The C-type lectin DCIR contributes to the immune response and pathogenesis of colorectal cancer.

Development and progression of malignancies are accompanied and influenced by alterations in the surrounding immune microenvironment. Understanding the cellular and molecular interactions between immune cells and cancer cells has not only provided important fundamental insights into the disease, but has also led to the development of new immunotherapies. The C-type lectin Dendritic Cell ImmunoReceptor (DCIR) is primarily expressed by myeloid cells and is an important regulator of immune homeostasis, as demonstrated in various autoimmune, infectious and inflammatory contexts. Yet, the impact of DCIR on cancer development remains largely unknown. Analysis of available transcriptomic data of colorectal cancer (CRC) patients revealed that high DCIR gene expression is associated with improved patients' survival, immunologically "hot" tumors and high immunologic constant of rejection, thus arguing for a protective and immunoregulatory role of DCIR in CRC. In line with these correlative data, we found that deficiency of DCIR1, the murine homologue of human DCIR, leads to the development of significantly larger tumors in an orthotopic murine model of CRC. This phenotype is accompanied by an altered phenotype of tumor-associated macrophages (TAMs) and a reduction in the percentage of activated effector CD4+ and CD8+ T cells in CRC tumors of DCIR1-deficient mice. Overall, our results show that DCIR promotes antitumor immunity in CRC, making it an attractive target for the future development of immunotherapies to fight the second deadliest cancer in the world.

L-DOPA causes mitochondrial dysfunction in vitro: A novel mechanism of L-DOPA toxicity uncovered.

In Parkinson's disease (PD), as in many other neurodegenerative disorders, mitochondrial dysfunction, protein misfolding, and proteotoxic stress underly the disease process. For decades, the primary symptomatic treatment for PD has been the dopamine precursor L-DOPA (Levodopa). L-DOPA however can initiate protein misfolding through its ability to mimic the protein amino acid L-tyrosine, resulting in random errors in aminoacylation and L-DOPA becoming mistakenly inserted into the polypeptide chain of proteins in place of L-tyrosine. In the present study we examined the impact that the generation of DOPA-containing proteins had on human neuroblastoma cell (SH-SY5Y) function in vitro. We showed that even in the presence of antioxidants there was a significant accumulation of cytosolic ubiquitin in DOPA-treated cells, an upregulation in the endosomal-lysosomal degradation system, deleterious changes to mitochondrial morphology and a marked decline in mitochondrial function.The effects of L-DOPA on mitochondrial function were not observed with D-DOPA, the stereoisomer of L-DOPA that cannot be inserted into proteins so did not result from oxidative stress. We could fully protect against these effects by co-treatment with L-tyrosine, supporting the view that misincorporation of L-DOPA into proteins contributed to these cytotoxic effects, leading us to suggest that co-treatment with L-tyrosine could be beneficial therapeutically.

Extracellular DNA release from the genome-reduced pathogen Mycoplasma hyopneumoniae is essential for biofilm formation on abiotic surfaces.

Mycoplasma hyopneumoniae is an economically devastating, globally disseminated pathogen that can maintain a chronic infectious state within its host, swine. Here, we depict the events underpinning M. hyopneumoniae biofilm formation on an abiotic surface and demonstrate for the first time, biofilms forming on porcine epithelial cell monolayers and in the lungs of pigs, experimentally infected with M. hyopneumoniae. Nuclease treatment prevents biofilms forming on glass but not on porcine epithelial cells indicating that extracellular DNA (eDNA), which localises at the base of biofilms, is critical in the formation of these structures on abiotic surfaces. Subpopulations of M. hyopneumoniae cells, denoted by their ability to take up the dye TOTO-1 and release eDNA, were identified. A visually distinct sub-population of pleomorphic cells, that we refer to here as large cell variants (LCVs), rapidly transition from phase dark to translucent "ghost" cells. The translucent cells accumulate the membrane-impermeable dye TOTO-1, forming readily discernible membrane breaches immediately prior to lysis and the possible release of eDNA and other intracellular content (public goods) into the extracellular environment. Our novel observations expand knowledge of the lifestyles adopted by this wall-less, genome-reduced pathogen and provide further insights to its survival within farm environments and swine.

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.

Exploitation of plasmin(ogen) by bacterial pathogens of veterinary significance.

The plasminogen (Plg) system plays an important homeostatic role in the degradation of fibrin clots, extracellular matrices and tissue barriers important for cellular migration, as well as the promotion of neurotransmitter release. Plg circulates in plasma at physiologically high concentrations (150-200µg ml(-1)) as an inactive proenzyme. Proteins enriched in lysine and other positively charged residues (histidine and arginine) as well as glycosaminoglycans and gangliosides bind Plg. The binding interaction initiates a structural adjustment to the bound Plg that facilitates cleavage by proteases (plasminogen activators tPA and uPA) that activate Plg to the active serine protease plasmin. Both pathogenic and commensal bacteria capture Plg onto their cell surface and promote its conversion to plasmin. Many microbial Plg-binding proteins have been described underpinning the importance this process plays in how bacteria interact with their hosts. Bacteria exploit the proteolytic capabilities of plasmin by (i) targeting the mammalian fibrinolytic system and degrading fibrin clots, (ii) remodeling the extracellular matrix and generating bioactive cleavage fragments of the ECM that influence signaling pathways, (iii) activating matrix metalloproteinases that assist in the destruction of tissue barriers and promote microbial metastasis and (iv) destroying immune effector molecules. There has been little focus on the exploitation of the fibrinolytic system by veterinary pathogens. Here we describe several pathogens of veterinary significance that possess adhesins that bind plasmin(ogen) onto their cell surface and promote its activation to plasmin. Cumulative data suggests that these attributes provide pathogenic and commensal bacteria with a means to colonize and persist within the host environment.

Evaluation of recombinant Mycoplasma hyopneumoniae P97/P102 paralogs formulated with selected adjuvants as vaccines against mycoplasmal pneumonia in pigs.

Pig responses to recombinant subunit vaccines containing fragments of eight multifunctional adhesins of the Mycoplasma hyopneumoniae (Mhp) P97/P102 paralog family formulated with Alhydrogel(®) or Montanide™ Gel01 were compared with a commercial bacterin following experimental challenge. Pigs, vaccinated intramuscularly at 9, 12 and 15 weeks of age with either of the recombinant formulations (n=10 per group) or Suvaxyn(®) M. hyo (n=12), were challenged with Mhp strain Hillcrest at 17 weeks of age. Unvaccinated, challenged pigs (n=12) served as a control group. Coughing was assessed daily. Antigen-specific antibody responses were monitored by ELISA in serum and tracheobronchial lavage fluid (TBLF), while TBLF was also assayed for cytokine responses (ELISA) and bacterial load (qPCR). At slaughter, gross and histopathology of lungs were quantified and damage to epithelial cilia in the porcine trachea was evaluated by scanning electron microscopy. Suvaxyn(®) M. hyo administration induced significant serological responses against Mhp strain 232 whole cell lysates (wcl) and recombinant antigen F3P216, but not against the remaining vaccine subunit antigens. Alhydrogel(®) and Montanide™ Gel01-adjuvanted antigen induced significant antigen-specific IgG responses, with the latter adjuvant eliciting comparable Mhp strain 232 wcl specific IgG responses to Suvaxyn(®) M. hyo. No significant post-vaccination antigen-specific mucosal responses were detected with the recombinant vaccinates. Suvaxyn(®) M. hyo was superior in reducing clinical signs, lung lesion severity and bacterial load but the recombinant formulations offered comparable protection against cilial damage. Lower IL-1ß, TNF-α and IL-6 responses after challenge were associated with reduced lung lesion severity in Suvaxyn(®) M. hyo vaccinates, while elevated pathology scores in recombinant vaccinates corresponded to cytokine levels that were similarly elevated as in unvaccinated pigs. This study highlights the need for continued research into protective antigens and vaccination strategies that will prevent Mhp colonisation and establishment of infection.

Cilium adhesin P216 (MHJ_0493) is a target of ectodomain shedding and aminopeptidase activity on the surface of Mycoplasma hyopneumoniae.

MHJ_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.

Characterization of cleavage events in the multifunctional cilium adhesin Mhp684 (P146) reveals a mechanism by which Mycoplasma hyopneumoniae regulates surface topography.

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.

Mhp182 (P102) binds fibronectin and contributes to the recruitment of plasmin(ogen) to the Mycoplasma hyopneumoniae cell surface.

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.