Apigenin suppresses mycoplasma-induced alveolar macrophages necroptosis via enhancing the methylation of TNF-α promoter by PPARγ-Uhrf1 axis.

BACKGROUND: Mycoplasma-associated pneumonia is characterized by severe lung inflammation and immunological dysfunction. However, current anti-mycoplasma agents used in clinical practice do not prevent dysfunction of alveolar macrophages caused by the high level of the cytokine tumor necrosis factor-α (TNF-α) after mycoplasma infection. Apigenin inhibits the production of TNF-α in variet inflammation associated disease. PURPOSE: This study aimed to investigate apigenin's effect on mycoplasma-induced alveolar immune cell injury and the mechanism by which it inhibits TNF-α transcription. METHODS: In this study, we performed a mouse model of Mycoplasma hyopneumoniae infection to evaluate the effect of apigenin on reducing mycoplasma-induced alveolar immune cell injury. Furthermore, we carried out transcriptome analysis, RNA interference assay, methylated DNA bisulfite sequencing assay, and chromatin immunoprecipitation assay to explore the mechanism of action for apigenin in reducing TNF-α. RESULTS: We discovered that M. hyopneumoniae infection-induced necroptosis in alveolar macrophages MH-S cells and primary mouse alveolar macrophages, which was activated by TNF-α autocrine. Apigenin inhibited M. hyopneumoniae-induced elevation of TNF-α and necroptosis in alveolar macrophages. Apigenin inhibited TNF-a mRNA production via increasing ubiquitin-like with PHD and RING finger domains 1 (Uhrf1)-dependent DNA methylation of the TNF-a promotor. Finally, we demonstrated that apigenin regulated Uhrf1 transcription via peroxisome proliferator activated receptor gamma (PPARγ) activation, which acts as a transcription factor binding to the Uhrf1 promoter and protected infected mice's lungs, and promoted alveolar macrophage survival. CONCLUTSION: This study identified a novel mechanism of action for apigenin in reducing alveolar macrophage necroptosis via the PPARγ/ Uhrf1/TNF-α pathway, which may have implications for the treatment of Mycoplasma pneumonia.

Identification of the multiple roles of enolase as an plasminogen receptor and adhesin in Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the etiological agent underlying porcine enzootic pneumonia, a chronic respiratory disease worldwide. The recruitment of plasminogen to the surface and subsequently promotion of plasmin conversion by the surface-located receptor, have been reported to assist the adhesion and invasion of Mycoplasmas. The surface localization and plasminogen-binding ability of M. hyopneumoniae enolase were previously confirmed; however, the biological functions were not be determined, especially the role as a plasminogen receptor. Here, using ELISA and SPR analyses, we confirmed the stable binding of M. hyopneumoniae enolase to plasminogen in a dose-dependent manner. The facilitation of the activation of plasminogen in the presence of tPA and direct activation of plasminogen at low efficiency without tPA addition by M. hyopneumoniae enolase were also determined using a plasmin-specific chromogenic substrate. Notably, the C-terminal and N-terminal regions located in M. hyopneumoniae enolase play an important role in plasminogen binding and activation. Additionally, we demonstrate that M. hyopneumoniae enolase can competitively inhibit the adherence of M. hyopneumoniae to PK15 cells. These results provide insight into the role of enolase in M. hyopneumoniae infection, a mechanism that manipulates the proteolytic system of the host.

Genotyping and biofilm formation of Mycoplasma hyopneumoniae and their association with virulence.

Mycoplasma hyopneumoniae, the causative agent of swine respiratory disease, demonstrates differences in virulence. However, factors associated with this variation remain unknown. We herein evaluated the association between differences in virulence and genotypes as well as phenotype (i.e., biofilm formation ability). Strains 168 L, RM48, XLW-2, and J show low virulence and strains 232, 7448, 7422, 168, NJ, and LH show high virulence, as determined through animal challenge experiments, complemented with in vitro tracheal mucosa infection tests. These 10 strains with known virulence were then subjected to classification via multilocus sequence typing (MLST) with three housekeeping genes, P146-based genotyping, and multilocus variable-number tandem-repeat analysis (MLVA) of 13 loci. MLST and P146-based genotyping identified 168, 168 L, NJ, and RM48 as the same type and clustered them in a single branch. MLVA assigned a different sequence type to each strain. Simpson's index of diversity indicates a higher discriminatory ability for MLVA. However, no statistically significant correlation was found between genotypes and virulence. Furthermore, we investigated the correlation between virulence and biofilm formation ability. The strains showing high virulence demonstrate strong biofilm formation ability, while attenuated strains show low biofilm formation ability. Pearson correlation analysis revealed a significant positive correlation between biofilm formation ability and virulence. To conclude, there was no association between virulence and our genotyping data, but virulence was found to be significantly associated with the biofilm formation ability of M. hyopneumoniae.

Phenotypic characteristics and protective efficacy of an attenuated Mycoplasma hyopneumoniae vaccine by aerosol administration.

With the improvement of large-scale breeding in pig farms, conventional head-by-head immunization has disadvantages with low efficiency and high cost. Considering that most pathogens leading to pulmonary diseases circulate from the respiratory mucosa, immunization through the respiratory tract route has been a highly attractive vaccine delivery strategy. In this study, to develop an effective Mycoplasma hyopneumoniae (Mhp) aerosol vaccine, a customized ultrasonic atomizer was developed. The aerodynamic diameter, activity, and content of the Mhp aerosol vaccine were measured. In addition, piglets were immunized with the Mhp aerosol vaccine, and the immunity of the animal challenge protection test was evaluated. At the end of nebulization, the mass median aerodynamic diameters (MMAD) and geometric standard deviation (GSD) of the aerosol were 2.98 ± 0.02 µm and 1.51 ± 0.02, respectively. Moreover, 10 min after nebulization, the MMAD and GSD of the aerosol were 2.76 ± 0.02 µm and 1.51 ± 0.01, respectively, which were hardly changed. Compared with theoretical value, the actual titer of aerosol vaccines presented in 50% color changing unit (CCU50) after nebulization decreased 0.6. The shape, size, and uniformity of collected aerosols are relatively stable. The proportion of Mhp in aerosol produced by vaccine stock solution and 10 times diluted vaccine solution was 76.52% and 58.82%, respectively, and the average number of Mhp in a single aerosol was 3.06 and 1.51, respectively. In addition, the aerosol vaccine antigen particles could be transported to the lower respiratory tract, a local mucosal immune response was induced in piglets. The vaccine colonized the respiratory tract and significantly decline the lung lesion index after aerosol vaccination. In conclusion, an effective aerosol vaccine against Mhp infection was developed. And this is the first effective assessment for Mhp live vaccine with aerosolization against infection in piglets.

Characterization of Mutations in DNA Gyrase and Topoisomerase IV in Field Strains and In Vitro Selected Quinolone-Resistant Mycoplasma hyorhinis Mutants.

Mycoplasma hyorhinis is ubiquitous in swine, and it is a common pathogen of swine that causes polyserositis, arthritis, and maybe pneumonia. Fluoroquinolones are effective antimicrobials used for the treatment of mycoplasmal infection. However, a decrease in fluoroquinolones susceptibility in mycoplasma was observed. The molecular mechanisms have been studied in many mycoplasma species, while the mechanism in M. hyorhinis is still unknown. This study aimed to illustrate the in vitro development of fluoroquinolone resistance in M. hyorhinis and unveil the resistance mechanisms in both in vitro selected mutants and field strains. Seven ciprofloxacin-sensitive M. hyorhinis isolates were chosen to induce the fluoroquinolone resistance in vitro, and the point mutations in the quinolone resistance-determining regions (QRDRs) were characterized. The substitutions first occurred in ParC, resulting in a 2- to 8-fold increase in resistance, followed by additional mutations in GyrA and/or ParE to achieve a 32-fold increase. The mutations occurred in hot spots of QRDRs, and they were diverse and variable, including five in ParC (Ser80Phe, Ser80Tyr, Phe80Tyr, Glu84Gly, and Glu84Lys), four in GyrA (Ala83Val, Ser84Pro, Asp87Tyr, and Asp87Asn) and one in ParE (Glu470Lys). Target mutations in field strains were observed in the ParC (Ser80Phe, Ser81Pro, and Glu84Gln) of isolates with MICCIP = 2 µg/mL. This study characterized the point mutations in the QRDRs of M. hyorhinis and could be useful for the rapid detection of fluoroquinolone resistance in M. hyorhinis field isolates.

NADH oxidase of Mycoplasma hyopneumoniae functions as a potential mediator of virulence.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: NOX is considered to be a potential virulence factor of M. hyopneumoniae and may play a significant role in mediating its pathogenesis.

A multifunctional enolase mediates cytoadhesion and interaction with host plasminogen and fibronectin in Mycoplasma hyorhinis.

Mycoplasma hyorhinis may cause systemic inflammation of pigs, typically polyserositis and arthritis, and is also associated with several types of human cancer. However, the pathogenesis of M. hyorhinis colonizing and breaching the respiratory barrier to establish systemic infection is poorly understood. Glycolytic enzymes are important moonlighting proteins and virulence-related factors in various bacteria. In this study, we investigated the functions of a glycolytic critical enzyme, enolase in the infection and systemic spread of M. hyorhinis. Bacterial surface localization of enolase was confirmed by flow cytometry and colony hybridization assay. Recombinant M. hyorhinis enolase (rEno) was found to adhere to pig kidney (PK-15) cells, and anti-rEno serum significantly decreased adherence. The enzyme was also found to bind host plasminogen and fibronectin, and interactions were specific and strong, with dissociation constant (KD) values of 1.4 nM and 14.3 nM, respectively, from surface plasmon resonance analysis. Activation of rEno-bound plasminogen was confirmed by its ability to hydrolyze plasmin-specific substrates and to degrade a reconstituted extracellular matrix. To explore key sites during these interactions, C-terminal lysine residues of enolase were replaced with leucine, and the resulting single-site and double-site mutants show significantly reduced interaction with plasminogen in far-Western blotting and surface plasmon resonance tests. The binding affinities of all mutants to fibronectin were reduced as well. Collectively, these results imply that enolase moonlights as an important adhesin of M. hyorhinis, and interacts with plasminogen and fibronectin. The two lysine residues in the C-terminus are important binding sites for its multiple binding activities.

DnaK Functions as a Moonlighting Protein on the Surface of Mycoplasma hyorhinis Cells.

Mycoplasma hyorhinis is a common pathogen of swine and is also associated with various human tumors. It causes systemic inflammation, typically polyserositis and polyarthritis, in some infected pigs. However, the pathogenic mechanism of M. hyorhinis remains unclear. DnaK is a highly conserved protein belonging to the heat-shock protein 70 family of molecular chaperones, which plays important roles as a moonlighting protein in various bacteria. In the present study, we identified the surface exposure of M. hyorhinis DnaK. Two virulent strains expressed more DnaK on their surface than the avirulent strain. Thereafter, the potential moonlighting functions of DnaK were investigated. Recombinant M. hyorhinis DnaK (rMhr-DnaK) was found to be able to adhere to swine PK-15 cells and human NCI-H292 cells. It also bound to four extracellular matrix components-fibronectin, laminin, type IV collagen, and vitronectin-in a dose-dependent manner. ELISA demonstrated an interaction between rMhr-DnaK and plasminogen, which was significantly inhibited by a lysine analog, ε-aminocaproic acid. rMhr-DnaK-bound plasminogen was activated by tissue-type plasminogen activator (tPA), and the addition of rMhr-DnaK significantly enhanced the activation. Finally, a DnaK-specific antibody was detected in the serum of pigs immunized with inactivated vaccines, which indicated good immunogenicity of it. In summary, our findings imply that DnaK is an important multifunctional moonlighting protein in M. hyorhinis and likely participates extensively in the infection and pathogenesis processes of M. hyorhinis.

Development of an indirect competitive enzyme linked immunosorbent assay for the quantitative detection of Mycoplasma hyopneumoniae during the vaccine production process.

Inactivated Mycoplasma hyopneumoniae vaccine is used extensively to control M. hyopneumoniae infection worldwide. Quantification techniques are essential in the process of standardizing and validating vaccines. In this study, we developed and optimized an indirect competitive enzyme linked immunosorbent assay (ic-ELISA) for the rapid quantification of M. hyopneumoniae antigen during vaccine production. Briefly, whole M. hyopneumoniae antigen was coated onto microtiter plates, and a polyclonal antibody against M. hyopneumoniae recombinant elongation factor thermo unstable (EF-Tu) protein was prepared and added with the samples to be tested. The methods were optimized and showed significant reproducibility, with coefficients of variation of 4.01% and 6.14% for the intra-and inter-assays, respectively. Quantification of M. hyopneumoniae cultures at different growth stages using the ic-ELISA test showed a similar curve to that of the traditional color changing units (CCU) assay, with a delay in the time when the amount reached the peak and started to fall. In the inactivated vaccine production process, the cultures could be harvested later than that for the live vaccine, at about 12 h after the end of the logarithmic growth phase. Different batches of cultures were measured for their relative potency value compared with the in-house reference vaccine, which was used to determine whether the cultures met the antigen amount requirements for vaccine preparation. The curves of the CCU titer and ic-ELISA titer in the logarithmic phase correlated strongly and a linear regression equation was established to calculate the CCU values rapidly using the ic-ELISA results. In conclusion, an ic-ELISA method was established to rapidly assess the amount of antigen in an M. hyopneumoniae culture during the vaccine production process.

Nicotinamide Adenine Dinucleotide-Dependent Flavin Oxidoreductase of Mycoplasma hyopneumoniae Functions as a Potential Novel Virulence Factor and Not Only as a Metabolic Enzyme.

Mycoplasma hyopneumoniae (Mhp) is the main pathogen that causes enzootic pneumonia, a disease that has a significant impact on the pig industry worldwide. The pathogenesis of enzootic pneumonia, especially possible virulence factors of Mhp, has still not been fully elucidated. The transcriptomic and proteomic analyses of different Mhp strains reported in the literature have revealed differences in virulence, and differences in RNA transcription 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 from Mhp were performed, a rabbit-derived polyclonal antibody against NFOR was prepared, and multiple sequence alignment and evolutionary analyses 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 to the cell surface and could adhere to immortalized porcine bronchial epithelial cells (hTERT-PBECs). Adhesion to hTERT-PBECs could be specifically inhibited by an anti-NFOR polyclonal antibody, and the rates of adhesion to both high- and low-virulence strains, 168 and 168L, significantly decreased by more than 40%. Moreover, Mhp NFOR not only recognized and interacted with host fibronectin and plasminogen but also induced cellular oxidative stress and apoptosis in hTERT-PBECs. The release of lactate dehydrogenase by hTERT-PBECs incubated with Mhp NFOR was significantly positively correlated with the virulence of Mhp. 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 of Mhp; these findings provide new ideas and theoretical support for studying the pathogenic mechanisms of other mycoplasmas.

Long-Residence Pneumonia Vaccine Developed Using PEG-Grafted Hybrid Nanovesicles from Cell Membrane Fusion of Mycoplasma and IFN-γ-Primed Macrophages.

CD8+ T cell responses play a critical regulatory role in protection against mycoplasma infection-related respiratory diseases. Nanovesicles derived from cell membranes have been shown to induce CD8+ T cell responses. Moreover, the short residence time of mycoplasma membrane-related vaccines in local lymph nodes limits the efficacy of current mycoplasma vaccines. Here, a long-residence pneumonia vaccine is developed using nanovesicles prepared by cell membrane fusion of Mycoplasma hyopneumoniae and interferon-γ (IFN-γ  )-primed macrophages, which are grafted with polyethylene glycol to increase residence time in the lymph nodes. Upregulation of intercellular adhesion molecule-1 (ICAM-1) on the membrane of IFN-γ-primed macrophages increases the targeting of the hybrid nanovesicle vaccine to the local lymph nodes, with increased CD8+ T cell activation. A mechanistic study reveals that CD8+ T cell activation is achieved via a pathway involving upregulation of C-C motif chemokine ligand 2/3 expression by E26 transformation-specific sequences, followed by increased immune-stimulatory activity of dendritic cells. In vivo, prophylactic testing reveals that the hybrid nanovesicle vaccine triggers a long-term immune response, as evidenced by a memory CD8+ T cell response against mycoplasma infection. The current study provides a new design strategy for mycoplasma vaccines that involves a hybrid method using biological sources and artificial modification.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) moonlights as an adhesin in Mycoplasma hyorhinis adhesion to epithelial cells as well as a plasminogen receptor mediating extracellular matrix degradation.

Mycoplasma hyorhinis infects pigs causing polyserositis and polyarthritis, and has also been reported in a variety of human tumor tissues. The occurrence of disease is often linked with the systemic invasion of the pathogen. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), one of the key enzymes of glycolysis, was reported as a surface multifunctional molecule in several bacteria. Here, we investigated whether GAPDH could manifest binary functions; as an adhesin to promote colonization as well as a plasminogen receptor functioning in extracellular matrix (ECM) degradation to promote systemic invasion. The surface localization of GAPDH was observed in M. hyorhinis with flow cytometry and colony blot analysis. Recombinant GAPDH (rGAPDH) was found to be able to bind porcine-derived PK-15 and human-derived NCI-H292 cells. The incubation with anti-GAPDH antibody significantly decreased the adherence of M. hyorhinis to both cell lines. To investigate its function in recruiting plasminogen, firstly, the interaction between rGAPDH and plasminogen was demonstrated by ELISA and Far-Western blot assay. The activation of the rGAPDH-bound plasminogen into plasmin was proved by using a chromogenic substrate, and furtherly confirmed to degrade extracellular matrix by using a reconstituted ECM. Finally, the ability of rGAPDH to bind different ECM components was demonstrated, including fibronectin, laminin, collagen type IV and vitronectin. Collectively, our data imply GAPDH as an important adhesion factor of M. hyrohinis and a receptor for hijacking host plasminogen to degrade ECM. The multifunction of GAPDH to bind both plasminogen and ECM components is believed to increase the targeting of proteolysis and facilitate the dissemination of M. hyorhinis.

Paracellular Pathway-Mediated Mycoplasma hyopneumoniae Migration across Porcine Airway Epithelial Barrier under Air-Liquid Interface Conditions.

Mycoplasma hyopneumoniae is an important respiratory pathogen of pigs that causes persistent and secondary infections. However, the mechanisms by which this occurs are unclear. In this study, we established air-liquid interface culture systems for pig bronchial epithelial cells (ALI-PBECs) that were comparable to the conditions in the native bronchus in vivo We used this ALI-PBECs model to study the infection and migration characteristics of M. hyopneumoniaein vitro Based on the results, we confirmed that M. hyopneumoniae was able to adhere to ALI-PBECs and disrupt mucociliary function. Importantly, M. hyopneumoniae could migrate to the basolateral chamber through the paracellular route but not the transcellular pathway, and this was achieved by reversibly disrupting tight junctions (TJs) and increasing the permeability and damaging the integrity of the epithelial barrier. We examined the migration ability of M. hyopneumoniae using an ALI-PBECs model for the first time. The disruption of the epithelial barrier allowed M. hyopneumoniae to migrate to the basolateral chamber through the paracellular route, which may be related to immune evasion, extrapulmonary dissemination, and persistent infection of M. hyopneumoniae.

Fructose-1,6-bisphosphate aldolase encoded by a core gene of Mycoplasma hyopneumoniae contributes to host cell adhesion.

Mycoplasma hyopneumoniae is an important respiratory pathogen that causes great economic losses to the pig industry worldwide. Although some putative virulence factors have been reported, pathogenesis remains poorly understood. Herein, we evaluated the relative abundance of proteins in virulent 168 (F107) and attenuated 168L (F380) M. hyopneumoniae strains to identify virulence-associated factors by two-dimensional electrophoresis (2-DE). Seven proteins were found to be ≥ 1.5-fold more abundant in 168, and protein-protein interaction network analysis revealed that all seven interact with putative virulence factors. Unexpectedly, six of these virulence-associated proteins are encoded by core rather than accessory genomic elements. The most differentially abundant of the seven, fructose-1,6-bisphosphate aldolase (FBA), was successfully cloned, expressed and purified. Flow cytometry demonstrated the surface localisation of FBA, recombinant FBA (rFBA) mediated adhesion to swine tracheal epithelial cells (STEC), and anti-rFBA sera decreased adherence to STEC. Surface plasmon resonance showed that rFBA bound to fibronectin with a moderately strong KD of 469 nM. The results demonstrate that core gene expression contributes to adhesion and virulence in M. hyopneumoniae, and FBA moonlights as an important adhesin, mediating binding to host cells via fibronectin.

Application of a sIgA-ELISA method for differentiation of Mycoplasma hyopneumoniae infected from vaccinated pigs.

In order to evaluate the sIgA-ELISA method reported previously for differentiating Mycoplasma hyopneumoniae (M. hyopneumoniae) infected from vaccinated pigs, dynamics of anti-M. hyopneumoniae secretory IgA (sIgA) antibody secretion in nasal mucus and IgG antibodies in serum from 10 pigs experimentally infected with M. hyopneumoniae or vaccinated with an inactivated vaccine were examined using sIgA-ELISA and a commercial M. hyopneumoniae antibody detection kit (IgG-ELISA), respectively. In addition, nasal swabs and serum samples from 2368 pigs of different ages originating from 10 pig farms with different M. hyopneumoniae infection and vaccination status were examined using the two ELISA. In the experimental model, anti-M. hyopneumoniae IgG antibodies were detected in both, the challenge group and the vaccine group. Anti-M. hyopneumoniae sIgA antibodies were detected in the challenge group from 7 days post challenge onwards, but not in the vaccine group. According to the data obtained from pig farms maintaining administration of inactivated vaccine, the prevalence of anti-M. hyopneumoniae sIgA antibody positive pigs was significantly lower than that of IgG antibody positive pigs. In non-vaccinating herds, the prevalence of sIgA antibodies was correlated with the severity of clinical symptoms typical for porcine enzootic pneumonia. In all suckling pigs, no matter vaccinated or not, the prevalence of anti-M. hyopneumoniae sIgA antibody positives was significantly lower than that of IgG antibody positives. These results prove that the sIgA-ELISA is a valuable method enabling the surveillance of M. hyopneumoniae infections in pig herds without interference due to maternally derived antibodies or antibodies induced by administration of inactivated vaccines.

Hsp90/Sec22b promotes unconventional secretion of mature-IL-1ß through an autophagosomal carrier in porcine alveolar macrophages during Mycoplasma hyopneumoniae infection.

Interleukin-1ß (IL-1ß) is a critical inflammatory regulator in response to Mycoplasma hyopneumoniae infection. However, the mechanism involved in the secretion of IL-1ß during Mycoplasma hyopneumoniae infection is unclear. In this study, we demonstrated that Mycoplasma hyopneumoniae infection increased the secretion of mature-IL-1ß (m-IL-1ß), but not pro-IL-1ß, in porcine alveolar macrophages. Moreover, Mycoplasma hyopneumoniae infection promoted the generation of autophagosomes, which attributed to the unconventional secretion of m-IL-1ß. Further results revealed that Hsp90 was required for the entry of m-IL-1ß into autophagosomes during Mycoplasma hyopneumoniae infection. The fusion of m-IL-1ß-containing autophagosome and plasma membranes was regulated by Sec22b and independent of lysosomal dysfunction. In conclusion, we provide evidence that Hsp90/Sec22b promotes the unconventional secretion of IL-1ß through an autophagosomal carrier during Mycoplasma hyopneumoniae infection. The elucidation of the molecular and cellular machinery in Mycoplasma hyopneumoniae infected mammalian cells in this study suggests avenues for further study and applications and paves the way for novel therapeutic strategies to prevent tissue damage in mycoplasma-associated diseases.

The immune mechanism of Mycoplasma hyopneumoniae 168 vaccine strain through dendritic cells.

BACKGROUND: Mycoplasma hyopneumoniae (Mhp) causes porcine enzootic pneumonia, a disease that cause major economic losses in the pig industry. Dendritic cells (DCs), the most effective antigen-presenting cells, are widely distributed beneath respiratory epithelium, DCs uptake and present antigens to T cells, to initiate protective immune responses in different infections. In this study, we investigated the role of porcine DCs in vaccine Mhp-168 exposure. RESULTS: The antigen presenting ability of DCs were improved by vaccine Mhp-168 exposure. DCs could activate T-cell proliferation by up-regulating the antigen presenting molecule MHCII expression and co-stimulatory molecule CD80/86. However, the up-regulation of IL-10 and accompany with down-regulation of IFN-γ gene level may account for the limitation of attenuated Mhp-168 strain use as vaccine alone. CONCLUSION: These findings are benefit for exploring the protection mechanisms and the possible limitations of this attenuated Mhp-168 vaccine.

Effects of Mycoplasma hyopneumoniae on porcine nasal cavity dendritic cells.

Mycoplasma hyopneumoniae (Mhp) is the primary etiological agent responsible for swine enzootic pneumonia (EP), a disease that cause tremendous economic losses all over the swine industry. Dendritic cells (DCs), the most effective antigen-presenting cells, are widely distributed beneath respiratory epithelium. DCs uptake and present antigens to T cells, to initiate protective immune responses or generate immune-mediated pathology in different infections. In this study, we investigated the changes in the different DCs subpopulations, T cells and SIgA positive cells counts in porcine nasal cavity after long time Mhp infection. We further evaluated the role of porcine DCs in Mhp exposure. Our results showed that the number of SLA-II-DR+SWC3a+DCs, SLA-II-DR+CD11b+ DCs, T cells, SIgA positive cells in nasal cavity were decreased after Mhp 28 days infection in vivo experiment. The antigen presenting ability of DCs were inhibited by Mhp exposure. DCs couldn't activate T-cell proliferation by down-regulating the antigen presenting molecule CD1a expression and promoting high level of IL-10 production. Further more, the expression levels of IL-12 and IFN-γ in DCs were decreased, suggesting that DCs favour for Th2 immune response development after Mhp exposure in vitro. Taken together, Mhp infection impairs the immune function which allows the persistence of Mhp and cause predispose pigs to secondary infections. The decline of DCs presentation ability is the reason why dysfunction and persistence in Mhp infection. These findings are benefit for exploring the pathogenic mechanisms of Mhp in pigs.

Characterization of the role in adherence of Mycoplasma hyorhinis variable lipoproteins containing different repeat unit copy numbers.

Mycoplasma hyorhinis (M. hyorhinis) is an important pathogen of pigs. In previous studies, the variable lipoprotein (Vlp) family has been shown to play a role in mediating M. hyorhinis cytoadhesion. Herein, we performed several experiments to study the function of each Vlp family member in detail, especially examining the cytoadhesion functional domain and how the repeat unit copy number impacts on function. Recombinant proteins rVlpII, composed of region II from all seven Vlp members; rVlpIII, composed of repeat peptides from region III of all of Vlp members; as well as a series of recombinant rVlp proteins for each member containing different repeat unit copy numbers were constructed. All of the proteins were expressed in Escherichia coli and purified by affinity chromatography. The recombinant proteins, as well as seven keyhole limpet hemocyanin-conjugated Vlp peptides containing two copies of the repeat unit, were analyzed for their adherence to swine tracheal epithelial cells using a microtiter plate adherence assay. Both rVlpII and rVlpIII proteins were able to bind to cell membrane proteins. Among the repeat unit peptides, only PepVlpB and PepVlpG were able to bind to cell membrane proteins. All of the Vlp members had cytoadhesion capability. The adhesion abilities of the proteins containing 0 or 3 copies of the repeat unit were stronger than those of the proteins containing 12 copies. For rVlpA, rVlpB, rVlpD, rVlpF and rVlpG, the proteins containing no copies bound stronger than the proteins containing 3 copies. In contrast, the adherence of rVlpC3 was stronger than that of rVlpC0. There was no significant difference between the adherence of rVlpE3 and that of rVlpE0. Our results suggest that the major cytoadhesion sites of Vlps are mainly contained in region II, the function of which would be blocked by region III when region III is longer.

The effects of Mycoplasma hyopneumoniae on porcine circovirus type 2 replication in vitro PK-15 cells.

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS). Mycoplasma hyopneumoniae (Mhp) is a very well-known co-factor that potentially enhances PCV2 replication and thus the development of PMWS. However, co-infection with Mhp and PCV2 in vivo under different conditions can produce divergent clinical signs and lesions. In this study, PCV2 replication could be enhanced by subsequent co-inoculation with Mhp (PCV2+Mhp) in a time and dose dependent method, but not by prior (Mhp+PCV2) or simultaneous (Mhp/PCV2) co-inoculation. Furthermore, different magnitudes of PCV2-infected cells, varying from 150% ± 14% to 351% ± 28%, were detected when co-infected with different Mhp strains. The relative percentage of PCV2-infected cells greatly decreased from 351% ± 28 to 141% ± 18 when the Mhp strain was treated with UV light for 12 h. These results offer the evidences to better understand the complex clinical syndromes in Mhp/PCV2 co-infection cases, and the occurrence of PMWS.