necrosis, apoptosis is not a passive process but a phenomenon that induces autologous injury through a series of signal activations, protein expressions, and other regulations . Research has demonstrated that ROS deposition is considered to be a direct cause of apoptosis because ROS have strong cytotoxicity to cells . When intracellular ROS rise sharply, they stimulate oxidative stress and apoptosis . We found that rNOX could induce obvious oxidative stress damage in hTERT-PBECs. In addition, virulence-attenuated M. hyopneumoniae strain 168L induced ROS release that was significantly lower than the other three virulent strains (JS, 168 and J). More importantly, rNOX alone produced significantly lower levels of oxidative stress than the attenuated 168L strain. This result may indicate that M. hyopneumoniae contains other factors that lead to higher oxidative stress in infected host cells. Moreover, flow cytometry results indicated that rNOX could induce apoptosis in hTERT-PBECs as well, and the late-stage apoptosis rate was upregulated by 87.4% compared with the negative control group (4.9%) after 12 h. Late-stage apoptosis induced by high virulence M. hyopneumoniae strain 168 or low virulence strain 168L in porcine bronchial epithelial cells was significantly decreased when rNOX was blocked with anti-rNOX antibody, with the late-stage apoptotic cell percentage decreasing by 58.7% and 46.2%, respectively. In conclusion, the above results further indicate that NOX, like the positive control (various M. hyopneumoniae strains), may function as a mediator of virulence in M. hyopneumoniae strains.
The study showed that significant differences exist in the transcription levels of nox genes between high and low virulence strains, suggesting that NADH oxidase may be related with M. hyopneumoniae virulence. In addition, NADH oxidase was found in both the cytoplasm and cell membrane of M. hyopneumoniae and it has strong binding affinity with fibronectin. Besides, NADH oxidase had the ability to adhere to immortalized porcine bronchial epithelial cells and it could also induce host cell cellular oxidative stress, cytotoxicity, and apoptosis. In conclusion, NADH oxidase is considered to be a potential virulence factor candidate of M. hyopneumoniae, which may play a pivotal role in mediating its pathogenesis.
Bacterial strains, cell lines, and growth/ culture conditions
All M. hyopneumoniae strains in this study were thawed from frozen stocks and subcultured three times before use. M. hyopneumoniae strain J (ATCC25934), which was subcultured once to establish frozen stocks from ATCC. Strain 168 was isolated from a pig exhibiting typical characteristics of EP in Gansu Province, China . This field strain was gradually attenuated by continuous subculturing to the 350th passage through KM2 cell-free liquid medium (modified Friis medium) containing 20% (v/v) swine serum at 37 °C, to yield strain 168L . Thus, the strain 168L used in our work was derived from subculture 353. Strain JS is a virulent strain that can induce typical characteristics of EP with a lung lesion score of approximately 15, as described previously . Strain LH was also a virulent clinical strain isolated in our lab and capable of inducing typical characteristics of EP (GenBank accession number: CP079799) . These five M. hyopneumoniae strains were cultured in modified Friis’ medium designated KM2 cell-free medium containing 20% (v/v) swine serum (produced in our lab from a clean snatch-farrowed, porcine-colostrum-deprived (sF-pCD) piglet after irradiation sterilization) at 37 °C in a humidified incubator. Titers of M. hyopneumoniae strains were quantified using a CCU50 assay , which was modified based on the color changing unit (CCU) assay  in culture medium and confirmed by quantitative PCR.
Immortalized porcine bronchial epithelial cells (hTERT-PBECs) were established for M. hyopneumoniae infection in vitro by our laboratory . The cells were grown in Dulbecco's modified Eagle’s medium: nutrient mixture F-12 (DMEM/F12) supplemented with 2% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/mL penicillin (HyClone, Carlsbad, CA, USA), 0.1 mg/mL streptomycin (HyClone, Carlsbad, CA, USA), 15 μg/mL enrofloxacin (Aladdin, Shanghai, China), 0.1 mg/mL gentamicin, and 5 μg/mL amphotericin (Dingguo, Beijing, China), as well as additional epithelial cell growth factors (Cat No. CC-4175, Lonza, Basel, Switzerland).
Transcriptional analysis of NOX gene expression levels of different M. hyopneumoniae strains
Five M. hyopneumoniae strains (strain 168, JS, LH, J and 168L) were cultured in KM2 cell-free liquid medium containing 20% (v/v) swine serum at 37 °C for 48 h, and the titer of these five M. hyopneumoniae strains were adjusted to 1 × 108 CCU/mL before the next experiment. And then total RNA was extracted using a Total RNA Extraction Kit (Omega). No less than 1 μg of total RNA was reverse transcribed in a 10 µL reaction volume using HiScript® III RT SuperMix for qPCR (+gDNA wiper) (Cat No. R323-01, Vazyme, Nanjing, China) before running on an ABI 7500 Real Time PCR System using the ChamQ Universal SYBR qPCR Master Mix (Cat No. Q711-02, Vazyme, Nanjing, China). The reverse transcription reaction was followed the manufacturer’s protocol, and the qRT-PCR was performed using cDNA under specific conditions for the nox gene with the following procedure: samples were heated at 95 °C for 30 s, and a two-step cycle (10 s at 95 °C, 30 s at 60 °C) was repeated for 40 cycles. The MHP7448_0333 gene of M. hyopneumoniae was used as the internal control [38, 39]. The PCR primers used in the quantitative assays are listed in Supplementary Table S1. The fold change of mRNA expression of nox gene between different M. hyopneumoniae strains was determined using the 2-ΔΔCT method .
Bioinformatic analysis, cloning, expression and purification of rNOX
Eighteen amino acid sequences of NOX from M. hyopneumoniae were retrieved from the National Center for Biotechnology Information (NCBI) and UniProt protein databases, and the homologies among them were analyzed. All sequences were aligned with CLUSTAL W program (open source). Molecular Evolutionary Genetics Analysis version 10 (MAGE10) was used for assessing phylogenetic inference according to the neighbor-joining criterion. The robustness of the hypothesis was evaluated with 1000 nonparametric bootstrap analyses.
The M. hyopneumoniae nox gene (MHP168_RS00400) was synthesized by GenScript Biotech Corp. (Nanjing, China) and then expressed using the pET-21a vector in the BL21(DE3) E. coli strain. The rNOX protein was purified using high affinity Ni-Charged resin and identified by Western blot analysis. Detailed protocols are provided in supplementary file 1 (S1).
Preparation of polyclonal antibody recognizing rNOX
Two 1-month-old New Zealand white rabbits were immunized with rNOX protein. After three immunizations at 2-week intervals, sera were collected and purified using a HiTrap Protein G HP antibody purification column. Detailed protocols are provided in supplementary file 1 (S1).
Enzymatic activity assays
The enzymatic activity of purified rNOX was determined by measuring the oxidation of NADH to NAD+ at a temperature of 25 °C as previously described with modifications [7, 41]. The reaction system contained 0.1 M potassium phosphate buffer (pH 7.5) and 1 mM dithiothreitol. The reaction system had a volume of 2 mL, purified rNOX was added at a final concentration of 5 μg/mL, and 10 μM flavin adenine dinucleotide (FAD) was added (Cat No. F100990, Aladdin, Shanghai, China). NADH (0.5 mM) Cat No. N106933, Aladdin, Shanghai, China) was added followed by incubation for 5 min at 25 °C. The optical density (OD) at 340 nm (OD340) was measured. The specific activity of rNOX was calculated as follows.
Detection of surface‑exposed NOX by flow cytometry
To investigate whether NOX is present on the surface of M. hyopneumoniae strains and to probe NOX surface distribution differences between highly virulent strain 168 and virulence-attenuated strain 168L, fluorescence intensity was measured using a flow cytometer as previously described with modifications . Briefly, M. hyopneumoniae strains 168 and 168L (each 1 × 108 CCU/mL) were incubated with anti-rNOX serum at a 1:100 dilution (1:100 diluted pre-immune serum was used as a negative control). M. hyopneumoniae strains were then stained with fluorescein isothiocyanate (FITC)-conjugated anti-IgG (Cat No. BA1105, Boster, Wuhan, China), and the fluorescence intensity was measured using a BD Accuri C6 flow cytometer.
Subcellular localization of NOX in different virulent M. hyopneumoniae strains
Membrane proteins and cytoplasmic proteins of M. hyopneumoniae were obtained using a membrane protein and cytoplasmic protein extraction kit (Cat No. BB-3111, Bestbio, Shanghai, China) according to the manufacturer’s instructions. At the same time, the washed bacterial precipitate was resuspended in PBS and sonicated to prepare whole bacterial protein. Protein concentration was determined by the BCA Protein Assay Kit (Cat No. P0012S, Beyotime, Shanghai, China). Subcellular localization of NADH oxidase in various M. hyopneumoniae strains that differed in virulence was assessed by Western blot. Detailed protocols are provided in supplementary file 1 (S1).
Adherence of rNOX to hTERT-PBECs
hTERT-PBECs were grown to confluence in 24-well plates with DMEM/F12 medium supplemented with 2% (v/v) fetal bovine serum plus the abovementioned growth factors. Cultured hTERT-PBECs were washed with PBS three times and fixed with 4% paraformaldehyde for 10 min at room temperature. Subsequently, the cells were treated with 0.1% Triton X-100 and then blocked for 2 h in 3% (w/v) BSA in PBS. After incubation with different concentrations of purified rNOX (from 5 to 500 μg), the cells were washed three times with PBS and incubated with a 1:250 dilution anti-rNOX antibody for 2 h at 37 °C. Following three PBS washes, cells were incubated with tetraethyl rhodamine isothiocyanate (TRITC)-tagged anti-IgG (Cat No. SA00007-2, Proteintech, Rosemont, IL, USA) at a 1:100 dilution for 1 h at 37 °C. Finally, nuclei were stained with DAPI (Cat No. D8417, Sigma-Aldrich, St Louis, MO, USA) before cells were observed using a fluorescence microscope (Zeiss). BSA and rGroEL (the cytoplasmic chaperone protein of Aeromonas hydrophila)  were used instead of rNOX as the negative control.
Inhibition of adherence using antibody recognizing rNOX
M. hyopneumoniae bacteria (strains 168 and 168L, 1 × 107 CCU/mL) were collected and washed three times with PBS before resuspension in 500 µL PBS. The samples were preincubated with polyclonal antibody raised against rNOX or pre-immune sera (1:20 dilution) at 37 °C for 30 min. hTERT-PBECs with a confluence of 85% in 24-well cell plates were washed with PBS three times before the experiment. Bacteria suspended in DMEM/F12 medium were added to cells seeded in 24-well cell plates, and the cell plates were centrifuged at 800 × g for 10 min and then incubated for 2 h at 4 °C. After washing with PBS three times, the cells in each group were treated with lysis buffer containing 0.1% trypsin. Bacterial counting, including bacterial genome extraction and real-time PCR, was performed according to a previous method . The real-time PCR primers used in the quantitative assays are listed in Supplementary Table S1. In addition, the titers of different M. hyopneumoniae were quantified using a CCU50 assay as mentioned above. These tests were performed in triplicate, and data were analyzed using GraphPad Prism 6 and SPSS 20.0.
Identification of rNOX-binding ligands
As previously described with some modifications , the recombinant protein rNOX and the negative control proteins rGroEL were serially diluted twofold from 2 μg/ml to 0.5 μg/mL, and PBS was used as a blank control rather than rNOX. For each dilution, 100 μL per well was coated (in triplicate) in a 96-well plate. The wells were blocked with 5% BSA (w/v) in PBS containing 0.5% Tween 20 (PBST) for 2 h at 37 °C. Two hundred micrograms of fibronectin (Cat No. F1056, Sigma–Aldrich, St Louis, MO, USA) in 100 μL of PBST was applied to the wells, and ELISA plates were incubated at 37 °C for 1 h. Wells were washed three times with PBST and incubated with a 1:2000 dilution of anti-fibronectin antibody (Cat No. ab299, Abcam, Cambridge, UK) for 1 h at 37 °C. Following three washes with PBST, the wells were incubated with HRP-conjugated goat anti-rabbit IgG antibody (Cat No. BA1055, Boster, Wuhan, China, 1:5000 in PBST) at 37 °C for 1 h. Finally, a 3,3',5,5'-Tetramethylbenzidine (TMB) (Cat No. P0209, Beyotime, Shanghai, China) substrate was applied at 37 °C, and the wells were treated with the stop solution (2 M H2SO4). OD readings were obtained at a wavelength of 450 nm with an ELISA microplate reader (Bio-Tek, Winooski, VT, USA).
Each 20 μg sample of rNOX was resolved by 10% SDS-PAGE and then transferred to a PVDF membrane (Cat No. IPFL00010, Millipore, Darmstadt, Germany). The membranes were washed three times with PBS and then blocked with 5% skimmed milk in TBST (TBS containing 0.5% Tween 20) at 37 °C for 2 h. Subsequently, the membranes were incubated with 15 μg/mL fibronectin (Cat No. F1056, Sigma-Aldrich, St Louis, MO, USA) for 2 h at 37 °C. Following three washes using TBST, the membranes were incubated with anti-fibronectin antibody (Cat No. ab299, Abcam, Cambridge, UK, 1:1000 dilution) in blocking solution at 37 °C for 2 h, after which they were washed three times with TBST followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cat No. BA1055, Boster, Wuhan, China) at 37 °C for 2 h. Finally, the membranes were developed with Electro-Chemi-Luminescence (ECL, Cat No. 32109, ThermoFisher, Rockford, IL, USA) substrate using a ChemiDoc XRS+ system (Bio–Rad). BSA was used as a negative control rather than rNOX.
Quantification of lactate dehydrogenase (LDH) Release
hTERT-PBECs were incubated for 6 h and grown to confluence in 24-well plates. After three washes with PBS, the cells were then incubated with purified rNOX protein at different concentrations (5 μg, 10 μg, 15 μg, and 20 μg). PBS was used as a negative control rather than rNOX, and LPS (5 μg/mL), DMSO (1% diluted in DMEM/F12 medium), and four M. hyopneumoniae strains (strains JS, 168, J and 168L, titers of which were 1 × 108 CCU/mL) were used as positive controls. All groups were performed with three independent replicates. After 6 h, culture supernatants were collected, and LDH activity was measured using a CytoTox 96® Non-Radioactive Cytotoxicity Assay (Cat No. G1780, Promega, Madison, WI, USA) according to the manufacturer’s instructions.
hTERT-PBECs were seeded and grown to confluence in 24-well plates. The cells were washed with PBS followed by incubation with purified rNOX protein of different concentrations (from 5 to 20 μg) at 37 °C for 6 h. At the same time, PBS was used as a negative control, and cells treated with LPS (5 μg/mL), DMSO (1%), and four M. hyopneumoniae strains (strain JS, 168, J and 168L, 1 × 108 CCU/mL) were used as positive controls. All tests were performed with five independent replicates. Reactive oxygen species (ROS) were measured using a ROS-Glo™ H2O2 Assay (Cat No. G8820, Promega, Madison, WI, USA) according to the manufacturer’s instructions, and RLUs were recorded using a plate reader.
Induced apoptosis in hTERT-PBECs measured by flow cytometry
hTERT-PBECs were seeded in 24-well plates at 2×105 cells/well in 500 µL of culture medium. Then, hTERT-PTECs were incubated with 20 μg purified rNOX at 37 °C for 12 h. Cells cultured in DMEM/F12 plus 2% FBS culture medium supplemented with epithelial growth factors F12 without the reagents mentioned above were used as a negative control, and cells incubated with M. hyopneumoniae strains (strains 168 and 168L, 1 × 108 CCU/mL) were used as positive controls. At the same time, to explore whether antiserum to rNOX, which could block the apoptosis induced by M. hyopneumoniae in hTERT-PBECs, we pre-incubated M. hyopneumoniae strains (168 and 168L) with anti-rNOX antibody (1:20 dilution) at 37 °C for 30 min before inoculation with hTERT-PBEC seeds in 24-well plates at 37 °C for 12 h. All groups were performed with three independent replicates. Apoptosis rates were detected with the dual staining Annexin V- fluorescein isothiocyanate / propidium iodide (Annexin V- FITC/PI) method using an Annexin V-FITC/PI Apoptosis Detection Kit (Cat No. A211, Vazyme, Nanjing, China).
All experiments were repeated in triplicate, except for the detection of ROS, which was conducted in five independent replicates. Data represent the mean ± standard deviation (SD) of three or five individual experiments. Relative mRNA expression differences of the nox gene between the virulence-attenuated strain 168L and the virulent Mhp strains 168, JS, LH and J were assessed via one-way analysis of variance (ANOVA) using SPSS Statics v20.0 software and GraphPad Prism 6 software. The adhesion rates between anti-NOX serum or negative serum and groups of the high-virulence strain 168 as well as the adhesion rates between the low-virulence strain 168L and anti-NOX serum or negative serum were compared by Student’s t test. P < 0.05 was considered statistically significant, P < 0.01 was considered highly significant, and P < 0.001 was considered extremely significant.
Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files.
analysis of variance
bovine serum albumin
color changing unit
- CCU50 :
50% color changing unit
Dulbecco's modified Eagle’s medium nutrient mixture F-12
flavin adenine dinucleotide
heat shock protein Hsp60
immortalized porcine bronchial epithelial cells
Indirect immunofluorescence assay
intracellular enzymes or intracellular / surface moonlighting proteins
Molecular Evolutionary Genetics Analysis version 10
the mean fluorescence intensity
nicotinamide adenine dinucleotide
National Center for Biotechnology Information
NADH-dependent flavin oxidoreductase
phosphate buffered saline
phosphate buffered saline containing 0.5% Tween 20
porcine respiratory disease complex
porcine reproductive and respiratory syndrome virus
quantitative polymerase chain reaction
recombinant NOX protein
relative luminescence units
reactive oxygen species
reverse transcription polymerase chain reaction
tetraethyl rhodamine isothiocyanate
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We thank Jin Wang of Nanjing Agricultural University for assistance in performing cell apoptosis analysis. We are grateful for the editing of our manuscript by an English professional with a scientific background at American Journal Experts, which can be verified at www.aje.com/certificate (the verification code: C9DD-5034-0731-8FAD-F354).
This work was supported by Jiangsu Agriculture Science and Technology Innovation Fund (CX(21)3124); the Independent Research Project Program of Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base, Ministry of Science and Technology, China (2019sy004); National Natural Science Foundation of China (31800161; 31800160); Natural Science Foundation of Jiangsu Province (BK20180297).
Ethics approval and consent to participate
The animal experimental procedure, including sample collection, was performed in strict accordance with the animal regulations of Jiangsu Province (Government Decree No. 45). All animal experiments were approved by the Committee on the Ethics of Animal Experiments (Protocol # PDC2018005) and performed at Jiangsu Academy of Agricultural Sciences (License No. SYXK (Su) 2015-0019, SYXK (Su) 2020-0023). The study was carried out in compliance with the ARRIVE guidelines.
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Table S1. Primers used in this study. Fig. S1. (A) Multiple sequence alignment of proteins NOX from different M. hyopneumoniae strains. (B) Phylogenetic analysis based on the NOX proteins of different M. hyopneumoniae strains by the neighbor-joining (NJ) method with the sequences available in GenBank and Uniprot Database. The numbers at the phylogenetic branches indicated the bootstrap values (1000 replicates) in percentage supporting each group. The bar represents the genetic distance. Fig. S2. Prokaryotic expression and purification of recombinant protein, WB verification of prepared polyclonal antibody. “a”, Lane M is prestained protein mass markers, with recombinant protein rNOX size of 50.6 KDa. The prepared polyclonal antibody effect of the recombinant protein rNOX was tested by Western Blot. “b”, a clear band appeared at position of 50.6 KDa. Lane 1, purified recombinant protein rNOX. Lane 2, unpurified E. coli whole bacteria protein before purification. Lane M, prestained protein mass marker. Fig. S3. SDS–PAGE analysis of purified recombinant protein of rNOX, other proteins of M. hyopneumoniae (Mhp) and BSA. Lane M, prestained protein mass marker. Lane 1, purified recombinant protein rNOX. Lane 2, bovine serum albumin (BSA). Lane 3, purified recombinant nicotinamide adenine dinucleotide-dependent flavin oxidoreductase (NFOR) protein of Mhp. Lane 4 and 6, blank. Lane 5 and 7, purified recombinant leucine aminopeptidase (LAP) protein of Mhp. Fig. S4. M. hyopneumoniae proteins with fibronectin interaction analysis by far Western blot. Lane M, prestained protein mass marker. Lane 1, purified recombinant protein rNFOR. PVDF membrane with transferred Mhp NFOR protein incubated with fibronectin and the anti-fibronectin antibody. Lane 2, 4 and 8, BSA, PVDF membrane with transferred BSA (negative control) incubated with fibronectin and the anti-fibronectin antibody. Lane 3, purified recombinant protein rNOX. PVDF membrane with transferred Mhp NOX protein incubated with fibronectin and the anti-fibronectin antibody. Lane 5, 6, 7 and 9, purified recombinant protein rLAP. PVDF membrane with transferred Mhp LAP protein incubated with fibronectin and the anti-fibronectin antibody.
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Hao, F., Xie, X., Feng, Z. et al. NADH oxidase of Mycoplasma hyopneumoniae functions as a potential mediator of virulence. BMC Vet Res 18, 126 (2022). https://doi.org/10.1186/s12917-022-03230-7