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.

Isolation of Polystyrene Bead-Induced Phagosomes for Western Blotting.

The engulfment of "self" and "non-self" particles by immune and non-immune cells is crucial for maintaining homeostasis and combatting infection. Engulfed particles are contained within vesicles termed phagosomes that undergo dynamic fusion and fission events, which ultimately results in the formation of phagolysosomes that degrade the internalized cargo. This process is highly conserved and plays an important role in maintaining homeostasis, and disruptions in this are implicated in numerous inflammatory disorders. Given its broad role in innate immunity, it is important to understand how different stimuli or changes within the cell can shape the phagosome architecture. In this chapter, we describe a robust protocol for the isolation of polystyrene bead-induced phagosomes using sucrose density gradient centrifugation. This process results in a highly pure sample that can be used in downstream applications, namely, Western blotting.

C-type Lectins in Immunity to Lung Pathogens.

The respiratory tract is tasked with responding to a constant and vast influx of foreign agents. It acts as an important first line of defense in the innate immune system and as such plays a crucial role in preventing the entry of invading pathogens. While physical barriers like the mucociliary escalator exert their effects through the clearance of these pathogens, diverse and dynamic cellular mechanisms exist for the activation of the innate immune response through the recognition of pathogen-associated molecular patterns (PAMPs). These PAMPs are recognized by pattern recognition receptors (PRRs) that are expressed on a number of myeloid cells such as dendritic cells, macrophages, and neutrophils found in the respiratory tract. C-type lectin receptors (CLRs) are PRRs that play a pivotal role in the innate immune response and its regulation to a variety of respiratory pathogens such as viruses, bacteria, and fungi. This chapter will describe the function of both activating and inhibiting myeloid CLRs in the recognition of a number of important respiratory pathogens as well as the signaling events initiated by these receptors.

Vibrio cholerae residing in food vacuoles expelled by protozoa are more infectious in vivo.

Vibrio cholerae interacts with many organisms in the environment, including heterotrophic protists (protozoa). Several species of protozoa have been reported to release undigested bacteria in expelled food vacuoles (EFVs) when feeding on some pathogens. While the production of EFVs has been reported, their biological role as a vector for the transmission of pathogens remains unknown. Here we report that ciliated protozoa release EFVs containing V. cholerae. The EFVs are stable, the cells inside them are protected from multiple stresses, and large numbers of cells escape when incubated at 37 °C or in the presence of nutrients. We show that OmpU, a major outer membrane protein positively regulated by ToxR, has a role in the production of EFVs. Notably, cells released from EFVs have growth and colonization advantages over planktonic cells both in vitro and in vivo. Our results suggest that EFVs facilitate V. cholerae survival in the environment, enhancing their infectious potential and may contribute to the dissemination of epidemic V. cholerae strains. These results improve our understanding of the mechanisms of persistence and the modes of transmission of V. cholerae and may further apply to other opportunistic pathogens that have been shown to be released by protists in EFVs.

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.

The Effect of Collimating Lens Focusing on Laser Beam Shape in Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS).

Tissue imaging using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a well-established technique that, in recent years, has seen wider adoption and novel application. Applications such imaging mass spectrometry (IMS) and biotyping are beginning to gain greater exposure and use; however, with limitations in optimization methods, producing the best result often relies on the ability to customize the physical characteristics of the instrumentation, a task that is challenging for most mass spectrometry laboratories. With this in mind, we have described the effect of making simple adjustments to the laser optics at the final collimating lens area, to adjust the laser beam size and shape in order to allow greater customization of the instrument for improving techniques such as IMS. We have therefore been able to demonstrate that improvements can be made without requiring the help of an electrical engineer or external funding in a way that only costs a small amount of time. Graphical Abstract ᅟ.

N-terminomics identifies widespread endoproteolysis and novel methionine excision in a genome-reduced bacterial pathogen.

Proteolytic processing alters protein function. Here we present the first systems-wide analysis of endoproteolysis in the genome-reduced pathogen Mycoplasma hyopneumoniae. 669 N-terminal peptides from 164 proteins were identified, demonstrating that functionally diverse proteins are processed, more than half of which 75 (53%) were accessible on the cell surface. Multiple cleavage sites were characterised, but cleavage with arginine in P1 predominated. Putative functions for a subset of cleaved fragments were assigned by affinity chromatography using heparin, actin, plasminogen and fibronectin as bait. Binding affinity was correlated with the number of cleavages in a protein, indicating that novel binding motifs are exposed, and protein disorder increases, after a cleavage event. Glyceraldehyde 3-phosphate dehydrogenase was used as a model protein to demonstrate this. We define the rules governing methionine excision, show that several aminopeptidases are involved, and propose that through processing, genome-reduced organisms can expand protein function.

A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis.

Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O'Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single 'spots' in a polyacrylamide gel, allowing the quantitation of changes in a proteoform's abundance to ascertain changes in an organism's phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the 'Top-Down'. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O'Farrell's paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism's proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer.

The Characterization of Laser Ablation Patterns and a New Definition of Resolution in Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS).

Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) is a technique that has seen a sharp rise in both use and development. Despite this rapid adoption, there have been few thorough investigations into the actual physical mechanisms that underlie the acquisition of IMS images. We therefore set out to characterize the effect of IMS laser ablation patterns on the surface of a sample. We also concluded that the governing factors that control spatial resolution have not been correctly defined and therefore propose a new definition of resolution. Graphical Abstract ᅟ.

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.

A versatile cost-effective method for the analysis of fresh frozen tissue sections via matrix-assisted laser desorption/ionisation imaging mass spectrometry.

RATIONALE: There are currently multiple methods available for the preparation of fresh frozen tissue samples for analysis via matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) imaging mass spectrometry (IMS). Although these methods report excellent results, many are expensive automated approaches. With no published attempt to standardise less expensive manual processes, our work aims to provide a robust and repeatable method of sample preparation for MALDI-TOF-IMS that is applicable to a variety of tissue types, well explained, simple and cost effective. METHODS: Fresh frozen tissue was sectioned at 12 µm and mounted onto liquid nitrocellulose coated slides, washed in a graded alcohol series and then mounted into a modified sublimation apparatus. Matrix is deposited onto the slide to achieve a desired coating of 0.2 mg/cm(2). Once coated, the slide is mounted into a custom-built vapor chamber and recrystallised with 50% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) for 1 h at 37°C. The slide is then analysed using MALDI-IMS. RESULTS: We have successfully implemented this method for a host of tissue samples, including brain, liver, kidney and heart, with no variation in relative spectra or processing method required. When the protocol is followed correctly, sublimations and recrystallisations are highly predictable with limited variation between samples and a very low failure rate. Additional apparatuses can be easily constructed by following the included instructions, that perform as per specifications with no variation. CONCLUSIONS: We believe that we have described a complete protocol for MALDI-IMS that is easy to use and highly reproducible. The lack of expensive commercially available equipment makes this process very cheap with a relatively low initial outlay and our hope is that more laboratories will begin IMS-based avenues of research based on the work we have performed.

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.

MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae.

Aminopeptidases are part of the arsenal of virulence factors produced by bacterial pathogens that inactivate host immune peptides. Mycoplasma hyopneumoniae is a genome-reduced pathogen of swine that lacks the genetic repertoire to synthesize amino acids and relies on the host for availability of amino acids for growth. M. hyopneumoniae recruits plasmin(ogen) onto its cell surface via the P97 and P102 adhesins and the glutamyl aminopeptidase MHJ_0125. Plasmin plays an important role in regulating the inflammatory response in the lungs of pigs infected with M. hyopneumoniae. We show that recombinant MHJ_0461 (rMHJ_0461) functions as a leucine aminopeptidase (LAP) with broad substrate specificity for leucine, alanine, phenylalanine, methionine and arginine and that MHJ_0461 resides on the surface of M. hyopneumoniae. rMHJ_0461 also binds heparin, plasminogen and foreign DNA. Plasminogen bound to rMHJ_0461 was readily converted to plasmin in the presence of tPA. Computational modelling identified putative DNA and heparin-binding motifs on solvent-exposed sites around a large pore on the LAP hexamer. We conclude that MHJ_0461 is a LAP that moonlights as a multifunctional adhesin on the cell surface of M. hyopneumoniae.

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.

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.