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.

Identification of a novel TLR5 agonist derived from the P97 protein of Mycoplasma hyopneumoniae.

By modulating specific immune responses against antigens, adjuvants are used in many vaccine preparations to enhance protective immunity. The C-terminal domain of the protein P97 (P97c) of Mycoplasma hyopneumoniae, which is the etiologic agent of porcine enzootic pneumonia, has been shown to increase the specific humoral response against an antigen when this antigen is merged with P97c and delivered by adenovectors. However, the immunostimulating mechanism of this protein remains unknown. In the present study, recombinantly expressed P97c triggered a concentration-dependent TLR5 activation and stimulates the production of interleukin-8 from HEK-Blue mTLR5 cells. Circular dichroism spectroscopy and prediction of 3-dimensional conformation exposed a relevant secondary and tertiary structural homology between P97c and flagellin, the known potent TLR5 agonist. P97c adjuvanticity was evaluated by fusing the conserved epitope of the ectodomain matrix 2 protein (M2e) of the influenza A virus to the protein. Mice immunized with P97c-3M2e revealed a high antibody titer against the M2e epitope associated with a mixed Th1/Th2 immune response. Overall, this study identifies a novel agonist of the pattern recognition receptor TLR5 and reveals that P97c is a potential adjuvant through the activation of the innate immune system.

Mycoplasma hyopneumoniae resides intracellularly within porcine epithelial cells.

Enzootic pneumonia incurs major economic losses to pork production globally. The primary pathogen and causative agent, Mycoplasma hyopneumoniae, colonises ciliated epithelium and disrupts mucociliary function predisposing the upper respiratory tract to secondary pathogens. Alleviation of disease is reliant on antibiotics, vaccination, and sound animal husbandry, but none are effective at eliminating M. hyopneumoniae from large production systems. Sustainable pork production systems strive to lower reliance on antibiotics but lack of a detailed understanding of the pathobiology of M. hyopneumoniae has curtailed efforts to develop effective mitigation strategies. M. hyopneumoniae is considered an extracellular pathogen. Here we show that M. hyopneumoniae associates with integrin ß1 on the surface of epithelial cells via interactions with surface-bound fibronectin and initiates signalling events that stimulate pathogen uptake into clathrin-coated vesicles (CCVs) and caveosomes. These early events allow M. hyopneumoniae to exploit an intracellular lifestyle by commandeering the endosomal pathway. Specifically, we show: (i) using a modified gentamicin protection assay that approximately 8% of M. hyopneumoniae cells reside intracellularly; (ii) integrin ß1 expression specifically co-localises with the deposition of fibronectin precisely where M. hyopneumoniae cells assemble extracellularly; (iii) anti-integrin ß1 antibodies block entry of M. hyopneumoniae into porcine cells; and (iv) M. hyopneumoniae survives phagolysosomal fusion, and resides within recycling endosomes that are trafficked to the cell membrane. Our data creates a paradigm shift by challenging the long-held view that M. hyopneumoniae is a strict extracellular pathogen and calls for in vivo studies to determine if M. hyopneumoniae can traffic to extrapulmonary sites in commercially-reared pigs.

The effect of polyclonal antibody on intracellular calcium increase induced by Mycoplasma hyopneumoniae in porcine tracheal cells.

An investigation was undertaken to assess whether polyclonal convalescent and hyperimmune sera obtained from pigs inhibit Mycoplasma hyopneumoniae induced increases in intracellular calcium [Ca2+](i) in ciliated porcine tracheal cells. Basal [Ca2+](i) in the tracheal cells was 97+/-13 nM (n=22 cells in four experiments) and after exposure to M. hyopneumoniae (300 micro g/mL or 10(11) CCU/mL), [Ca2+](i) increased by 246+/-56 nM within 100 s. After pre-treatment with hyperimmune or convalescent serum, M. hyopneumoniae increased [Ca2+](i) by 196+/-43 and 223+/-65 nM, respectively. It was found that neither hyperimmune nor convalescent serum significantly prevented the increase in [Ca2+](i) compared with M. hyopneumoniae alone. It was concluded that polyclonal antibodies produced by mycoplasma vaccination or exposure to the pathogen do not prevent M. hyopneumoniae-induced increase in [Ca2+](i).

Decreased protein accretion in pigs with viral and bacterial pneumonia is associated with increased myostatin expression in muscle.

Chronic respiratory infections reduce growth in pigs but protein accretion (PA) during an ongoing multifactorial respiratory infection has not been determined, and the mechanisms underlying growth inhibition are largely unknown. The objectives of this study were to determine whether viral and bacterial pneumonia in young pigs decrease PA, increase serum IL-1beta and IL-6, and increase myostatin (MSTN) mRNA in biceps femoris and triceps muscles. Mycoplasma hyopneumoniae (Mh) or medium was given intratracheally at 4 wk of age, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) or medium was given intranasally at 6 wk of age, and pigs were killed 7 or 14 d after PRRSV inoculation for body composition analysis. PRRSV but not Mh induced a marked increase (P < 0.01) in IL-1beta, IL-6, and MSTN mRNA and a decrease (P < 0.01) in food intake, daily weight gain, PA, and lipid accretion. PRRSV also reduced (P < 0.01) myofiber area in the biceps femoris. Food intake, weight gain, PA, and weight of biceps femoris and triceps muscles were negatively correlated (r = -0.4 to -0.8, P < 0.05) with serum IL-1beta and IL-6 and with MSTN mRNA in muscle. These results suggest that the magnitude of increases in inflammatory cytokines during a respiratory infection may be predictive of decreases in PA and growth. They further suggest that during infection growth of skeletal muscle is limited in part by myostatin.

Immunohistochemical labelling of cytokines in lung lesions of pigs naturally infected with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (Mh) is the primary agent of porcine enzootic pneumonia (PEN), a chronic respiratory disease endemic to pig farms, and characterized histologically by infiltration of mononuclear cells in airways and prominent hyperplasia of the bronchus-associated lymphoid tissue (BALT). To gain further insight into the pathogenesis of PEN, cytokine expression in the lung, with particular attention to the BALT, was examined immunohistochemically in pigs naturally infected with Mh. An increase (P < 0.05) in proinflammatory and immunoregulatory cytokines (especially interleukin [IL]-2, IL-4 and tumour necrosis factor [TNF]-alpha, and to a lesser extent IL-1 [alpha and beta] and IL-6) was detected in the BALT, which showed intense lymphoid hyperplasia. IL-1beta and TNF-alpha were also detected in the bronchoalveolar exudate of infected pigs, and IL-6 and IL-8 were demonstrated in mononuclear cells of the alveolar septa. The results showed that in Mh infection, macrophage and lymphocyte activation results in the expression of a number of cytokines capable of inducing lung lesions and lymphoreticular hyperplasia of the BALT.