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Mesomycoplasma hyopneumoniae is the etiological agent of mycoplasmal pneumonia of swine (MPS), which causes substantial economic losses to the world's swine industry. Moonlighting proteins are increasingly being shown to play a role in the pathogenic process of M. hyopneumoniae. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, displayed a higher abundance in a highly virulent strain of M. hyopneumoniae than in an attenuated strain, suggesting that it may have a role in virulence. The mechanism by which GAPDH exerts its function was explored. Flow cytometry and colony blot analysis showed that GAPDH was partly displayed on the surface of M. hyopneumoniae. Recombinant GAPDH (rGAPDH) was able to bind PK15 cells, while the adherence of a mycoplasma strain to PK15 was significantly blocked by anti-rGAPDH antibody pretreatment. In addition, rGAPDH could interact with plasminogen. The rGAPDH-bound plasminogen was demonstrated to be activated to plasmin, as proven by using a chromogenic substrate, and to further degrade the extracellular matrix (ECM). The critical site for GAPDH binding to plasminogen was K336, as demonstrated by amino acid mutation. The affinity of plasminogen for the rGAPDH C-terminal mutant (K336A) was significantly decreased according to surface plasmon resonance analysis. Collectively, our data suggested that GAPDH might be an important virulence factor that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the tissue ECM barrier. IMPORTANCE Mesomycoplasma hyopneumoniae is a specific pathogen of pigs that is the etiological agent of mycoplasmal pneumonia of swine (MPS), which is responsible for substantial economic losses to the swine industry worldwide. The pathogenicity mechanism and possible particular virulence determinants of M. hyopneumoniae are not yet completely elucidated. Our data suggest that GAPDH might be an important virulence factor in M. hyopneumoniae that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the extracellular matrix (ECM) barrier. These findings will provide theoretical support and new ideas for the research and development of live-attenuated or subunit vaccines against M. hyopneumoniae., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could appear to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The unfolded protein response (UPR) plays a crucial role in Mycoplasma hyopneumoniae (M. hyopneumoniae) pathogenesis. We previously demonstrated that M. hyopneumoniae interferes with the host UPR to foster bacterial adhesion and infection. However, the underlying molecular mechanism of this UPR modulation is unclear. Here, we report that M. hyopneumoniae membrane protein Mhp271 interacts with host GRP78, a master regulator of UPR localized to the porcine tracheal epithelial cells (PTECs) surface. The interaction of Mhp271 with GRP78 reduces the porcine beta-defensin 2 (PBD-2) production, thereby facilitating M. hyopneumoniae adherence and infection. Furthermore, the R1-2 repeat region of Mhp271 is crucial for GRP78 binding and the regulation of PBD-2 expression. Intriguingly, a coimmunoprecipitation (Co-IP) assay and molecular docking prediction indicated that the ATP, rather than the substrate-binding domain of GRP78, is targeted by Mhp271 R1-2. Overall, our findings identify host GRP78 as a target for M. hyopneumoniae Mhp271 modulating the host UPR to facilitate M. hyopneumoniae adherence and infection., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

A highly immunogenic nucleotide fragment (195bp) was selected from the Mhp183 gene of Mycoplasma hyopneumoniae using information software technology and was named Mhp183195bp. Three Mhp183195bp were linked to form a new nucleotide sequence called Mhp183615bp. Mhp183615bp was directly synthesized and cloned into a pET100 vector and expressed in Escherichia coli. After purification, the proteins were successfully validated using SDS-PAGE and Western blot. BALB/c mice were injected with purified proteins on the first, eighth, and fifteenth days of feeding, respectively; serum samples were collected from mice on the day of immunization and on the 22nd day after immunization. The antibody level in mouse serum was detected by Western blotting using purified expressed proteins as antigens. IL-2, TNF-α and IFN-γ were simultaneously detected in mouse serum by ELISA. The 30 kDa protein was successfully expressed and reacted specifically with the specific serum Mhp His-Tag mouse monoclonal antibody and pig antibody. The expressed recombinant protein was immunogenic. The expression levels of IFN-γ, IL-2 and TNF-α were found to be significantly higher on day 22 than in the control group. This study suggests that the expressed recombinant protein could be used as one of the novel vaccine candidates for Mhp., (Copyright© by the Polish Academy of Sciences.)

Mycoplasmas persist in the host for a long time, suggesting that they possess mechanisms for immune evasion. Factor H is a negative regulator of the complement system, which binds to host cells to avoid unexpected complement activation. In this study, we revealed that many mycoplasmas, such as Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, Mycoplasma gallisepticum, Mycoplasma pneumoniae, Mycoplasma genitalium, Mycoplasma flocculare , and Mycoplasma bovis could hijack factor H such that they present themselves as a host tissue and thus escape from complement attack. Furthermore, the mechanism of recruiting factor H was identified in M. hyopneumoniae. M. hyopneumoniae binds factor H via factor H binding proteins, such as elongation factor thermo unstable (EF-Tu), P146, pyruvate dehydrogenase (acetyl-transferring) E1 component subunit alpha (PdhA), P46, Pyruvate dehydrogenase E1 component subunit beta (PdhB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and three different hypothetical proteins. The binding of factor H by EF-Tu further contributes to decreased C3 deposition on the M. hyopneumoniae surface and ultimately blocks further complement activation. In fact, binding of factor H occurs in a multifactorial manner; factor H is not only exploited by M. hyopneumoniae via its regulator activity to help mycoplasmas escape from complement killing, but also increases M. hyopneumoniae adhesion to swine tracheal epithelial cells, partially through EF-Tu. Meanwhile, the high sequence identity among EF-Tu proteins in the above-mentioned mycoplasmas implied the universality of the mechanism. This is the first report that mycoplasmas can escape complement killing by binding to factor H.

Mycoplasma hyopneumoniae is the most costly pathogen for swine production. Although several studies have focused on the host-bacterium association, little is known about the changes in gene expression of swine cells upon infection. To improve our understanding of this interaction, we infected swine epithelial NPTr cells with M. hyopneumoniae strain J to identify differentially expressed mRNAs and miRNAs. The levels of 1,268 genes and 170 miRNAs were significantly modified post-infection. Up-regulated mRNAs were enriched in genes related to redox homeostasis and antioxidant defense, known to be regulated by the transcription factor NRF2 in related species. Down-regulated mRNAs were enriched in genes associated with cytoskeleton and ciliary functions. Bioinformatic analyses suggested a correlation between changes in miRNA and mRNA levels, since we detected down-regulation of miRNAs predicted to target antioxidant genes and up-regulation of miRNAs targeting ciliary and cytoskeleton genes. Interestingly, most down-regulated miRNAs were detected in exosome-like vesicles suggesting that M. hyopneumoniae infection induced a modification of the composition of NPTr-released vesicles. Taken together, our data indicate that M. hyopneumoniae elicits an antioxidant response induced by NRF2 in infected cells. In addition, we propose that ciliostasis caused by this pathogen is partially explained by the down-regulation of ciliary genes.

Mycoplasma hyopneumoniae ( M. hyopneumoniae ) is the causative agent of pandemic pneumonia among pigs, namely, swine enzootic pneumonia. Although M. hyopneumoniae was first identified in 1965, little is known regarding its metabolic pathways, which might play a pivotal role during disease pathogenesis. Lipoate is an essential cofactor for enzymes important for central metabolism. However, the lipoate metabolism pathway in M. hyopneumoniae is definitely unclear. Here, we identified a novel gene, lpl, encoding a lipoate protein ligase in the genome of M. hyopneumoniae (Mhp-Lpl). This gene contains 1,032 base pairs and encodes a protein of 343 amino acids, which is between 7.5 and 36.09% identical to lipoate protein ligases (Lpls) of other species. Similar to its homologs in other species, Mhp-Lpl catalyzes the ATP-dependent activation of lipoate to lipoyl-AMP and the transfer of the activated lipoyl onto the lipoyl domains of M. hyopneumoniae GcvH (Mhp H) in vitro . Enzymatic and mutagenesis analysis indicate that residue K56 within the SKT sequence of Mhp H protein is the lipoyl moiety acceptor site. The three-dimensional structure showed typical lipoate protein ligase folding, with a large N-terminal domain and a small C-terminal domain. The large N-terminal domain is responsible for the full enzymatic activity of Mhp-Lpl. The identification and characterization of Mhp-Lpl will be beneficial to our understanding of M. hyopneumoniae metabolism., Summary: Lipoic acid is an essential cofactor for the activation of some enzyme complexes involved in key metabolic processes. Lipoate protein ligases (Lpls) are responsible for the metabolism of lipoic acid. To date, little is known regarding the Lpls in M. hyopneumoniae . In this study, we identified a lipoate protein ligase of M. hyopneumoniae . We further analyzed the function, overall structure and ligand-binding site of this protein. The lipoate acceptor site on M. hyopneumoniae GcvH was also identified. Together, these findings reveal that Lpl exists in M. hyopneumoniae and will provide a basis for further exploration of the pathway of lipoic acid metabolism in M. hyopneumoniae ., (Copyright © 2020 Zhu, Chen, Jin, Wang, Ma, Huang, Feng, Xin, Zhang and Liu.)

Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetic similar bacteria that colonize the swine respiratory tract. However, while M. hyopneumoniae is a pathogen that causes porcine enzootic pneumonia, M. flocculare is a commensal. Adhesion to the respiratory epithelium is mediated by surface-displayed adhesins, and at least some M. hyopneumoniae adhesins are post-translational proteolytically processed, producing differential proteoforms with differential adhesion properties. Based on LC-MS/MS data, we assessed differential proteolytic processing among orthologs of the five most abundant adhesins (p97 and p216) or adhesion-related surface proteins (DnaK, p46, and ABC transporter xylose-binding lipoprotein) from M. hyopneumoniae strains 7448 (pathogenic) and J (non-pathogenic), and M. flocculare. Both surface and cytoplasmic non-tryptic cleavage events were mapped and compared, and antigenicity predictions were performed for the resulting proteoforms. It was demonstrated that not only bona fide adhesins, but also adhesion-related proteins undergo proteolytical processing. Moreover, most of the detected cleavage events were differential among M. hyopneumoniae strains and M. flocculare, and also between cell surface and cytoplasm. Overall, our data provided evidences of a complex scenario of multiple antigenic proteoforms of adhesion-related proteins, that is differential among M. hyopneumoniae strains and M. flocculare, altering the surface architecture and likely contributing to virulence and pathogenicity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the pathogen of swine enzootic pneumonia, a chronic respiratory disease affecting pigs of all ages. The ciliated epithelial cells of the respiratory tract are the main target invaded and colonized by M. hyopneumoniae. Therefore, the ideal vaccine would be mucosally administered and able to stimulate suitable mucosal immunity and prevent the adherence of pathogens to mucosal cell surfaces. Currently, Bacillus subtilis as a recombinant vaccine carrier has been used for antigen delivery and proved to be effectively enhancing the innate immunity of nasal mucosa. Here, our study attempts to construct recombinant Bacillus subtilis (B.S-P97R1, B.S-P46), which can express the P97R1 or P46 antigen of M. hyopneumoniae, and to evaluate the immune responses in BALB/c mice. Initially, we respectively successfully constructed recombinant B.S-P97R1, B.S-P46 and validated the expression of antigen proteins by Western analysis. Then, recombinant B.S-P97R1 or B.S-P46 were respectively intranasally (i.n.) immunized in mice. Both strong P97R1-specific and P46-specific immunoglobulin G (IgG), secretory immunoglobulin A (SIgA) antibodies were induced in sera, bronchoalveolar lavage fluids (BALs) by ELISA analysis. Moreover, the levels of specific IL-4, IFN-γ in the immunized mice were elevated, and the proliferation of lymphocytes was also enhanced. In general, intranasal inoculation of recombinant B.S-P97R1 or B.S-P46 resulted in strong mucosal immunity, cell-mediated and humoral immunity, which was a mixed Th1/Th2-type response. In addition, our results provided a potential novel strategy that may be applied to the development of vaccines against M. hyopneumoniae., (© 2019 The Author(s).)

Enolase is an evolutionarily conserved enzyme involved in the processes of glycolysis and gluconeogenesis. Mycoplasma hyopneumoniae belongs to Mycoplasma , whose species are wall-less and among the smallest self-replicating bacteria, and is an important colonizing respiratory pathogen in the pig industry worldwide. Mycoplasma hyopneumoniae enolase ( Mhp Eno) expression is significantly increased after infection and was previously found to be a virulence factor candidate. Our studies show that Mhp Eno is a cell surface-localized protein that can adhere to swine tracheal epithelial cells (STECs). Adhesion to STECs can be specifically inhibited by an Mhp Eno antibody. Mhp Eno can recognize and interact with plasminogen with high affinity. Here, the first crystal structure of the mycoplasmal enolase from Mycoplasma hyopneumoniae was determined. The structure showed unique features of Mhp Eno in the S3/H1, H6/S6, H7/H8, and H13 regions. All of these regions were longer than those of other enolases and were exposed on the Mhp Eno surface, making them accessible to host molecules. These results show that Mhp Eno has specific structural characteristics and acts as a multifunctional adhesin on the Mycoplasma hyopneumoniae cell surface.

Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar. However, M. hyopneumoniae causes porcine enzootic pneumonia, while M. flocculare is a commensal bacterium. M. hyopneumoniae and M. flocculare do not penetrate their host cells, and secreted proteins are important for bacterium-host interplay. Thus, the secretomes of a swine trachea cell line (NPTr) infected with M. hyopneumoniae 7448 (a pathogenic strain), M. hyopneumoniae J (a non-pathogenic strain) and M. flocculare were compared to shed light in bacterium-host interactions. Medium from the cultures was collected, and secreted proteins were identified by a LC-MS/MS. Overall numbers of identified host and bacterial proteins were, respectively, 488 and 58, for NPTr/M. hyopneumoniae 7448; 371 and 67, for NPTr/M. hyopneumoniae J; and 203 and 81, for NPTr/M. flocculare. The swine cells revealed different secretion profiles in response to the infection with each M. hyopneumoniae strain or with M. flocculare. DAMPs and extracellular proteasome proteins, secreted in response to cell injury and death, were secreted by NPTr cells infected with M. hyopneumoniae 7448. All three mycoplasmas secreted virulence factors during NPTr infection, but M. hyopneumoniae 7448 secreted higher number of adhesins and hypothetical proteins, that may be related with pathogenicity. SIGNIFICANCE: The enzootic pneumonia caused by mycoplasmas of swine respiratory tract has economic loss consequences in pig industry due to antibiotic costs and pig weight loss. However, some genetically similar mycoplasmas are pathogenic while others, such as Mycoplasma hyopneumoniae and Mycoplasma flocculare, are non-pathogenic. Here, we conducted an infection assay between swine cells and pathogenic and non-pathogenic mycoplasmas to decipher secreted proteins during host-pathogen interaction. Mycoplasma response to cell infection was also observed. Our study provided new insights on secretion profile of swine cells in response to the infection with pathogenic and non-pathogenic mycoplasmas. It was possible to observe that pathogenic M. hyopneumoniae 7448 secreted known virulence factors and swine cells responded by inducing cell death. Otherwise, M. hyopneumoniae J and M. flocculare, non-pathogenic mycoplasmas, secreted a different profile of virulence factors in response to swine cells. Consequently, swine cells altered their secretome profile, but the changes were not sufficient to cause disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Porcine circovirus and Mycoplasma hyopneumoniae can cause respiratory diseases in pigs, which cause serious economic loss in the worldwide pig industry. Currently, these infections are mainly prevented and controlled by vaccination. The new vaccines on the market are mainly composed of subunits and inactivated vaccines but usually have lower antigenicity than traditional live vaccines. Thus, there is an increasing need to develop new adjuvants that can cause rapid and long-lasting immunity to enhance the antigenic efficacy for vaccines. Studies have shown that meningococcal porin PorB can act as a ligand to combine with Toll-like receptors to activate the production of immunological projections and act as a vaccine immunological adjuvant., Objective: In this article, we expressed and purified the recombinant PorB protein and verified its immunogenicity against porcine circovirus type 2 and Mycoplasma hyopneumoniae genetically engineered vaccine., Methods: In this article, we used prokaryotic expression to express and purify recombinant PorB protein, four different concentrations of PorB protein, Freund's adjuvant with two genetically engineered vaccines were combined with subcutaneous immunization of mice., Results: Our study shows that the appropriate dose of the recombinant protein PorB can enhance the levels of humoral and cellular responses induced by two genetically engineered vaccines in a short period of time in mice. The PorB adjuvant group may cause statistically higher antibody titers for both genetically engineered vaccines compared to Freund's commercial adjuvant (P<0.001)., Conclusion: The recombinant protein PorB may be a good candidate adjuvant for improving the protective effect of vaccines against porcine circovirus type 2 and Mycoplasma hyopneumoniae, and the protein can be used for future practical applications., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia in swine, an important disease worldwide. It has finite biosynthetic capabilities, including a deficit in de novo nucleotide synthesis. The source(s) for nucleotides in vivo are unknown, but mycoplasmas are known to carry membrane-bound nucleases thought to participate in the acquisition of nucleotides from host genomic DNA. Recent research has demonstrated that neutrophils can produce extracellular traps (NETs), chromatin NETs decorated with granular proteins to interact with and eliminate pathogens. We hypothesized that M. hyopneumoniae could utilize its membrane nuclease to obtain nucleotides from extracellular traps to construct its own DNA. Using the human monocytic cell line THP-1, we induced macrophage extracellular traps (METs), which are structurally similar to NETs. The thymidine analogue ethynyl deoxyuridine (EdU) was incorporated into THP-1 DNA and METs were induced. When incubated with M. hyopneumoniae, METs were degraded and the modified nucleotide label could be co-localized within M. hyopneumoniae DNA. When the nucleases were inhibited, MET degradation and nucleotide transfer were also inhibited. Controls confirmed that the EdU originated directly from the METs and not from free nucleotides arising from intracellular pools released during extrusion of the chromosomal DNA. M. hyopneumoniae incorporated labelled nucleotides more efficiently when 'fed' on METs than from free nucleotides in the medium, suggesting a tight linkage between nuclease degradation of DNA and nucleotide transport. These results strongly suggest that M. hyopneumoniae could degrade extracellular traps formed in vivo during infection and incorporate those host nucleotides into its own DNA.

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia. In our previous work, we reconstructed the metabolic models of this species along with two other mycoplasmas from the respiratory tract of swine: Mycoplasma hyorhinis, considered less pathogenic but which nonetheless causes disease and Mycoplasma flocculare, a commensal bacterium. We identified metabolic differences that partially explained their different levels of pathogenicity. One important trait was the production of hydrogen peroxide from the glycerol metabolism only in the pathogenic species. Another important feature was a pathway for the metabolism of myo-inositol in M. hyopneumoniae. Here, we tested these traits to understand their relation to the different levels of pathogenicity, comparing not only the species but also pathogenic and attenuated strains of M. hyopneumoniae. Regarding the myo-inositol metabolism, we show that only M. hyopneumoniae assimilated this carbohydrate and remained viable when myo-inositol was the primary energy source. Strikingly, only the two pathogenic strains of M. hyopneumoniae produced hydrogen peroxide in complex medium. We also show that this production was dependent on the presence of glycerol. Although further functional tests are needed, we present in this work two interesting metabolic traits of M. hyopneumoniae that might be directly related to its enhanced virulence., (© 2018 John Wiley & Sons Ltd.)

Mycoplasma hyopneumoniae , an agriculturally important porcine pathogen, disrupts the mucociliary escalator causing ciliostasis, loss of cilial function, and epithelial cell death within the porcine lung. Losses to swine production due to growth rate retardation and reduced feed conversion efficiency are severe, and antibiotics are used heavily to control mycoplasmal pneumonia. Notably, little is known about the repertoire of host receptors that M. hyopneumoniae targets to facilitate colonization. Here we show, for the first time, that actin exists extracellularly on porcine epithelial monolayers (PK-15) using surface biotinylation and 3D-Structured Illumination Microscopy (3D-SIM), and that M. hyopneumoniae binds to the extracellular β-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i) monoclonal antibodies that target β-actin significantly block the ability of M. hyopneumoniae to adhere and colonize PK-15 cells; (ii) microtiter plate binding assays show that M. hyopneumoniae cells bind to monomeric G-actin in a dose dependent manner; (iii) more than 100 M. hyopneumoniae proteins were recovered from affinity-chromatography experiments using immobilized actin as bait; and (iv) biotinylated monomeric actin binds directly to M. hyopneumoniae proteins in ligand blotting studies. Specifically, we show that the P97 cilium adhesin possesses at least two distinct actin-binding regions, and binds monomeric actin with nanomolar affinity. Taken together, these observations suggest that actin may be an important receptor for M. hyopneumoniae within the swine lung and will aid in the future development of intervention strategies against this devastating pathogen. Furthermore, our observations have wider implications for extracellular actin as an important bacterial receptor.

Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar bacteria, which coinhabit the porcine respiratory tract. These mycoplasmas share most of the known virulence factors, but, while M. hyopneumoniae causes porcine enzootic pneumonia (PEP), M. flocculare is a commensal species. To identify potential PEP determinants and provide novel insights on mycoplasma-host interactions, the whole cell proteomes of two M. hyopneumoniae strains, one pathogenic (7448) and other non-pathogenic (J), and M. flocculare were compared. A cell fractioning approach combined with mass spectrometry (LC-MS/MS) proteomics was used to analyze cytoplasmic and surface-enriched protein fractions. Average detection of ~ 50% of the predicted proteomes of M. hyopneumoniae 7448 and J, and M. flocculare was achieved. Many of the identified proteins were differentially represented in M. hyopneumoniae 7448 in comparison to M. hyopneumoniae J and M. flocculare, including potential PEP determinants, such as adhesins, proteases, and redox-balancing proteins, among others. The LC-MS/MS data also provided experimental validation for several genes previously regarded as hypothetical for all analyzed mycoplasmas, including some coding for proteins bearing virulence-related functional domains. The comprehensive proteome profiling of two M. hyopneumoniae strains and M. flocculare provided tens of novel candidates to PEP determinants or virulence factors, beyond those classically described.

Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC-MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity., (© 2016 The Authors.)

Lipid-associated membrane proteins (LAMPs) are important in the pathogenicity of the Mycoplasma genus of bacteria. We investigated whether Mycoplasma hyopneumoniae LAMPs have pathogenic potential by inducing apoptosis in a St. Jude porcine lung epithelial cell line (SJPL). LAMPs from a pathogenic strain of M. hyopneumoniae (strain 232) were used in the research. Our investigation made use of diamidino-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) staining, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) analysis, and Annexin-V-propidium iodide staining. After LAMP treatment for 24 h, typical changes were induced, chromosomes were concentrated, apoptotic bodies were observed, the 3'-OH groups of cleaved genomes were exposed, and the percentage of apoptotic cells reached 36.5 ± 11.66%. Caspase 3 and caspase 8 were activated and cytochrome c (cyt c) was released from the mitochondria into the cytoplasm; poly ADP ribose polymerase (PARP) was digested into two fragments; p38 mitogen-activated protein kinase (MAPK) was phosphorylated; and the expression of pro-apoptosis protein Bax increased while the anti-apoptosis protein Bcl-2 decreased. LAMPs also stimulated SJPL cells to produce nitric oxide (NO) and superoxide. This study demonstrated that LAMPs from M. hyopneumoniae can induce apoptosis in SJPL cells through the activation of caspase 3, caspase 8, cyt c, Bax, and p38 MAPK, thereby contributing to our understanding of the pathogenesis of M. hyopneumoniae, which should improve the treatment of M. hyopneumoniae infections.

Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ&#95;0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non-ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized; however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C-terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M. hyopneumoniae and fibronectin and show that M. hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells., (© 2014 John Wiley & Sons Ltd.)

Mycoplasma hyopneumoniae is the causative agent of swine enzootic pneumonia (EP), a disease that causes considerable economic losss in swine industry. Lipid-associated membrane proteins (LAMPs) of mycoplasma play important roles in causing mycoplasma diseases. The present study explores the pathogenic mechanisms of M. hyopneumoniae LAMPs by elucidating their role in modulating the inflammation, apoptosis, and relevant signaling pathways of peripheral blood mononuclear cells (PBMCs) of pig. LAMP treatment inhibited the growth of PBMCs. Up-regulation of cytokines, such as IL-6 and IL-1β, as well as increased production of nitric oxide (NO) and superoxide anion were all detected in the supernatant of LAMPs-treated PBMCs. Furthermore, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMPs of M. hyopneumoniae induced a time-dependent apoptosis in lymphocyts and monocytes from PBMCs, which was blocked by NOS inhibitor or antioxidant. In addition, LAMPs induced the phosphorylation of p38, the ratio of pro-apoptotic Bax protein to anti-apoptotic Bcl-2, activation of caspase-3 and caspase-8, and poly ADP-ribose polymerase (PARP) cleavage in PBMCs. These findings demonstrated that M. hyopneumoniae LAMPs induced the production of proinflammatory cytokines, NO and reactive oxygen species (ROS), and apoptosis of PBMCs in vitro through p38 MAPK and Bax/Bcl-2 signaling pathways, as well as caspase activation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The characterization of the repertoire of proteins exposed on the cell surface by Mycoplasma hyopneumoniae (M. hyopneumoniae), the etiological agent of enzootic pneumonia in pigs, is critical to understand physiological processes associated with bacterial infection capacity, survival and pathogenesis. Previous in silico studies predicted that about a third of the genes in the M. hyopneumoniae genome code for surface proteins, but so far, just a few of them have experimental confirmation of their expression and surface localization. In this work, M. hyopneumoniae surface proteins were labeled in intact cells with biotin, and affinity-captured biotin-labeled proteins were identified by a gel-based liquid chromatography-tandem mass spectrometry approach. A total of 20 gel slices were separately analyzed by mass spectrometry, resulting in 165 protein identifications corresponding to 59 different protein species. The identified surface exposed proteins better defined the set of M. hyopneumoniae proteins exposed to the host and added confidence to in silico predictions. Several proteins potentially related to pathogenesis, were identified, including known adhesins and also hypothetical proteins with adhesin-like topologies, consisting of a transmembrane helix and a large tail exposed at the cell surface. The results provided a better picture of the M. hyopneumoniae cell surface that will help in the understanding of processes important for bacterial pathogenesis. Considering the experimental demonstration of surface exposure, adhesion-like topology predictions and absence of orthologs in the closely related, non-pathogenic species Mycoplasma flocculare, several proteins could be proposed as potential targets for the development of drugs, vaccines and/or immunodiagnostic tests for enzootic pneumonia.

Currently available ELISAs used to diagnose Mycoplasma hyopneumoniae infection in pigs have high specificity but low sensitivity. To develop more sensitive assays, the kinetics of specific serum IgG and respiratory mucosal sIgA responses against three M. hyopneumoniae antigens, namely, P97R1 (an adhesin protein), P46 (a membrane protein), and P36 (a cytosolic protein), were characterised over 133 days following experimental infection. Immunoglobulin G against the three proteins remained at high concentrations from 28 to 133 days post-infection (dpi), although IgG against P97R1 was detected earlier and was more reactive than the other two antigens under assessment. Mucosal sIgA appeared earlier than serum IgG but did not persist as long; sIgA concentrations against P97R1 were the highest. Seroconversion was detected 2 weeks earlier with the P97R1-based ELISA than with a commercially available ELISA. On analysis of serum samples from five pig farms that did not use a M. hyopneumoniae vaccine, the P97R1-based IgG ELISA demonstrated a 73.6% coincidence rate with the commercial kit. Moreover, this more specific P97R1-based ELISA detected more positive samples than the commercial kit (52.8% vs. 39.2%). It was concluded that the systemic immune response to M. hyopneumoniae infection in pigs was delayed in onset but persistent whereas the mucosal response developed more rapidly but was less sustained. The P97R1 antigen was identified as a suitable serological marker for diagnosing M. hyopneumoniae infection in pigs, particularly early stage infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

MHJ&#95;0493 (P216) is a highly expressed cilium adhesin in Mycoplasma hyopneumoniae. P216 undergoes cleavage at position 1074 in the S/T-X-F↓-X-D/E-like motif (1072)T-N-F↓Q-E(1076) generating N-terminal and C-terminal fragments of 120 kDa (P120) and 85 kDa (P85) on the surface of M. hyopneumoniae. Here we show that several S/T-X-F↓X-D/E-like motifs exist in P216 but only (1072)T-N-F↓Q-E(1076) and (1344)I-T-F↓A-D-Y(1349) were determined to be bona fide processing sites by identifying semitryptic peptides consistent with cleavage at the phenylalanine residue. The location of S/T-X-F↓-X-D/E-like motifs within or abutting regions of protein disorder greater than 40 consecutive amino acids is consistent with our hypothesis that site access influences the cleavage efficiency. Approximately 20 cleavage fragments of P216 were identified on the surface of M. hyopneumoniae by LC-MS/MS analysis of biotinylated proteins and 2D SDS-PAGE. LC-MS/MS analysis of semitryptic peptides within P216 identified novel cleavage sites. Moreover, detection of a series of overlapping semitryptic peptides that differed by the loss a single amino acid at their N-terminus is consistent with aminopeptidase activity on the surface of M. hyopneumoniae. P120 and P85 and their cleavage fragments bind heparin and cell-surface proteins derived from porcine epithelial-like cells, indicating that P216 cleavage fragments retain the ability to bind glycosaminoglycans.

Background: Infection by Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae, either alone or together, causes serious respiratory diseases in pigs., Results: To develop an efficient multi-disease subunit vaccine against these pathogens, we produced a chimeric protein called Ap97, which comprises a deletion derivative of the N-terminal region of the A. pleuropneumoniae ApxIII toxin (ApxN) and the R1 and R2 repeats of M. hyopneumoniae P97 adhesin (P97C), using an E. coli expression system.The levels of both IgG1 and IgG2a isotypes specific for ApxN and P97C in the sera of Ap97-immunized mice increased, and Ap97 induced the secretion of IL-4 and IFN-γ by mouse splenocytes. Antisera from mice and pigs immunized with Ap97 readily reacted with both native ApxIII and P97 proteins. In addition, immunization with the Ap97 vaccine effectively protected pigs against challenge with both pathogens., Conclusions: These findings suggest that Ap97 confers immunogenicity, and is an effective vaccine that protects pigs against infection by M. hyopneumoniae and A. pleuropneumoniae.

Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.

Mycoplasma hyopneumoniae is a prevalent swine respiratory pathogen that is a major cause of economic loss to pig producers. Control is achieved by a combination of antimicrobials, vaccination and management practices, but current vaccines offer only partial control and there is a need for improved preventative strategies. A major barrier to advances in understanding the pathogenesis of M. hyopneumoniae and in developing new vaccines is the lack of tools to genetically manipulate the organism. We describe the development and optimisation of the first successful plasmid-based system for the genetic manipulation of M. hyopneumoniae. Our artificial plasmids contain the origin of replication (oriC) of M. hyopneumoniae along with tetM, conferring resistance to tetracycline. With these plasmids, we have successfully transformed M. hyopneumoniae strain 232 by electroporation, generating tetracycline resistant organisms. The persistence of extrachromosomal plasmid and maintenance of plasmid DNA over serial passages shows that these artificial plasmids are capable of self-replication in M. hyopneumoniae. In addition to demonstrating the amenability of M. hyopneumoniae to genetic manipulation and in optimising the conditions necessary for successful transformation, we have used this system to determine the minimum functional oriC of M. hyopneumoniae. In doing so, we have developed a plasmid with a small oriC that is stably maintained over multiple passages that may be useful in generating targeted gene disruptions. In conclusion, we have generated a set of plasmids that will be valuable in studies of M. hyopneumoniae pathogenesis and provide a major step forward in the study of this important swine pathogen.

In the present study, a computational comparative and subtractive genomic/proteomic analysis aimed at the identification of putative therapeutic target and vaccine candidate proteins from Kyoto Encyclopedia of Genes and Genomes (KEGG) annotated metabolic pathways of Mycoplasma hyopneumoniae was performed for drug design and vaccine production pipelines against M.hyopneumoniae. The employed comparative genomic and metabolic pathway analysis with a predefined computational systemic workflow extracted a total of 41 annotated metabolic pathways from KEGG among which five were unique to M. hyopneumoniae. A total of 234 proteins were identified to be involved in these metabolic pathways. Although 125 non homologous and predicted essential proteins were found from the total that could serve as potential drug targets and vaccine candidates, additional prioritizing parameters characterize 21 proteins as vaccine candidate while druggability of each of the identified proteins evaluated by the DrugBank database prioritized 42 proteins suitable for drug targets., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Bacterial aminopeptidases play important roles in pathogenesis by providing a source of amino acids from exogenous proteins, destroying host immunological effector peptides and executing posttranslational modification of bacterial and host proteins. We show that MHJ&#95;0125 from the swine respiratory pathogen Mycoplasma hyopneumoniae represents a new member of the M42 class of bacterial aminopeptidases. Despite lacking a recognizable signal sequence, MHJ&#95;0125 is detectable on the cell surface by fluorescence microscopy and LC-MS/MS of (i) biotinylated surface proteins captured by avidin chromatography and (ii) peptides released by mild trypsin shaving. Furthermore, surface-associated glutamyl aminopeptidase activity was detected by incubation of live M. hyopneumoniae cells with the diagnostic substrate H-Glu-AMC. MHJ&#95;0125 moonlights as a multifunctional adhesin, binding to both heparin and plasminogen. Native proteomics and comparative modelling studies suggest MHJ&#95;0125 forms a dodecameric, homopolymeric structure and provide insight into the positions of key residues that are predicted to interact with heparin and plasminogen. MHJ&#95;0125 is the first aminopeptidase shown to both bind plasminogen and facilitate its activation by tissue plasminogen activator. Plasmin cleaves host extracellular matrix proteins and activates matrix metalloproteases, generating peptide substrates for MHJ&#95;0125 and a source of amino acids for growth of M. hyopneumoniae. This unique interaction represents a new paradigm in microbial pathogenesis.

The severity of swine influenza is highly variable and can be exacerbated by many factors, such as a pre-infection of pigs with Mycoplasma hyopneumoniae (Mhp). The aim of this study was to investigate the oxidative stress induced by Mhp and the impact of this stress on the evolution of an infection with the European avian-like swine H1N1 influenza virus. Two experimental trials (E1 and E2), which differed only by the feed delivered to the animals, were conducted on SPF pigs. In each trial, one group of nine 6-week-old pigs was inoculated intra-tracheally with Mhp and H1N1 at 21 days intervals and a mock-infected group (8 pigs) was included. Clinical signs were observed, blood samples were collected throughout the study and pathogens were detected in nasal swabs and lung tissues. Results indicated that Mhp infection induced an oxidative stress in E1 and E2, but its level was more important in E2 than in E1 three weeks post-Mhp inoculation, before H1N1 infection. In both trials, a strong inflammatory response and a response to the oxidative stress previously induced by Mhp appeared after H1N1 infection. However, the severity of influenza disease was significantly more marked in E2 as compared to E1, as revealed by prolonged hyperthermia, stronger reduction in mean daily weight gain and earlier viral shedding. These results suggested that severity of flu syndrome and reduction in animal performance may vary depending on the level of oxidative stress at the moment of the influenza infection, and that host responses could be influenced by the feed., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This study investigated the efficacy of a single intramuscular injection of a new formulation of florfenicol to treat clinical respiratory disease following experimental Mycoplasma hyopneumoniae infection. M. hyopneumoniae-free piglets were allocated to three groups, namely, a treatment group (TG) and a positive control group (PCG), which were both inoculated endotracheally with a highly virulent isolate of M. hyopneumoniae, and a negative control group. At the onset of clinical disease, the TG received a single injection of florfenicol (30 mg/kg). All pigs were euthanased 4 weeks post-infection. Clinical symptoms were significantly reduced in the TG in comparison with the PCG. Average daily gain, feed conversion ratio, mortality and lung lesions were improved in the TG compared to the PCG, but the differences were not statistically significant., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Enzootic pneumonia caused by Mycoplasma hyopneumoniae is a major constraint to efficient pork production throughout the world. This pathogen has a small genome with 716 coding sequences, of which 418 are homologous to proteins with known functions. However, almost 42% of the 716 coding sequences are annotated as hypothetical proteins. Alternative methodologies such as threading and comparative modeling can be used to predict structures and functions of such hypothetical proteins. Often, these alternative methods can answer questions about the properties of a model system faster than experiments. In this study, we predicted the structures of seven proteins annotated as hypothetical in M. hyopneumoniae, using the structure-based approaches mentioned above. Three proteins were predicted to be involved in metabolic processes, two proteins in transcription and two proteins where no function could be assigned. However, the modeled structures of the last two proteins suggested experimental designs to identify their functions. Our findings are important in diminishing the gap between the lack of annotation of important metabolic pathways and the great number of hypothetical proteins in the M. hyopneumoniae genome.

Unlabelled: Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50(P146), P40(P146), and P85(P146) that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence (672)ATEF↓QQ(677), consistent with a cleavage motif resembling S/T-X-F↓X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1(P146)-F3(P146) that mimic P50(P146), P40(P146), and P85(P146) were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3(P146) generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography., Importance: Vaccines used to control Mycoplasma hyopneumoniae infection provide only partial protection. Proteins of the P97/P102 families are highly expressed, functionally redundant molecules that are substrates of endoproteases that generate multifunctional adhesin fragments on the cell surface. We show that P146 displays a chimeric structure consisting of an N terminus, which shares sequence identity with P97, and novel central and C-terminal regions. P146 is endoproteolytically processed at multiple sites, generating at least nine fragments on the surface of M. hyopneumoniae. Dominant cleavage events occurred at S/T-X-F↓X-D/E-like sites generating P50(P146), P40(P146), and P85(P146). Recombinant proteins designed to mimic the major cleavage fragments bind porcine cilia, heparin, and plasminogen. P146 undergoes endoproteolytic processing events at multiple sites and with differential processing efficiency, generating combinatorial diversity on the surface of M. hyopneumoniae.

P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of Mycoplasma hyopneumoniae that bind glycosaminoglycans, plasminogen, and fibronectin and perform essential roles in colonization of ciliated epithelia. We show that the P102 paralogue Mhp384 is efficiently cleaved at an S/T-X-F↓X-D/E-like site, creating P60(384) and P50(384). The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115(385)) and 88 kDa (P88(385)) and 27 kDa (P27(385)) cleavage fragments identified by LC-MS/MS. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry. The semitryptic peptide (752)IQFELEPISLNV(763) denotes the C-terminus of P88(385) and defines the novel cleavage site (761)L-N-V↓A-V-S(766) in Mhp385. P115(385), P88(385), P27(385), P60(384), and P50(384) were shown to reside extracellularly, though it is unknown how the fragments remain attached to the cell surface. Heparin- and cilium-binding sites were identified within P60(384), P50(384), and P88(385). No primary function was attributed to P27(385); however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (α3 chain). P97 and P102 paralogue families are adhesins targeted by several proteases with different cleavage efficiencies, and this process generates combinatorial complexity on the surface of M. hyopneumoniae.

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), a chronic respiratory disease which causes significant economic losses to the swine industry worldwide. More efficient strategies for controlling this disease are necessary. In this study, we cloned17 genes coding for transmembrane proteins from M. hyopneumoniae, among which six were successfully expressed in Escherichia coli and had their immunogenic and antigenic properties evaluated. All proteins were immunogenic in mice and sera from naturally infected pigs reacted with the recombinant proteins, suggesting that they are expressed during infection. These antigens may contribute for the development of new recombinant vaccines and diagnostic tests against EP., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Type I signal peptidase (SPase I) is a membrane-anchored protease of the general secretory pathway, which is encoded by the sipS gene in Mycoplasma hyopneumoniae, the etiological agent of porcine enzootic pneumonia (PEP). In this study, the expression of the M. hyopneumoniae SPase I (MhSPase I) was analyzed in virulent and avirulent strains, and the recombinant protein (rMhSPase I), expressed in Escherichia coli, was evaluated regarding its potential as an immunodiagnostic antigen. It was demonstrated that the sipS coding DNA sequence (CDS) is most likely part of an operon, being co-transcribed along with four other CDSs. Quantitative reverse transcriptase PCR and immunoblot assays showed that MhSPase I is expressed by all three strains analyzed, with no transcriptional difference, but with evidence of a higher protein level in a pathogenic strain (7422), in comparison to another pathogenic (7448) and a non-pathogenic (J) strain. rMhSPase I was strongly immunogenic for mice, and the MhSPase I antigenicity was confirmed. Polyclonal serum anti-rMhSPase I presented no detectable cross-reaction with Mycoplasma flocculare and Mycoplasma hyorhinis. Moreover, phylogenetic analysis demonstrated a low conservation between MhSPase I and orthologous proteins from other porcine respiratory disease complex-related bacteria, Firmicutes and other Mycoplasma species. The potential of an rMhSPase I-based ELISA for PEP immunodiagnosis was demonstrated. Overall, we investigated the expression of sipS and the encoded MhSPase I in three M. hyopneumoniae strains and showed that this protein is a good antigen for use in PEP serodiagnosis and possibly vaccination, as well as a potential target for antibiotic development., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mycoplasma hyopneumoniae is a major, economically damaging respiratory pathogen. Although M. hyopneumoniae cells bind plasminogen, the identification of plasminogen-binding surface proteins and the biological ramifications of acquiring plasminogen requires further investigation. mhp182 encodes a highly expressed 102 kDa protein (P102) that undergoes proteolytic processing to generate surface-located N-terminal 60 kDa (P60) and C-terminal 42 kDa (P42) proteins of unknown function. We show that recombinant P102 (rP102) binds plasminogen at physiologically relevant concentrations (K(D) ~ 76 nM) increasing the susceptibility of plasmin(ogen) to activation by tissue-specific plasminogen activator (tPA). Recombinant proteins constructed to mimic P60 (rP60) and P42 (rP42) also bound plasminogen at physiologically significant levels. M. hyopneumoniae surface-bound plasminogen was activated by tPA and is able to degrade fibrinogen, demonstrating the biological functionality of M. hyopneumoniae-bound plasmin(ogen) upon activation. Plasmin(ogen) was readily detected in porcine ciliated airways and plasmin levels were consistently higher in bronchoalveolar lavage fluid from M. hyopneumoniae-infected animals. Additionally, rP102 and rP42 bind fibronectin with K(D) s of 26 and 33 nM respectively and recombinant P102 proteins promote adherence to porcine kidney epithelial-like cells. The multifunctional binding ability of P102 and activation of M. hyopneumoniae-sequestered plasmin(ogen) by an exogenous activator suggests P102 plays an important role in virulence., (© 2011 Blackwell Publishing Ltd.)

Mycoplasma hyopneumoniae colonizes the ciliated respiratory epithelium of swine, disrupting mucociliary function and inducing chronic inflammation. P97 and P102 family members are major surface proteins of M. hyopneumoniae and play key roles in colonizing cilia via interactions with glycosaminoglycans and mucin. The p102 paralog, mhp683, and homologs in strains from different geographic origins encode a 135-kDa pre-protein (P135) that is cleaved into three fragments identified here as P45(683), P48(683), and P50(683). A peptide sequence (TTKF↓QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48(683) and P50(683), determined by Edman degradation and mass spectrometry, confirmed cleavage after the phenylalanine residue. A similar proteolytic cleavage site was identified by mass spectrometry in another paralog of the P97/P102 family. Trypsin digestion and surface biotinylation studies showed that P45(683), P48(683), and P50(683) reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1(683)-F5(683), spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1(683)-F5(683) also bound porcine epithelial cilia, and antisera to F2(683) and F5(683) significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45(683), P48(683), and P50(683) each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family.

Mycoplasma hyopneumoniae is the causative pathogen of porcine enzootic pneumonia, an economically significant disease that disrupts the mucociliary escalator in the swine respiratory tract. Expression of Mhp107, a P97 paralog encoded by the gene mhp107, was confirmed using ESI-MS/MS. To investigate the function of Mhp107, three recombinant proteins, F1(Mhp107), F2(Mhp107), and F3(Mhp107), spanning the N-terminal, central, and C-terminal regions of Mhp107 were constructed. Colonization of swine by M. hyopneumoniae requires adherence of the bacterium to ciliated cells of the respiratory tract. Recent studies have identified a number of M. hyopneumoniae adhesins that bind heparin, fibronectin, and plasminogen. F1(Mhp107) was found to bind porcine heparin (K(D) ∼90 nM) in a dose-dependent and saturable manner, whereas F3(Mhp107) bound fibronectin (K(D) ∼180 nM) at physiologically relevant concentrations. F1(Mhp107) also bound porcine plasminogen (K(D) = 24 nM) in a dose-dependent and physiologically relevant manner. Microspheres coated with F3(Mhp107) mediate adherence to porcine kidney epithelial-like (PK15) cells, and all three recombinant proteins (F1(Mhp107)-F3(Mhp107)) bound swine respiratory cilia. Together, these findings indicate that Mhp107 is a member of the multifunctional M. hyopneumoniae adhesin family of surface proteins and contributes to both adherence to the host and pathogenesis.

Limited reports are available on the growth response of Mycoplasma hyopneumoniae in Friis medium and the routinely used color changing units (CCU) assay has not yet been profoundly compared with other titration methods. Firstly, growth kinetics of 7 diverse M. hyopneumoniae isolates were followed by ATP luminometry in five Friis medium batches. Secondly, results of the CCU and ATP assays were compared hereby evaluating the methods. Growth curves of all isolates had log, stationary and senescence phases, and reached similar maximal titres when cultured in the same batch of Friis medium. Doubling times (Tds) of the isolates grown in slowly shaken cultures varied between 4.8 and 7.8 h. Maximal titres, Tds, growth phase in which the phenol red indicator turned from red to yellow due to acidification by mycoplasmal metabolism, and the length of the stationary phase varied depending on the Friis medium batch. The effect of static vs. shaking culture conditions on the Td depended on the isolate. ATP and CCU assays obtained similar growth curves, but when maximal levels were reached the CCU titre dropped earlier than the ATP titre. During log phase, CCU and ATP titres were strongly linearly linked. We developed a model enabling transformation of ATP into CCU titres or vice versa. The calculated amount of ATP per CCU (1.77 amol ATP/ml) indicated that the CCU assay likely underestimates the actual cell concentration. When titres were determined as means of 3 measurements, the ATP assay was 7 times more accurate and had 11-fold lower outliers than the CCU assay. Unlike the CCU assay, luminometry only requires one measurement to obtain sufficient accuracy. It was concluded that the ATP assay constitutes a valuable robust alternative for reproducible real-time titre assessment of freshly grown M. hyopneumoniae cultures. It is faster, more accurate and time, work and cost efficient compared to the CCU assay. The assay is preferred to better standardise and describe M. hyopneumoniae cultures used in various experiments., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Porcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 kDa in size. A variety of pathogenic bacterial species have been shown to bind the extracellular matrix component fibronectin as an adherence mechanism. M. hyopneumoniae cells were found to bind fibronectin in a dose-dependent and saturable manner. Surface plasmon resonance was used to show that a recombinant C-terminal domain of P116 bound fibronectin at physiologically relevant concentrations (K(D) 24 ± 6 nm). Plasmin(ogen)-binding proteins are also expressed by many bacterial pathogens, facilitating extracellular matrix degradation. M. hyopneumoniae cells were found to also bind plasminogen in a dose-dependent and saturable manner; the C-terminal domain of P116 binds to plasminogen (K(D) 44 ± 5 nm). Plasminogen binding was abolished when the C-terminal lysine of P116 was deleted, implicating this residue as part of the plasminogen binding site. P116 fragments adhere to the PK15 porcine kidney epithelial-like cell line and swine respiratory cilia. Collectively these data suggest that P116 is an important adhesin and virulence factor of M. hyopneumoniae.

Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, adheres to ciliated respiratory epithelia resulting in ciliostasis and epithelial cell death. The cilium adhesin P97 (Mhp183) contains two repeat regions, designated R1 and R2, that play key roles in adherence. Eight pentapeptide repeats in R1 are sufficient to bind porcine cilia; however, both R1 and R2 are needed to bind heparin. Mhp271, a paralogue of P97, is the only other M. hyopneumoniae protein to contain both R1 and R2 repeats. These repeats are arranged as a set of three pentapeptide repeats (designated R1A₂₇₁), two decapeptide repeats (designated R2₂₇₁), and a second set of six pentapeptide repeats (designated R1B₂₇₁). To determine their function, recombinant proteins containing R1A₂₇₁) (F1₂₇₁) and R2₂₇₁-R1B₂₇₁ (F2₂₇₁) were constructed and used in in vitro binding assays. F2₂₇₁, but not F1₂₇₁, bound heparin (K(D)=8.1 ± 0.4 nM), fibronectin (K(D)=174 ± 13 nM) and porcine cilia. Pre-incubation of F2₂₇₁ with 100 µM heparin blocked cilium binding by ~69%. Cell surface shaving with trypsin combined with two-dimensional liquid chromatography coupled to tandem mass spectrometry analysis identified Mhp271 as surface-exposed. Our data suggest that both R1 and R2 in Mhp271 are involved in binding to host molecules., (© 2010 Blackwell Publishing Ltd.)

Mycoplasma hyopneumoniae colonizes at the porcine respiratory-ciliated epithelial cells and causes the enzootic pneumonia of swine. The adhesion step is crucial in the colonization process. A few adhesion molecules have been characterized, and the concurrent receptors from the porcine ciliated cells have also been suggested to recognize the adhesion molecules. In the present study, the interactions between M. hyopneumoniae and porcine tracheal ciliated cells were investigated by employing the Far-Western blotting method. The results indicate that aconitase, lamin A/C, and peroxiredoxin of the porcine tracheal ciliated cell may interact specifically with the mycoplasmal proteins. We speculate that these mycoplasmal proteins could be secreted cleavage products, and their relative small size may enable them to penetrate into ciliated cells interfering with important metabolic pathways and other critical cellular processes.

Mycoplasma hyopneumoniae, the etiological agent of swine enzootic pneumonia, is an important pathogen in the swine industry worldwide. Vaccination is the most cost-effective strategy for controlling and prevention of this disease. However, investigations on pathogenicity mechanisms as well as current serological detection methods and the development of new recombinant subunit vaccines are hampered by the lack of known and well characterized species-specific M. hyopneumoniae antigens. In this work, 54 predicted genes encoding proteins with potential to be used as subunit vaccine or antigens in diagnostic tests were selected, amplified by PCR and cloned into Escherichia coli expression vectors. Recombinant protein expression, solubility and yields were analyzed. The majority of the recombinant proteins were expressed in inclusion bodies. After solubilization with urea or N-lauroyl sarcosine, recombinant proteins were purified by Ni(2+) affinity chromatography. This approach allowed purification of thirty recombinant M. hyopneumoniae proteins which will be evaluated as vaccine candidates and/or as antigens to be used in diagnostic tests.

Objective: To construct recombinant adenovirus carrying C-Terminal of the adhesion factor gene p97 of Mycoplasma hyopneumoniae (Mhp) so as to provide basis for further studying new type Mhp vaccine., Methods: We amplified p97 gene from the genome of Mhp and cloned into pShuttle-CMV plasmid. The correctly identified recombinant plasmid was linearized with Pme I and transformed into E. coli BJ5183-AD-1 competent cells containing adenovirus backbone vector to produce recombinant adenovirus DNA by homologous recombination. Purified recombinant adenovirus plasmid was linearized with Pac I, and transfected into AD293 cells to obtain recombinant adenovirus. The recombinant adenovirus was identified by RT-PCR, indirect immunofluorescence assay and Western blot, and purified by cesium chloride density centrifugation kit, then its titer was determined. Balb/c mice were immunized with recombinant adenovirus via intramuscular and intranasal routes and analysis the immunity results through humoral immunity,mucosal immunity and cell-mediated immunity aspect., Results: Digestion by Pac I proved successful homologous recombination. RT-PCR, Indirect immunofluorescence assay and Western Blot showed that the recombinant adenovirus transcribed and expressed P97 C-terminal protein successfully, the titer could achieve to 5 x 10(11)TCID50/mL after purification. Inoculation with the recombinant adenovirus by each route elicited P97 C-terminal protein specific serum and lung homogenate IgG and SIgA was induced by intranasal route, but the special lymphocyte proliferation was not induced by each route., Conclusion: The recombinant adenovirus expressing p97-Terminal gene was successfully constructed and it induced special humoral and mucosal immunity but no cell-mediated immunity.

Mycoplasma hyopneumoniae induces respiratory disease in swine by colonizing cilia causing ciliostasis, cilial loss and epithelial cell death. Heparin binds to M. hyopneumoniae cells in a dose-dependent manner and blocks its ability to adhere to porcine cilia. We show here that Mhp493 (P216), a paralogue of the cilium adhesin P97 (Mhp183), is cleaved between amino acids 1040 and 1089 generating surface-accessible, heparin-binding proteins P120 and P85. Antiphosphoserine antibodies recognized P85 in 2-D immunoblotting studies and TiO(2) chromatography of trypsin digests of P85 isolated a single peptide with an m/z of 917.3. A phosphoserine residue in the tryptic peptide (90)VSELpSFR(96) (position 94 in P85) was identified by MALDI-MS/MS. Polyhistidine fusion proteins (F1(P216), F2(P216), F3(P216)) spanning Mhp493 bound heparin with biologically significant Kd values, and heparin, fucoidan and mucin inhibited this interaction. Latex beads coated with F1(P216), F2(P216) and F3(P216) adhered to and entered porcine kidney epithelial-like (PK15) cell monolayers. Microtitre plate-based assays showed that sequences within P120 and P85 bind to porcine cilia and are recognized by serum antibodies elicited during infection by M. hyopneumoniae. Mhp493 contributes significantly to the surface architecture of M. hyopneumoniae and is the first cilium adhesin to be described that lacks an R1 cilium-binding domain.

Mycoplasma hyopneumoniae is an important pathogen of pigs causing enzootic pneumonia of swine. The pathogen remains largely enigmatic as far as the host-pathogen interactions are concerned. In the present study, the protein profiles of two strains of M. hyopneumoniae were compared by two-dimensional gel electrophoresis and mass spectrometry. The results indicate that the major adhesin P97, the 50-kDa protein derived from P159 adhesin, and the 43-kDa cleavage product of P102 are expressed at much higher levels in the pathogenic strain 232. In contrast, the avirulent strain J switches its focus to metabolism and expresses more glyceraldehyde 3-phosphate dehydrogenase in gluconeogenesis and lactate dehydrogenase, pyruvate dehydrogenase, and phosphate acetyltransferase in the pyruvate metabolism pathway. We speculate that the avirulent strain may have developed better capabilities to cope with the rich environment during repeated inoculations. Simultaneously, the capability to infect host cells may become less important so that the adhesion-related protein genes are down-regulated.

Objective: To determine whether commercial Mycoplasma hyopneumoniae bacterins sold for use in swine contain porcine torque teno virus (TTV)., Sample Population: 22 commercially available M hyopneumoniae bacterins., Procedures: Direct and nested PCR assays for genogroup-specific TTV DNAs were performed on serials of M hyopneumoniae bacterins by use of published and custom-designed primer pairs at 3 laboratories in North America and Europe., Results: Of the 22 bacterins tested by use of direct and nested PCR assays, 7 of 9 from the United States, 2 of 5 from Canada, and 4 of 8 from Europe contained genogroup 1- and genogroup 2-TTV DNAs. In some bacterins, the TTV DNAs were readily detected by use of direct PCR assays., Conclusions and Clinical Relevance: Analysis of these data indicated that many of the commercially available M hyopneumoniae bacterins were contaminated with TTV DNA. It is possible that some of these bacterins could inadvertently transmit porcine TTV infection to TTV-naïve swine.

Mycoplasma hyopneumoniae, the causative agent of swine enzootic pneumonia, colonizes the cilia of swine lungs, causing ciliostasis and cell death. M. hyopneumoniae is a component of the porcine respiratory disease complex (PRDC) and is especially problematic for the finishing swine industry, causing the loss of hundreds of millions of dollars in farm revenues worldwide. For successful infection, M. hyopneumoniae must effectively resist oxidative stresses due to the release of oxidative compounds from neutrophils and macrophages during the host's immune response. However, the mechanism that M. hyopneumoniae uses to avert the host response is still unclear. To gain a better understanding of the transcriptional responses of M. hyopneumoniae under oxidative stress, cultures were grown to early exponential phase and exposed to 0.5% hydrogen peroxide for 15 min. RNA samples from these cultures were collected and compared to RNA samples from control cultures using two-colour PCR-based M. hyopneumoniae microarrays. This study revealed significant downregulation of important glycolytic pathway genes and gene transcription proteins, as well as a protein known to activate oxidative stressor cascades in neutrophils. Sixty-nine per cent of the upregulated genes were hypothetical with no known function. This study has also revealed significantly differentially expressed genes common to other environmental stress responses, indicating that further investigation of universal stress response genes of M. hyopneumoniae is merited.

Mycoplasma hyopneumoniae is an important pathogen for pigs, being the causative agent of enzootic pneumonia. Recently, the genome sequences of three strains, J, 7448 and 232 have been reported. Here, we describe the results of a proteomic analysis, based on two-dimensional gel electrophoresis of soluble protein extracts, immunoblot and mass spectrometry, which was carried out aiming the identification of gene products and antigenic proteins from the M. hyopneumoniae pathogenic strain 7448. A preliminary M. hyopneumoniae proteome map in two pH ranges (3-10 and 4-7) was produced. A total of 31 different coding DNA sequences (CDSs), including three hypothetical ones, were experimentally verified with the identification of the corresponding protein products by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. According to the Clusters of Orthologous Groups (COG) functional classification, the identified proteins were assigned to the groups of metabolism (13), cellular processes (5) and information and storage processing (4). Nine of the identified proteins were not classifiable by COG, including some related to cytoadherence and possibly involved in pathogenicity. Moreover, at least five highly antigenic proteins of M. hyopneumoniae were identified by immunoblots, including four novel ones (a heat shock protein 70, an elongation factor Tu, a pyruvate dehydrogenase E1-beta subunit and the P76 membrane protein). The now available proteome map is expected to serve as a reference for comparative analyses between M. hyopneumoniae pathogenic and non-pathogenic strains, and for methabolic studies based on cells cultured under modified conditions.

Mycoplasma hyopneumoniae, the etiological agent of swine enzootic pneumonia, is an important pathogen in the swine industry worldwide. Investigations on pathogenicity mechanisms as well as current serological detection methods and the development of new recombinant subunit vaccines are hampered by the lack of known and well characterized, species-specific M. hyopneumoniae antigens. As a first step to solve these problems membrane and membrane-associated proteins were enriched from M. hyopneumoniae cells by Triton X-114 fractionation and further analyzed by 2D gel electrophoresis and Western blot analyses using convalescent sera. Two previously unknown immunogenic proteins were identified by quadrupole time-of-flight mass spectrometry and database analyses as the conserved putative lipoproteins, Mhp378 and Mhp651. Both proteins were expressed as recombinant GST fusion proteins and reacted with sera from convalescent pigs. Coated as solid-phase antigen, Mhp651 showed a distinct cross-reaction only with Mycoplasma flocculare specific rabbit hyperimmune serum, whereas Mhp378 was only recognized by the positive control serum directed against M. hyopneumoniae, thereby indicating its species specificity.