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 β1 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 β1 expression specifically co-localises with the deposition of fibronectin precisely where M. hyopneumoniae cells assemble extracellularly; (iii) anti-integrin β1 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.

Combinations of these strategies are effective, however, there remains a pressing need to lower reliance on antibiotics to control infection in intensively reared animal production systems [22][23][24][25] . Sound herd management is challenged by the need to identify pig production operations free of M. hyopneumoniae and it remains a challenge to identify subclinically-infected and carrier animals. Ultimately, further investigation into the survival mechanisms of this important porcine pathogen is required to aid in the development of future strategies to prevent and control transmission. It is well known that numerous mycoplasma species can invade host cells [26][27][28][29] , and although it has historically been characterised as a strict extracellular pathogen, M. hyopneumoniae has been cultured from the liver, spleen, kidneys and bronchial lymph nodes of pigs infected experimentally with M. hyopneumoniae [30][31][32] . However, it is not known if M. hyopneumoniae colonises tissue sites distal to the respiratory tract in commercially-reared herds. Interestingly, M. hyopneumoniae has been isolated in pure culture from both pericardial and synovial joint fluids in slaughter-age commercial pigs with fibrinous pericarditis 33 . It is not known how M. hyopneumoniae traffics to these sites nor is it known if M. hyopneumoniae can invade epithelial cells and trigger cellular uptake pathways.
In this study for the first time, we show that M. hyopneumoniae cells interact with integrin β1 via fibronectin and colocalise in a manner that promotes cellular uptake via caveosomes and clathrin-coated vesicles. We monitored the cellular events that depict M. hyopneumoniae trafficking via the endocytic pathway, and escaping phagolysosomal fusion, before residing free in the cytoplasm. Collectively, our data have significant implications for detecting animals infected with M. hyopneumoniae and for development of therapies to eliminate this difficult-to-control pathogen.

M. hyopneumoniae resides intracellularly within epithelial cells.
In order to gather insight into how M. hyopneumoniae colonises host epithelial cell surfaces, scanning electron microscopy (SEM) was used to visualise the pattern of adherence to porcine kidney epithelial cells (PK-15) after 16 h. PK-15 cells have been used extensively as a model system for studying host-M. hyopneumoniae interactions 14,[34][35][36] . SEM images showed both small clusters and individual M. hyopneumoniae cells associated intimately with the cell surface of PK-15 cell monolayers (Fig. 1A-D). These adhering bacterial cells are encapsulated by cell surface projections via a process that resembles macropinocytosis (Fig. 1A-E), which occasionally leads to the complete engulfment of the  (Fig. 1F). Using immunofluorescence microscopy, we were able to confirm that these engulfed bacteria were indeed M. hyopneumoniae cells using anti-F2 P94-J antiserum, which is specific for M. hyopneumoniae. We double-labelled M. hyopneumoniae-infected PK-15 monolayers using F2 P94-J antisera conjugated to either CF 488 or CF 568 before and after permeabilisation, respectively. From this, extracellularly adhering bacteria were double-labelled (appearing yellow/green, Supplementary Fig. S1), while those cells residing intracellularly were singularly labelled (appearing red, Supplementary Fig. S1). Once we confirmed that these intracellular bacteria were M. hyopneumoniae, we replaced the secondary labelling step with the membrane impermeable dye DAPI, as it reliably stains M. hyopneumoniae nucleic acids 35 while also reducing background staining. Extracellularly adhering M. hyopneumoniae cells (labelled with anti-F2 P94-J antisera; magenta in Fig. 1G,H) were readily distinguishable from those residing intracellularly (stained with DAPI; cyan in Fig. 1G,H). Confocal laser scanning microscopy (CLSM) and 3D-Structured Illumination Microscopy (3D-SIM) images of these samples depict extracellular, F2 P94-J -labelled M. hyopneumoniae cells adhering to PK-15 cells, and numerous intracellular bacteria stained solely with DAPI (Fig. 1G,H). In uninfected control monolayers that were stained with DAPI post-permeabilisation, only the nuclei of the PK-15 cells were visible ( Supplementary Fig. S2). This confirmed that the staining technique did not stain nucleic acids in the cytoplasm of PK-15 monolayers and could be used to distinguish between extracellular and intracellular bacteria. To determine the number of intracellular M. hyopneumoniae cells, we applied a modified and optimised version of the gentamicin protection assay 37 . This showed that approximately 1 in every 12 (8%) M. hyopneumoniae cells that adhered to PK-15 cells reside intracellularly and are potentially invasive (data not shown). Although it is important to note that the gentamicin protection assay is a crude quantitative measure of cell invasion, we have routinely observed M. hyopneumoniae cells residing intracellularly using CLSM and 3D-SIM. Our observations presented here, are representative images of a minimum of 20 biological replicate experiments performed on different days over the course of several years.  32,38 . The electron-dense cells appear to be intact and do not seem to be associated with any vesicle-like structures ( Supplementary Fig. S3). To our knowledge, this is the first experimental evidence depicting mycoplasma-like organisms residing within swine respiratory epithelial cells.

M. hyopneumoniae cells are engulfed by clathrin-and caveolae-mediated endocytosis and are trafficked intracellularly via the complete endocytic pathway.
Clathrin-and caveolae-mediated endocytosis represent two endocytic pathways involved in the uptake of bacteria by eukaryotes 39 . We used confocal microscopy with monoclonal antibodies that recognise clathrin (mAb clath. ) and caveolin-1 (using mAb cav ) to show M. hyopneumoniae cells residing within clathrin-coated vesicles (CCVs) and caveosomes (Fig. 3), respectively, after 16 h incubation with PK-15 monolayers. Notably, we observed CCVs in direct contact with the PK-15 membrane in close proximity to M. hyopneumoniae cells on the extracellular side of the membrane in the process of being engulfed ( Supplementary Fig. S4). Furthermore, structures resembling caveolae in areas where M. hyopneumoniae cells were adhering to the PK-15 cell surface were observed via SEM ( Supplementary Fig. S5).
To investigate the association of M. hyopneumoniae cells with the endocytic pathway, we stained infected PK-15 cells with a panel of antibodies specific for early, late and recycling endosomes, and lysosomes. M. hyopneumoniae cells residing within early endosomes that display the markers EEA1 and RAB5 were observed using confocal microscopy ( Fig. 3). M. hyopneumoniae cells were also observed within endosomes displaying RAB11 suggesting that M. hyopneumoniae can influence the endosomal pathway and be exported back to the extracellular milieu ( Fig. 3). Early endosomes mature into late endosomes displaying RAB7, and M. hyopneumoniae cells were also observed within these vesicles (Fig. 3). Typically, late endosomes fuse with lysosomes to allow the delivery of hydrolytic enzymes to degrade the engulfed cargo. Using LAMP1 as a marker of the lysosomal membrane, we detected numerous lysosomes within the cytoplasm of PK-15 cells containing intracellular M. hyopneumoniae cells (Fig. 3). In these examples, numerous extracellularly adhering M. hyopneumoniae cells could be seen in the vicinity of the vesicle-bound intracellular bacteria, further supporting our differential staining protocol ( Supplementary Fig. S6). Notably, M. hyopneumoniae cells were identified using 3D-SIM in the immediate vicinity of LAMP1-labelled lysosomal membrane fragments ( Supplementary Fig. S7) and M. hyopneumoniae cells were identified in the cytoplasm of PK-15 cells (Fig. 2F-H) using TEM. To our knowledge, this is the first depiction of Mycoplasma spp. being trafficked intracellularly via the complete endocytic pathway. of the leading edge, often along the length of fibres that are consistent with fibronectin ( Supplementary Fig. S8). This was confirmed using confocal microscopy which showed M. hyopneumoniae cells adhering to fibronectin plumes secreted by PK-15 cells at the leading edge ( Supplementary Fig. S8).
In a previous study, M. hyopneumoniae cells were shown to sequester fibronectin to their cell surface 14 , a finding mirrored in this work (Fig. 4, panels B,E). In the extracellular matrix fibronectin connects with the actin cytoskeleton via the bridging molecule, integrin β1 40 . Numerous bacterial pathogens hijack these molecules to initiate internalisation 41 . Confocal microscopy was used to investigate co-localisation of fibronectin and integrin β1 in PK-15 cells infected with M. hyopneumoniae ( Fig. 4A-F). Abundant fibronectin staining was observed to associate with regions on PK-15 cells that were colonised with M. hyopneumoniae (Fig. 4B,E), an observation that is consistent with these bacterial cells sequestering fibronectin onto their cell surface. In addition, it was noted that integrin β1 staining was abundant in the same areas, forming "pockets" around the fibronectin-bound M. hyopneumoniae cells (Fig. 4C-F). Consistent with this observation was significantly higher integrin β1 staining of M. hyopneumoniae-infected PK-15 cells compared to the uninfected control (Fig. 4G). Collectively, these data suggest that M. hyopneumoniae cells sequester fibronectin that is expressed early in response to infection and co-localises with integrin β1. To determine if integrin β1 plays a role in intracellular uptake of M. hyopneumoniae, PK-15 cells were pre-incubated with a neutralising integrin β1 antibody (mAb ITGβ1 ) for 2 h prior to infection with M. hyopneumoniae. Our modified gentamicin protection assay showed that the antibody blocked (~75% reduction in viable colonies) the ability of M. hyopneumoniae to become internalised within PK-15 cells (Fig. 4H). This is the first time that integrin β1 has been shown to play a role during the initial phase of cellular invasion by M. hyopneumoniae, or any other Mycoplasma species. To examine this, we stained serial tracheal sections with mAb β-act and with F2 P94-J antisera. It was observed that serial tracheal sections from pigs infected experimentally with M. hyopneumoniae (diseased tissue) that were stained with F2 P94-J antisera contained bacterial cells adhering along the ciliary border of the epithelium (Fig. 5A) as expected. A serial section of the same diseased tissue stained with mAb β-act identified actin quite prominently in the subepithelial layer (Fig. 5B), whereas control uninfected tissues did not stain intensely with mAb β-act (Fig. 5C,D). In diseased tissue, lymphoid follicles and intraepithelial leukocytes that had infiltrated the tissue at the site of infection also stained intensely with mAb β-act (Fig. 5E,F). These data suggest that extensive cytoskeletal rearrangements may occur in the respiratory tract of swine infected with M. hyopneumoniae.

M. hyopneumoniae is trafficked alongside fibronectin and integrin β1.
Clathrin-and caveolinmediated endocytosis is also how the integrin heterodimer, α5β1, the primary receptor of fibronectin, is endocytosed 43,44 . Our hypothesis is that M. hyopneumoniae cells are endocytosed while bound to fibronectin, therefore we determined the spatial relationship between intracellular M. hyopneumoniae cells coated with fibronectin and integrin β1, caveosomes, CCVs, and lysosomes. To visualise intracellular fibronectin, we incubated M. hyopneumoniae-infected (16 h) PK-15 cells with anti-fibronectin (pAb Fn ) after membrane permeabilisation. It was found that M. hyopneumoniae cells associated both with CCVs ( Fig. 6A-D) and with caveosomes ( Fig. 6E-H) containing fibronectin. In one example, upwards of 20 M. hyopneumoniae cells were seen within a single CCV (Fig. 6C). Vesicle-like structures harbouring M. hyopneumoniae cells were also found to simultaneously stain with mAb ITGβ1 and fibronectin (Fig. 6I-L). Lysosomes containing fibronectin and M. hyopneumoniae cells were repeatedly observed ( Fig. 6M-P). Numerous lysosomes were observed surrounding vesicle-like structures that contain both fibronectin and M. hyopneumoniae cells (Fig. 6). Secondary antibody controls ensured that the observed association of fibronectin and these vesicles was not due to antibody cross-reactivity (data not shown).

Discussion
M. hyopneumoniae has historically been considered an extracellular pathogen despite numerous studies suggesting the contrary [30][31][32]45 . One study in particular, was able to recover M. hyopneumoniae from extrapulmonary sites during treatment with a therapeutic dose of marbofloxacin 30 . Notably, no extrapulmonary bacteria were In further support of this hypothesis, numerous studies have demonstrated that M. hyopneumoniae has the ability to sequester plasminogen to its cell surface and facilitate its activation to plasmin; a potent serine protease that can degrade a range of ECM and cellular junction components 9,16-18,46-48 . Additionally, elevated levels of plasmin have been detected in bronchialviolar lavage fluid of pigs infected with M. hyopneumoniae 18,49 . The ability to utilise host plasmin is a hallmark of pathogens that have developed sophisticated mechanisms to invade host cells and to disseminate to distal tissue sites [50][51][52][53][54] . For the first time we show M. hyopneumoniae has the capacity to enter host-derived epithelial cells (PK-15) via clathrin-and caveolae-mediated endocytosis. Membrane bound M. hyopneumoniae captured via these mechanisms are transported intracellularly via early, recycling and late endosomes (Fig. 3). Notably, a sub-population of cells appear to survive fusion with lysosomes and escape into the cytosol (Supplementary Fig. S7). However, it is not known how this invasive subpopulation of M. hyopneumoniae cells differs from the majority of adherent, non-invasive cells. Confocal microscopy studies shown here, the isolation of M. hyopneumoniae from infected PK-15 cells on Friis agar after treatment with a dose of gentamicin sufficient to kill extracellular populations of M. hyopneumoniae cells were allowed to adhere to PK-15 cells for 16 h after which they were fixed, permeabilised and incubated with pAb Fn which was conjugated to CF 488 (yellow). Samples were then incubated with mAbs targeting CCVs (mAb clath. ), caveosomes (mAb cav. ), integrin β1 (mAb ITGβ1 ) and lysosomes (LAMP1) and conjugated to CF 568 (magenta). PK-15 nuclei and M. hyopneumoniae nucleic acids were stained with DAPI (cyan). Each channel is presented as individual columns in addition to an overlay. Vesicles containing fibronectin are indicated by a white arrow and can be seen colocalising with CCVs, caveosomes, integrin β1, and lysosomes. M. hyopneumoniae cells can also be seen within each of these vesicles. These images indicate that M. hyopneumoniae cells are engulfed alongside fibronectin and integrin β1 by CCVs and caveosomes that eventually fuse with lysosomes. Fibronectin as part of the extracellular matrix can also be seen as yellow fibrous material. this pathogen, and 3D-SIM images depicting intracellular M. hyopneumoniae cells in the vicinity of lysosomal membrane remnants, all provide compelling evidence in support of a possible intracellular existence and lifestyle for this pathogen. Notably, intracellular membrane-bound M. hyopneumoniae were also observed to be trafficked via recycling endosomes, where they can re-enter the extracellular milieu. While we have attempted to identify M. hyopneumoniae residing within ciliated epithelium in the respiratory tract of pigs, the evidence presented is limited but sufficient to encourage further in vivo studies.
Integrins are highly abundant membrane bound receptors that link extracellular ligands, particularly from the extracellular matrix, to cytoskeletal actin in the cell. Endocytic trafficking has an important role in regulating the presentation of integrin receptors on the cell surface and has a direct impact on important intracellular signalling events 55 . Therefore, it is perhaps unsurprising that a central theme in bacterial pathogenesis involves the interaction between fibronectin-binding proteins on the surface of invasive bacterial pathogens, fibronectin and integrin α5β1 in a manner that influences integrin clustering, to elicit an intracellular signalling cascade 41,56 . M. hyopneumoniae cells display numerous fibronectin-binding adhesins on their surface 9,11,14,18 , an observation consistent with the co-localisation of fibronectin with M. hyopneumoniae during colonisation of PK-15 cells, as well as along the ciliated epithelial cell border of the lungs of pigs experimentally-infected with M. hyopneumoniae 14 . Here we extend these earlier observations and show that integrin β1 staining co-localises with the deposition of fibronectin, where M. hyopneumoniae cells adhere on the surface of PK-15 cells. We observed increased integrin β1 staining in PK-15 cells that were infected with M. hyopneumoniae as well as the subsequent co-localisation of fibronectin-coated extracellular M. hyopneumoniae cells to these areas (Fig. 4). Interestingly, unpublished data from our lab suggests that integrin β1 is not differentially expressed in infected monolayers, suggesting that the increased staining seen here may in fact be due to conformational changes in integrin β1 that enhance binding of mAb ITGβ1 after fixation 57 . This would possibly imply that these conformational changes are due to the presence of M. hyopneumoniae and its ability to sequester fibronectin, as it is a well-known phenomenon that pathogens utilise ECM proteins to interact with and activate integrins 58 . Previously, it has been shown that mAb ITGβ1 can activate integrin β1 59 , therefore this may explain why it was able to block invasion when incubated with live cells prior to infection with M. hyopneumoniae. Indeed, the ability of anti-integrin β1 antibodies to block cell invasion demonstrate the potentially significant role of integrin β1 in promoting the ability of M. hyopneumoniae to enter PK-15 cells. These observations led us to hypothesize that the uptake of M. hyopneumoniae into CCVs and caveosomes, and the subsequent fusion with lysosomes, is regulated by interactions between fibronectin recruited to the bacterial cell surface and integrin β1. Consistent with this view, we routinely observed M. hyopneumoniae cells residing within CCVs, caveosomes and lysosomes, in areas where fibronectin expression has been induced (Fig. 6). In some instances, we observed M. hyopneumoniae cells together with fibronectin residing within integrin β1-staining vesicles (Fig. 6I-L), suggesting that fibronectin-integrin complexes are trafficked with M. hyopneumoniae via the endosomal pathway. Integrin β1-fibronectin complexes are known to be readily engulfed via both clathrin-and caveolae-mediated endocytosis prior to degradation by lysosomes or recycling back onto the cell surface 43,55,60 . Although we did not observe M. hyopneumoniae co-localising with fibronectin within recycling endosomes, we do provide evidence that M. hyopneumoniae cells are exocytosed (Fig. 3).
M. hyopneumoniae expresses surface-accessible actin-binding proteins and may target extracellular actin as a receptor on the surface of epithelial cells 35 . As seen previously, sections of porcine lung tissue prepared from pigs infected with M. hyopneumoniae showed that the organism localised to the ciliated epithelial surface in the airway lumen (Fig. 5A) 14,18 . Similar sections stained with mAb β-act antibodies reveal deposition of actin in the sub-epithelial tissues beneath and surrounding infection foci (Fig. 5B) at levels that are absent from control uninfected porcine lung tissue (Fig. 5C,D). Cytoskeletal rearrangements in epithelial and subepithelial cells [61][62][63] are induced by proinflammatory cytokines TNF-α and IL-6, that are known to be up-regulated during infection caused by M. hyopneumoniae 32,64 . These same cytokines induce the recruitment of intraepithelial leukocytes via a process that requires cytoskeletal (actin) reorganisation. Leukocytes are an established portal for the dissemination of many pathogens to distal tissue sites [65][66][67][68] and provide a protective niche from the immune system. M. hyopneumoniae promotes the infiltration of leukocytes that stain with mAb β-act to foci of infection. The inflammatory response, a hallmark of lung infection caused by M. hyopneumoniae, may provide the organism with a mechanism to invade and survive professional phagocytic cells for dissemination to the liver, spleen, kidneys and lymph nodes [30][31][32] . However, further studies are needed to interrogate this hypothesis.
Collectively, our data suggests that M. hyopneumoniae potentially hijacks the intrinsic eukaryotic α5β1-fibronectin recycling pathway to gain access to host cells where it can persist intracellularly. This has important implications for the trafficking of M. hyopneumoniae from the respiratory tract to distal tissue sites and potentially vice versa after extended periods of dormancy. This study paves the way for the development of therapeutic strategies that seek to interfere or block the ability of this pathogen to persist in its only known host Sus scrofa.

Materials and Methods
All data generated or analysed during this study are included in this published article (and its Supplementary Information files).
Bacterial strains and culture. Mycoplasma hyopneumoniae strain 232 was grown in modified Friis medium as described previously 69 . Broth cultures were incubated at 37 °C for approximately 16 h until they reached mid-exponential phase and the medium turned orange. At this stage, cultures contain approximately 5 × 10 5 -1 × 10 6 CFU/mL. For growth on solid agar, cells were plated onto Friis agar using a method described previously 70 . Gentamicin protection assay. Porcine kidney epithelial-like monolayers (PK-15) were grown to semi-confluency and split into 12-well microtitre plates (~10 5 cells/well) and incubated overnight. For infection studies, a 16 h culture of M. hyopneumoniae strain 232 was pelleted, washed twice in PBS, and resuspended in 25 mM HEPES in DMEM containing 5% fetal bovine serum (infection medium) prior to incubation at 37 °C for 2 h. M. hyopneumoniae cells were added, so that each well received 0.5 mL representative volume (approximately 7.5 × 10 5 CFU/mL) of the initial culture, and allowed to adhere to PK-15 cells for 16 h at 37 °C/5% CO 2 . Non-adherent cells were removed by washing in PBS, followed by incubation in 300 µg ml −1 gentamicin in DMEM (filter sterilised through pore size 0.2 µm) for 4 h at 37 °C/5% CO 2 . Duplicate samples (positive controls) were not incubated with gentamicin. The gentamicin was removed and cells were washed 5 × in PBS to remove any residual antibiotic. The cell dissociation solution TrypLE (Gibco, ThermoFisher Scientific) was added to each well and incubated at 37 °C for 20 minutes. Cells were then removed by gently pipetting the TrypLE 3-4 times. The cell suspension was serially diluted in Friis broth to 10 −1 and 10 −2 . An aliquot (50 µl) of each dilution was pipetted onto 9 cm Friis agar plates, the liquid was allowed to air dry in a sterile environment for 10 minutes, and the plates were incubated at 37 °C/5% CO 2 for at least 7 days. M. hyopneumoniae colonies were counted using a stereomicroscope (×40 objective). Inhibition experiments were performed by pre-incubating PK-15 cells with a 1:100 dilution of monoclonal antibodies against integrin β1 (mAb ITGβ1. ; ~5 µg ml −1 mAb, Abcam) prior to the addition of M. hyopneumoniae cells.
Immunofluorescence microscopy of internalised M. hyopneumoniae cells. Experiments were performed as described previously 35 with some minor modifications. Once samples were fixed and blocked, M. hyopneumoniae cells were incubated with polycloncal F2 P94-J rabbit antisera at a dilution of 1:500 for 1 h at RT. A 1:1000 dilution of anti-rabbit CF 488-labelled secondary antibody (Biotium) was incubated for 1 h at RT. Cells were permeabilised in 0.5% (v/v) Triton X-100 in PBS for 5 min. Samples were then re-incubated with F2 P94-J rabbit antisera at a dilution of 1:500 for 1 h at RT, followed by incubating with a 1:1000 dilution of anti-rabbit CF 568-labelled secondary antibody (Biotium) for 1 h at RT. DAPI (Roche) was then added for 5 minutes at RT to stain nucleic acids. Samples were then prepared as previously described 35 .
Immunofluorescence of M. hyopneumoniae cells and clathrin, caveosomes, endosomes, integrin β1 and fibronectin. Experiments were performed identically to those described above except that intracellular M. hyopneumoniae cells were labelled exclusively with DAPI and not F2 P94-J antisera. Fibronectin was labelled prior to permeabilisation using polyclonal antisera raised in rabbit against fibronectin (pAb Fn ) as described previously 14  Image analysis. Images captured with the Nikon A1 Confocal microscope and those generated by the DeltaVision OMX 3D-SIM were processed using Bitplane, Imaris Scientific 3D/4D image processing software to create Maximum Intensity Projection (MIP) and slices images. M. hyopneumoniae-infected PK-cells and uninfected controls were labelled with integrin β1 (described above) and 10 random fields of view were captured using an Olympus BX51 Upright Epi Fluorescence Microscope at 20 × magnification and a constant exposure. The mean fluorescence of integrin β1-stained samples was calculated, after thresholding and binary conversion, using ImageJ. GraphPad Prism 7 was used to plot the data, including the standard error of the mean, and to perform the statistical analyses (unpaired t-test).
Infection of PK-15 cells for scanning electron microscopy. Experiments were performed as described previously with no modifications 35 .
Preparation of tracheal sections and infected monolayers for transmission electron microscopy Samples were fixed in a fixation solution containing 5% formaldehyde and 2% glutaraldehyde in cacodylate buffer (0.1 M cacodylate, 0.01 M CaCl2, 0.01 M MgCl2, 0.09 M sucrose, pH 6.9) and washed with cacodylate buffer. Samples were then osmificated with 1% aqueous osmium for 1 h at room temperature, washed and pellets of the samples were embedded in 2% water agar and cut into small cubes. Dehydration was achieved with a graded series of acetone (10%, 20%, 50%) for 30 min on ice followed by contrasting with 2% uranyl acetate in 70% acetone for overnight at 4 °C and further dehydrated with 90% and 100% acetone. Samples in 100% acetone were allowed to reach room temperature and were infiltrated with epoxy resin according to Spurr's formular for a medium resin 72 ; 1 part 100% acetone/1 part resin for overnight, 1 part 100% acetone/2 parts resin for 8 h, pure resin for overnight and several changes the following 2 days. Samples were then transferred to resin filled gelatine capsules and polymerized for 8 h at 75 °C. Ultrathin sections were cut with a diamond knife, picked up with formvar-coated copper grids (300 mesh) and counterstained with 2% aqueous uranyl acetate and lead citrate. After air-drying samples were examined in a Zeiss transmission electron microscope TEM910 at an acceleration voltage of 80 kV. Images were recorded digitally with a Slow-Scan CCD-Camera (ProScan, 1024 × 1024, Scheuring, Germany) with ITEM-Software (Olympus Soft Imaging Solutions, Münster, Germany). Brightness and contrast were adjusted with Adobe Photoshop CS4. Immunohistochemistry. All animal procedures were approved by the Animal Ethics Committee at the Elizabeth Macarthur Agricultural Institute and were in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Experiments were performed as previously described 14,32,38 with a minor modification. Briefly, 19 male weaner pigs, sourced from a commercial herd, were infected endotracheally, with M. hyopneumoniae strain Hillcrest and euthanized 6 weeks post-infection. Serial sections of lung lesions from pigs, as well as healthy lung tissue from control pigs were examined for actin distribution by staining with 1:200 mAb β-act for 1 hr at RT.