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Jarocki, VM, Steele, JR, Widjaja, M, Tacchi, JL, Padula, MP & Djordjevic, SP 2019, 'Formylated N-terminal methionine is absent from the Mycoplasma hyopneumoniae proteome: Implications for translation initiation.', International journal of medical microbiology : IJMM, vol. 309, no. 5, pp. 288-298.View/Download from: UTS OPUS or Publisher's site
N-terminal methionine excision (NME) is a proteolytic pathway that cleaves the N-termini of proteins, a process that influences where proteins localise in the cell and their turnover rates. In bacteria, protein biosynthesis is initiated by formylated methionine start tRNA (fMet-tRNAfMet). The formyl group is attached by formyltransferase (FMT) and is subsequently removed by peptide deformylase (PDF) in most but not all proteins. Methionine aminopeptidase then cleaves deformylated methionine to complete the process. Components of NME, particularly PDF, are promising therapeutic targets for bacterial pathogens. In Mycoplasma hyopneumoniae, a genome-reduced, major respiratory pathogen of swine, pdf and fmt are absent from its genome. Our bioinformatic analysis uncovered additional enzymes involved in formylated N-terminal methionine (fnMet) processing missing in fourteen mycoplasma species, including M. hyopneumoniae but not in Mycoplasma pneumoniae, a major respiratory pathogen of humans. Consistent with our bioinformatic studies, an analysis of in-house tryptic peptide libraries confirmed the absence of fnMet in M. hyopneumoniae proteins but, as expected fnMet peptides were detected in the proteome of M. pneumoniae. Additionally, computational molecular modelling of M. hyopneumoniae translation initiation factors reveal structural and sequence differences in areas known to interact with fMet-tRNAfMet. Our data suggests that some mycoplasmas have evolved a translation process that does not require fnMet.
Berry, IJ, Jarocki, VM, Tacchi, JL, Raymond, BBA, Widjaja, M, Padula, MP & Djordjevic, SP 2017, 'N-terminomics identifies widespread endoproteolysis and novel methionine excision in a genome-reduced bacterial pathogen.', Scientific Reports, vol. 7, no. 1, pp. 1-17.View/Download from: UTS OPUS or Publisher's site
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.
Widjaja, M, Harvey, KL, Hagemann, L, Berry, IJ, Jarocki, V, Raymond, BBA, Tacchi, JL, Gründel, A, Steele, JR, Padula, MP, Charles, IG, Dumke, R & Djordjevic, SP 2017, 'Elongation factor Tu is a multifunctional and processed moonlighting protein.', Scientific Reports, vol. 7, no. 1, pp. 1-17.View/Download from: UTS OPUS or Publisher's site
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.
Raymond, BBA, Jenkins, C, Seymour, LM, Tacchi, JL, Widjaja, M, Jarocki, VM, Deutscher, AT, Turnbull, L, Whitchurch, CB, Padula, MP & Djordjevic, SP 2015, 'Proteolytic processing of the cilium adhesin MHJ_0194 (P123(J)) in Mycoplasma hyopneumoniae generates a functionally diverse array of cleavage fragments that bind multiple host molecules', CELLULAR MICROBIOLOGY, vol. 17, no. 3, pp. 425-444.View/Download from: UTS OPUS or Publisher's site
Jarocki, VM, Santos, J, Tacchi, JL, Raymond, BBA, Deutscher, AT, Jenkins, C, Padula, MP & Djordjevic, SP 2015, 'MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae', Open Biology, vol. 5, no. 1, pp. 1-13.View/Download from: UTS OPUS or Publisher's site
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.
Jarocki, VM, Tacchi, JL & Djordjevic, SP 2015, 'Non-proteolytic functions of microbial proteases increases pathological complexity', PROTEOMICS, vol. 15, no. 5-6, pp. 1075-1088.View/Download from: UTS OPUS or Publisher's site
Proteases are enzymes that catalyse hydrolysis of peptide bonds thereby controlling the shape, size, function, composition, turnover and degradation of other proteins. In microbes, proteases are often identified as important virulence factors and as such have been targets for novel drug design. It is emerging that some proteases possess additional non-proteolytic functions that play important roles in host epithelia adhesion, tissue invasion and in modulating immune responses. These additional 'moonlighting' functions have the potential to obfuscate data interpretation and have implications for therapeutic design. Moonlighting enzymes comprise a subcategory of multifunctional proteins that possess at least two distinct biological functions on a single polypeptide chain. Presently, identifying moonlighting proteins relies heavily on serendipitous empirical data with clues arising from proteins lacking signal peptides that are localised to the cell surface. Here, we describe examples of microbial proteases with additional non-proteolytic functions, including streptococcal pyrogenic exotoxin B, PepO and C5a peptidases, mycoplasmal aminopeptidases, mycobacterial chaperones and viral papain-like proteases. We explore how these non-proteolytic functions contribute to host cell adhesion, modulate the coagulation pathway, assist in non-covalent folding of proteins, participate in cell signalling, and increase substrate repertoire. We conclude by describing how proteomics has aided in moonlighting protein discovery, focusing attention on potential moonlighters in microbial exoproteomes.
Jarocki, VM, Padula, MP & Djordjevic, S 2015, 'Mycoplasmal surface-associated aminopeptidases are multifunctional moonlighting proteins', Inflammation and Cell Signaling, vol. 2, no. 3, pp. 1-4.View/Download from: UTS OPUS or Publisher's site
Many bacterial pathogens require adhesion to the mucosal epithelium to establish colonisation and employ numerous strategies to then avoid clearance by the host immune system. One such strategy involves expressing plasminogen receptors on the cell surface. Recently we showed that Mycoplasma hyopneumoniae is adept at capturing porcine plasminogen onto cell surface adhesins. This interaction promotes the conversion of bound plasminogen to plasmin where it plays an important role in regulating lung inflammation. Cell surface plasmin triggers a proteolytic cascade that is thought to promote dissemination of the pathogen from the initial site of colonisation. M. hyopneumoniae is a genome-reduced pathogen that has lost the genes required to synthesise amino acids and is thus reliant on the host for amino acids for growth. We have shown M. hyopneumoniae expresses a glutamyl-aminopeptidase (MHJ_0125) and a leucyl-aminopeptidase (MHJ_0461) on the extracellular surface of the cell membrane and both are perceived as playing a key role in the generation of a pool of free amino acids for growth during pathogenesis. MHJ_0461 displays a catalytic preference for leucine, phenylalanine, and methionine, whilst MHJ_0125 demonstrates a preference for glutamic acid and alanine. In addition to their catalytic functions as aminopeptidases, both enzymes bind porcine plasminogen, promoting its conversion to plasmin by tPA, and display an affinity for highly sulphated glycosaminoglycans. MHJ_0461 was also shown to bind extracellular DNA. These studies highlight the multifunctional properties of surface proteins in M. hyopneumoniae and the increasing pool of evidence that moonlighting proteins play important roles during microbial pathogenesis.
Tacchi, JL, Raymond, BB, Jarocki, VM, Berry, IJ, Padula, M & Djordjevic, SP 2014, 'Cilium Adhesin P216 (MHJ_0493) Is a Target of Ectodomain Shedding and Aminopeptidase Activity on the Surface of Mycoplasma hyopneumoniae', Journal of Proteome Research, vol. Epub.View/Download from: UTS OPUS or Publisher's site
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 1072TN-F?Q-E1076 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 1072TN-F?Q-E1076 and 1344I-T-F?A-D-Y1349 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 LCMS/MS analysis of biotinylated proteins and 2D SDS-PAGE. LCMS/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.
Raymond, B, Tacchi, JL, Jarocki, VM, Minion, F, Padula, M & Djordjevic, SP 2013, 'P159 from Mycoplasma hyopneumoniae binds porcine cilia and heparin and is cleaved in a manner akin to ectodomain shedding', Journal of Proteome Research, vol. 12, no. 12, pp. 5891-5903.View/Download from: UTS OPUS or Publisher's site
Mycoplasma hyopneumoniae colonizes the ciliated epithelial lining of the upper respiratory tract of swine and results in chronic infection. Previously, we have observed that members of P97 and P102 paralog families of cilium adhesins undergo endoproteolytic processing on the surface of M. hyopneumoniae. We show that P159 (MHJ_0494), an epithelial cell adhesin unrelated to P97 and P102 paralog families, is a cilium adhesin that undergoes dominant cleavage events at S/T-X-F?X-D/E-like motifs located at positions 233F?Q234 and 981F?Q982, generating P27, P110, and P52. An unrelated cleavage site 738L-K-V?G-A-A743 in P110 shows sequence identity with a cleavage site (L-N-V?A-V-S) identified in the P97 paralog, Mhp385, and generates 76 (P76) and 35 kDa (P35) fragments. LCMS/MS analysis of biotinylated surface proteins identified six peptides with a biotin moiety on their N-terminus indicating novel, low abundance neo-N-termini. LCMS/MS of proteins separated by 2D-PAGE, 2D immunoblotting using monospecific antiserum raised against recombinant fragments spanning P159 (F1P159-F4P159), and proteins that bound to heparin-agarose were all used to map P159 cleavage fragments. P159 is the first cilium adhesin not belonging to the P97/P102 paralog families and is extensively processed in a manner akin to ectodomain shedding in eukaryotes.
Berry, IJ, Tacchi, JL, Jarocki, VM, Raymond, BBA, Padula, MP & Djordjevic, SP 2014, 'The Significance of Post-Translational Proteolysis in the Model Pathogen, Mycoplasma hyopneumoniae.', 2nd 'Proteomics & Beyond' Symposium, Proteomics & Beyond Symposium, Australian Proteomics Analysis Facility.
Proteolytic cleavage is one of the most ubiquitous post-translational modifications to proteins, responsible for protein signalling, activation, localisation and ultimately degradation. Due to a variety of experimental limitations this important physiological process has been largely understudied, particularly in prokaryotes and archaea. In order determine the scale and functional roles of protein processing in bacteria and to explore the underlying mechanisms in the production of mature proteins, a high-throughput systems wide approach is needed..
The primary focus of this project is to sequence the N-terminal sequences of mature protein products of prokaryotic pathogens. Using this data, we can identify true protein start sites and any downstream post-translational processing. The genome-reduced agriculturally-important pathogen Mycoplasma hyopneumoniae, was selected as a model organism for these studies due to the large body of literature demonstrating the extensive proteolytic processing of many highly expressed surface proteins of this organism, which are critical for pathogenesis.
Using a high-throughput methodology called Terminal Amine Isotopic Labelling of Substrates (TAILS), the data obtained from the analysis of M. hyopneumoniae has provided evidence confirming true start sites of protein translation and complementary data pinpointing the precise sites of proteolytic cleavage. Our data indicates that an unprecedented number of cell surface proteins are targets for posttranslational processing.