Proteomic investigation of the genome-reduced pathogen, Mycoplasma hyopneumoniae

University of Technology Sydney. Faculty of Science.Mycoplasma hyopneumoniae is a genome-reduced bacterium and an economically significant pathogen that chronically infects the respiratory tract of swine. This infection often leads to pneumonia and secondary infections, costing agricultural industries significantly in the use of antibiotics and vaccines, which are currently largely ineffective. An improved understanding of the molecular mechanisms behind the infection process is essential to our ability to rationally design better vaccine and therapeutic interventions. With fewer than 700 predicted protein coding sequences, M. hyopneumoniae possesses one of the smallest genomes of any free-living organism. As such, it lends itself well to thorough proteomic interrogation. In this thesis, a range of proteomic techniques have been used to investigate the M. hyopneumoniae global and surface proteome at the protein and peptide level, including surface shaving and labelling techniques, ligand and immuno-blotting and affinity chromatography, as well as N-terminal dimethyl labelling to determine true N-termini of mature proteins. This conceptually unbiased, function-oriented approach has revealed an unexpected level of complexity in the use of proteolytic processing, multifunctional proteins and moonlighting to compensate for reduced coding capacity at the genome level. While microarray and transcriptome studies suggest that under normal culture conditions, the majority of genes are transcribed; our analyses identified less than 400 detectable expressed protein products under similar conditions. A significant number of the expressed proteins were discovered to be multifunctional, post-translationally modified by proteolysis. Surface proteome analyses identified a range of proteins to be surface exposed, despite lacking known signal peptides. Even though many of these proteins had well-characterised functions in the cytoplasm, they were also identified to have secondary functions at the cell surface, a phenomenon known as moonlighting. Many of the proteins present at the cell surface were identified to be subjected to proteolytic cleavage events. These were predominantly cell surface adhesins, many of which have already been described in the literature, however a large number of cytoplasmic “housekeeping” proteins are also found to be post-translationally cleaved, multifunctional proteins or moonlighting proteins. These findings can be applied to improve the rational design and development of vaccines and therapeutics for the prevention and treatment of Mycoplasma hyopneumoniae, as well as having wider implications for the field of biology as a whole, if similar levels of post-translational regulation can be found in other bacterial pathogens

Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y

© 2019, The Author(s). Mycoplasma hyopneumoniae is an economically-devastating and geographically-widespread pathogen that colonises ciliated epithelium, and destroys mucociliary function. M. hyopneumoniae devotes ~5% of its reduced genome to encode members of the P97 and P102 adhesin families that are critical for colonising epithelial cilia, but mechanisms to impair mucociliary clearance and manipulate host immune response to induce a chronic infectious state have remained elusive. Here we identified two surface exposed M. hyopneumoniae proteases, a putative Xaa-Pro aminopeptidase (MHJ_0659; PepP) and a putative oligoendopeptidase F (MHJ_0522; PepF), using immunofluorescence microscopy and two orthogonal proteomic methodologies. MHJ_0659 and MHJ_0522 were purified as polyhistidine fusion proteins and shown, using a novel MALDI-TOF MS assay, to degrade four pro-inflammatory peptides that regulate lung homeostasis; bradykinin (BK), substance P (SP), neurokinin A (NKA) and neuropeptide Y (NPY). These findings provide insight into the mechanisms used by M. hyopneumoniae to influence ciliary beat frequency, impair mucociliary clearance, and initiate a chronic infectious disease state in swine, features that are a hallmark of disease caused by this pathogen

Proteome analysis of multidrug-resistant, breast cancer-derived microparticles

© 2014 Deep Pokharel et al. Cancer multidrug resistance (MDR) occurswhen cancer cells evade the cytotoxic actions of chemotherapeutics through the active efflux of drugs from within the cells. Our group have previously demonstrated that multidrug-resistant breast cancer cells spontaneously shed microparticles (MPs) and that these MPs can transfer resistance to drug-responsive cells and confer MDR on those cells in as little as 4 h. Furthermore, we also showed that, unlike MPs derived from leukaemia cells, breast cancer-derived MPs display a tissue selectivity in the transfer of P-glycoprotein (P-gp), transferring the resistance protein only to malignant breast cells. This study aims to define the proteome of breast cancer-derived MPs in order to understand the differences in protein profiles between those shed from drug-resistant versus drug-sensitive breast cancer cells. In doing so, we detail the protein cargo required for the intercellular transfer of MDR to drug-sensitive recipient cells and the factors governing the transfer selectivity to malignant breast cells. We describe the first proteomic analysis of MPs derived from human breast cancer cells using SDS PAGE and liquid chromatography-tandem mass spectrometry (LC/MS/MS), in which we identify 120 unique proteins found only in drug-resistant, breast cancer-derived MPs. Our results demonstrate that the MP-mediated transfer of P-gp to recipient cells occurs alongside CD44; the Ezrin, Radixin and Moesin protein family (ERM); and cytoskeleton motor proteins within the MP cargo

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

© 2017 The Author(s). 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

MHJ-0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae

© 2015 The Authors. Published. 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

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae

© 2016 The Authors. 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 endopro-teolytic 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

Elongation factor Tu is a multifunctional and processed moonlighting protein

© 2017 The Author(s). Many bacterial moonlighting proteins were originally described in medically, agriculturally, and commercially important members of the low G + C Firmicutes. We show Elongation factor Tu (Ef-Tu) moonlights on the surface of the human pathogens Staphylococcus aureus (SaEf-Tu) and Mycoplasma pneumoniae (MpnEf-Tu), and the porcine pathogen Mycoplasma hyopneumoniae (MhpEf-Tu). Ef-Tu is also a target of multiple processing events on the cell surface and these were characterised using an N-terminomics pipeline. Recombinant MpnEf-Tu bound strongly to a diverse range of host molecules, and when bound to plasminogen, was able to convert plasminogen to plasmin in the presence of plasminogen activators. Fragments of Ef-Tu retain binding capabilities to host proteins. Bioinformatics and structural modelling studies indicate that the accumulation of positively charged amino acids in short linear motifs (SLiMs), and protein processing promote multifunctional behaviour. Codon bias engendered by an A + T rich genome may influence how positively-charged residues accumulate in SLiMs

Comparing the transcriptomes of embryos from domesticated and wild Atlantic salmon (Salmo salar L.) stocks and examining factors that influence heritability of gene expression

Background  Due to selective breeding, domesticated and wild Atlantic salmon are genetically diverged, which raises concerns about farmed escapees having the potential to alter the genetic composition of wild populations and thereby disrupting local adaptation. Documenting transcriptional differences between wild and domesticated stocks under controlled conditions is one way to explore the consequences of domestication and selection. We compared the transcriptomes of wild and domesticated Atlantic salmon embryos, by using a custom 44k oligonucleotide microarray to identify perturbed gene pathways between the two stocks, and to document the inheritance patterns of differentially-expressed genes by examining gene expression in their reciprocal hybrids.  Results  Data from 24 array interrogations were analysed: four reciprocal cross types (W♀×W♂, D♀×W♂; W♀×D♂, D♀×D♂)×six biological replicates. A common set of 31,491 features on the microarrays passed quality control, of which about 62% were assigned a KEGG Orthology number. A total of 6037 distinct genes were identified for gene-set enrichment/pathway analysis. The most highly enriched functional groups that were perturbed between the two stocks were cellular signalling and immune system, ribosome and RNA transport, and focal adhesion and gap junction pathways, relating to cell communication and cell adhesion molecules. Most transcripts that were differentially expressed between the stocks were governed by additive gene interaction (33 to 42%). Maternal dominance and over-dominance were also prevalent modes of inheritance, with no convincing evidence for a stock effect.  Conclusions  Our data indicate that even at this relatively early developmental stage, transcriptional differences exist between the two stocks and affect pathways that are relevant to wild versus domesticated environments. Many of the identified differentially perturbed pathways are involved in organogenesis, which is expected to be an active process at the eyed egg stage. The dominant effects are more largely due to the maternal line than to the origin of the stock. This finding is particularly relevant in the context of potential introgression between farmed and wild fish, since female escapees tend to have a higher spawning success rate compared to males

Increasing female participation in municipal elections via the use of local radio in conflict-affected settings: The case of the West Bank municipal elections 2017

The 2017 West Bank Municipal elections were framed by locally-based non-governmental organisations (NGOs) and the Palestinian Authorities – albeit to a lesser extent – in terms of the desirability of increasing female participation in them in two particular ways: participation as representatives and participation as voters. Both aspects of participation were supported by extensive radio campaigns conducted by locally-based NGOs. The effectiveness of these campaigns and the approaches used form the basis of this article. Using a mixed methods approach consisting of both quantitative and qualitative data, it concludes that radio has endemic socio-technical advantages for reaching women, particularly in conflict-affected areas, and that broadcasting content aimed at women by women is essential in terms of increasing their representation and voting