Hijacking of Host Plasminogen by Mesomycoplasma (Mycoplasma) hyopneumoniae via GAPDH: an Important Virulence Mechanism To Promote Adhesion and Extracellular Matrix Degradation.

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.

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.

Mycoplasma hyopneumoniae membrane protein Mhp271 interacts with host UPR protein GRP78 to facilitate infection.

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.

Expression and immunogenicity study of a novel mhp183 gene fragment of Mycoplasma hyopneumoniae.

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.

Mycoplasma hyopneumoniae evades complement activation by binding to factor H via elongation factor thermo unstable (EF-Tu).

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 J elicits an antioxidant response and decreases the expression of ciliary genes in infected swine epithelial cells.

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.

Functional Identification and Structural Analysis of a New Lipoate Protein Ligase in Mycoplasma hyopneumoniae.

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.

Evidences of differential endoproteolytic processing on the surfaces of Mycoplasma hyopneumoniae and Mycoplasma flocculare.

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.

Mucosal and systemic immune responses induced by intranasal immunization of recombinant Bacillus subtilis expressing the P97R1, P46 antigens of Mycoplasma hyopneumoniae.

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.

Featured Species-Specific Loops Are Found in the Crystal Structure of Mhp Eno, a Cell Surface Adhesin From Mycoplasma hyopneumoniae.

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.

Immunoenhancement of Recombinant Neisseria meningitides PorB Protein on Porcine Circovirus Type 2 and Mycoplasma hyopneumoniae Genetically Engineered Vaccines.

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.

Differential secretome profiling of a swine tracheal cell line infected with mycoplasmas of the swine respiratory tract.

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.

Utilization of macrophage extracellular trap nucleotides by Mycoplasma hyopneumoniae.

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.

Comparative proteomics of two Mycoplasma hyopneumoniae strains and Mycoplasma flocculare identified potential porcine enzootic pneumonia determinants.

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.

Hydrogen peroxide production and myo-inositol metabolism as important traits for virulence of Mycoplasma hyopneumoniae.

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.

Extracellular Actin Is a Receptor for Mycoplasma hyopneumoniae.

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 ß-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i) monoclonal antibodies that target ß-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.

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae.

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.

Apoptosis induced by lipid-associated membrane proteins from Mycoplasma hyopneumoniae in a porcine lung epithelial cell line with the involvement of caspase 3 and the MAPK pathway.

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-derived lipid-associated membrane proteins induce inflammation and apoptosis in porcine peripheral blood mononuclear cells in vitro.

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-1ß, 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.

Proteolytic processing of the cilium adhesin MHJ_0194 (P123J ) in Mycoplasma hyopneumoniae generates a functionally diverse array of cleavage fragments that bind multiple host molecules.

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_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.