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.     

Survey of Surface Proteins from the Pathogenic Mycoplasma hyopneumoniae Strain 7448 Using a Biotin Cell Surface Labeling Approach

The characterization of the repertoire of proteins exposed on the cell surface by Mycoplasma hyopneumoniae (M. hyopneumoniae), the etiological agent of enzootic pneumonia in pigs, is critical to understand physiological processes associated with bacterial infection capacity, survival and pathogenesis. Previous in silico studies predicted that about a third of the genes in the M. hyopneumoniae genome code for surface proteins, but so far, just a few of them have experimental confirmation of their expression and surface localization. In this work, M. hyopneumoniae surface proteins were labeled in intact cells with biotin, and affinity-captured biotin-labeled proteins were identified by a gel-based liquid chromatography-tandem mass spectrometry approach. A total of 20 gel slices were separately analyzed by mass spectrometry, resulting in 165 protein identifications corresponding to 59 different protein species. The identified surface exposed proteins better defined the set of M. hyopneumoniae proteins exposed to the host and added confidence to in silico predictions. Several proteins potentially related to pathogenesis, were identified, including known adhesins and also hypothetical proteins with adhesin-like topologies, consisting of a transmembrane helix and a large tail exposed at the cell surface. The results provided a better picture of the M. hyopneumoniae cell surface that will help in the understanding of processes important for bacterial pathogenesis. Considering the experimental demonstration of surface exposure, adhesion-like topology predictions and absence of orthologs in the closely related, non-pathogenic species Mycoplasma flocculare, several proteins could be proposed as potential targets for the development of drugs, vaccines and/or immunodiagnostic tests for enzootic pneumonia.     

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.     

MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae

Bacterial aminopeptidases play important roles in pathogenesis by providing a source of amino acids from exogenous proteins, destroying host immunological effector peptides and executing posttranslational modification of bacterial and host proteins. We show that MHJ_0125 from the swine respiratory pathogen Mycoplasma hyopneumoniae represents a new member of the M42 class of bacterial aminopeptidases. Despite lacking a recognizable signal sequence, MHJ_0125 is detectable on the cell surface by fluorescence microscopy and LC-MS/MS of (i) biotinylated surface proteins captured by avidin chromatography and (ii) peptides released by mild trypsin shaving. Furthermore, surface-associated glutamyl aminopeptidase activity was detected by incubation of live M. hyopneumoniae cells with the diagnostic substrate H-Glu-AMC. MHJ_0125 moonlights as a multifunctional adhesin, binding to both heparin and plasminogen. Native proteomics and comparative modelling studies suggest MHJ_0125 forms a dodecameric, homopolymeric structure and provide insight into the positions of key residues that are predicted to interact with heparin and plasminogen. MHJ_0125 is the first aminopeptidase shown to both bind plasminogen and facilitate its activation by tissue plasminogen activator. Plasmin cleaves host extracellular matrix proteins and activates matrix metalloproteases, generating peptide substrates for MHJ_0125 and a source of amino acids for growth of M. hyopneumoniae. This unique interaction represents a new paradigm in microbial pathogenesis.     

Proteogenomic mapping of Mycoplasma hyopneumoniae virulent strain 232

Background

Mycoplasma hyopneumoniae causes respiratory disease in swine and contributes to the porcine respiratory disease complex, a major disease problem in the swine industry. The M. hyopneumoniae strain 232 genome is one of the smallest and best annotated microbial genomes, containing only 728 annotated genes and 691 known proteins. Standard protein databases for mass spectrometry only allow for the identification of known and predicted proteins, which if incorrect can limit our understanding of the biological processes at work. Proteogenomic mapping is a methodology which allows the entire 6-frame genome translation of an organism to be used as a mass spectrometry database to help identify unknown proteins as well as correct and confirm existing annotations. This methodology will be employed to perform an in-depth analysis of the M. hyopneumoniae proteome.

Results

Proteomic analysis indicates 483 of 691 (70%) known M. hyopneumoniae strain 232 proteins are expressed under the culture conditions given in this study. Furthermore, 171 of 328 (52%) hypothetical proteins have been confirmed. Proteogenomic mapping resulted in the identification of previously unannotated genes gatC and rpmF and 5-prime extensions to genes mhp063, mhp073, and mhp451, all conserved and annotated in other M. hyopneumoniae strains and Mycoplasma species. Gene prediction with Prodigal, a prokaryotic gene predicting program, completely supports the new genomic coordinates calculated using proteogenomic mapping.

Conclusions

Proteogenomic mapping showed that the protein coding genes of the M. hyopneumoniae strain 232 identified in this study are well annotated. Only 1.8% of mapped peptides did not correspond to genes defined by the current genome annotation. This study also illustrates how proteogenomic mapping can be an important tool to help confirm, correct and append known gene models when using a genome sequence as search space for peptide mass spectra. Using a gene prediction program which scans for a wide variety of promoters can help ensure genes are accurately predicted or not missed completely. Furthermore, protein extraction using differential detergent fractionation effectively increases the number of membrane and cytoplasmic proteins identifiable my mass spectrometry.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-576) contains supplementary material, which is available to authorized users.     

Updating the proteome of the uncultivable hemotrophic Mycoplasma suis in experimentally infected pigs

Mycoplasma suis belongs to the hemotrophic mycoplasmas that are associated with acute and chronic anemia in a wide range of livestock and wild animals. The inability to culture M. suis in vitro has hindered its characterization at the molecular level. Since the publication of M. suis genome sequences in 2011 only one proteome study has been published. Aim of the presented study was to significantly extend the proteome coverage of M. suis strain KI_3806 during acute infection by applying three different protein extraction methods followed by 1D SDS‐PAGE and LC‐MS/MS. A total of 404 of 795 M. suis KI_3806 proteins (50.8%) were identified. Data analysis revealed the expression of 83.7% of the predicted ORFs with assigned functions but also highlights the expression of 179 of 523 (34.2%) hypothetical proteins with unknown functions. Computational analyses identified expressed membrane‐associated hypothetical proteins that might be involved in adhesion or host–pathogen interaction. Furthermore, analyses of the expressed proteins indicated the existence of a hexose‐6‐phosphate‐transporter and an ECF transporter. In conclusion, our proteome study provides a further step toward the elucidation of the unique life cycle of M. suis and the establishment of an in vitro culture. All MS data have been deposited in the ProteomeXchange with identifier PXD002294 ( http://proteomecentral.proteomexchange.org/dataset/PXD002294 ).     

A Processed Multidomain Mycoplasma hyopneumoniae Adhesin Binds Fibronectin,Plasminogen, and Swine Respiratory Cilia

Porcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 kDa in size. A variety of pathogenic bacterial species have been shown to bind the extracellular matrix component fibronectin as an adherence mechanism. M. hyopneumoniae cells were found to bind fibronectin in a dose-dependent and saturable manner. Surface plasmon resonance was used to show that a recombinant C-terminal domain of P116 bound fibronectin at physiologically relevant concentrations (K_D 24 ± 6 nm). Plasmin(ogen)-binding proteins are also expressed by many bacterial pathogens, facilitating extracellular matrix degradation. M. hyopneumoniae cells were found to also bind plasminogen in a dose-dependent and saturable manner; the C-terminal domain of P116 binds to plasminogen (K_D 44 ± 5 nm). Plasminogen binding was abolished when the C-terminal lysine of P116 was deleted, implicating this residue as part of the plasminogen binding site. P116 fragments adhere to the PK15 porcine kidney epithelial-like cell line and swine respiratory cilia. Collectively these data suggest that P116 is an important adhesin and virulence factor of M. hyopneumoniae.     

Listeria monocytogenes Surface Proteins: from Genome Predictions to Function

Summary: The genome of the human food-borne pathogen Listeria monocytogenes is predicted to encode a high number of surface proteins. This abundance likely reflects the ability of this bacterium to survive in diverse environments, including soil, food, and the human host. This review focuses on the various mechanisms by which listerial proteins are attached at the bacterial surface and their many functions, including peptidoglycan metabolism, protein processing, adhesion to host cells, and invasion of host tissues. Extensive in silico analysis of the domains or motifs present in these mosaic proteins reveals that diverse structural features allow the surface proteome to interact with diverse bacterial or host components. This diversity offers new clues about the molecular bases of Listeria pathogenesis.     

Characterization of the Theileria parva sporozoite proteome

East Coast fever is a lymphoproliferative disease caused by the tick-borne protozoan parasite Theileria parva. The sporozoite stage of this parasite, harboured and released from the salivary glands of the tick Rhipicephalus appendiculatus during feeding, invades and establishes infection in bovine lymphocytes. Blocking this initial stage of invasion presents a promising vaccine strategy for control of East Coast fever and can in part be achieved by targeting the major sporozoite surface protein p67. To support research on the biology of T. parva and the identification of additional candidate vaccine antigens, we report on the sporozoite proteome as defined by LC–MS/MS analysis. In total, 4780 proteins were identified in an enriched preparation of sporozoites. Of these, 2007 were identified as T. parva proteins, representing close to 50% of the total predicted parasite proteome. The remaining 2773 proteins were derived from the tick vector. The identified sporozoite proteins include a set of known T. parva antigens targeted by antibodies and cytotoxic T cells from cattle that are immune to East Coast fever. We also identified proteins predicted to be orthologs of Plasmodium falciparum sporozoite surface molecules and invasion organelle proteins, and proteins that may contribute to the phenomenon of bovine lymphocyte transformation. Overall, these data establish a protein expression profile of T. parva sporozoites as an important starting point for further study of a parasitic species which has considerable agricultural impact.     

Experimental and computational analysis of the secretome of the hyperthermophilic archaeon Pyrococcus furiosus

Although Pyrococcus furiosus is one of the best studied hyperthermophilic archaea, to date no experimental investigation of the extent of protein secretion has been performed. We describe experimental verification of the extracellular proteome of P. furiosus grown on starch. LC–MS/MS-based analysis of culture supernatants led to the identification of 58 proteins. Fifteen of these proteins had a putative N-terminal signal peptide (SP), tagging the proteins for translocation across the membrane. The detected proteins with predicted SPs and known function were almost exclusively involved in important extracellular functions, like substrate degradation or transport. Most of the 43 proteins without predicted N-terminal signal sequences are known to have intracellular functions, mainly (70 %) related to intracellular metabolism. In silico analyses indicated that the genome of P. furiosus encodes 145 proteins with N-terminal SPs, including 21 putative lipoproteins and 17 with a class III peptide. From these we identified 15 (10 %; 7 SPI, 3 SPIII and 5 lipoproteins) under the specific growth conditions of this study. The putative lipoprotein signal peptides have a unique sequence motif, distinct from the motifs in bacteria and other archaeal orders.     

Comprehensive mapping of the bull sperm surface proteome

While the mechanisms that underpin maturation, capacitation, and sperm–egg interactions remain elusive it is known that these essential fertilisation events are driven by the protein complement of the sperm surface. Understanding these processes is critical to the regulation of animal reproduction, but few studies have attempted to define the full repertoire of sperm surface proteins in animals of agricultural importance. Recent developments in proteomics technologies, subcellular fractionation, and optimised solubilisation strategies have enhanced the potential for the comprehensive characterisation of the sperm surface proteome. Here we report the identification of 419 proteins from a mature bull sperm plasma membrane fraction. Protein domain enrichment analyses indicate that 67% of all the proteins identified may be membrane associated. A large number of the proteins identified are conserved between mammalian species and are reported to play key roles in sperm–egg communication, capacitation and fertility. The major functional pathways identified were related to protein catabolism (26S proteasome complex), chaperonin‐containing TCP‐1 (CCT) complex and fundamental metabolic processes such as glycolysis and energy production. We have also identified 118 predicted transmembrane proteins, some of which are implicated in cell adhesion, acrosomal exocytosis, vesicle transport and immunity and fertilisation events, while others have not been reported in mammalian LC‐MS‐derived sperm proteomes to date. Comparative proteomics and functional network analyses of these proteins expand our system's level of understanding of the bull sperm proteome and provide important clues toward finding the essential conserved function of these proteins.     

Cloning and purification of recombinant proteins of Mycoplasma hyopneumoniae expressed in Escherichia coli

Mycoplasma hyopneumoniae, the etiological agent of swine enzootic pneumonia, is an important pathogen in the swine industry worldwide. Vaccination is the most cost-effective strategy for controlling and prevention of this disease. However, investigations on pathogenicity mechanisms as well as current serological detection methods and the development of new recombinant subunit vaccines are hampered by the lack of known and well characterized species-specific M. hyopneumoniae antigens. In this work, 54 predicted genes encoding proteins with potential to be used as subunit vaccine or antigens in diagnostic tests were selected, amplified by PCR and cloned into Escherichia coli expression vectors. Recombinant protein expression, solubility and yields were analyzed. The majority of the recombinant proteins were expressed in inclusion bodies. After solubilization with urea or N-lauroyl sarcosine, recombinant proteins were purified by Ni²⁺ affinity chromatography. This approach allowed purification of thirty recombinant M. hyopneumoniae proteins which will be evaluated as vaccine candidates and/or as antigens to be used in diagnostic tests.     

Low Cell Number Proteomic Analysis Using In-Cell Protease Digests Reveals a Robust Signature for Cell Cycle State Classification

Comprehensive proteome analysis of rare cell phenotypes remains a significant challenge. We report a method for low cell number MS-based proteomics using protease digestion of mildly formaldehyde-fixed cells in cellulo, which we call the “in-cell digest.” We combined this with averaged MS1 precursor library matching to quantitatively characterize proteomes from low cell numbers of human lymphoblasts. About 4500 proteins were detected from 2000 cells, and 2500 proteins were quantitated from 200 lymphoblasts. The ease of sample processing and high sensitivity makes this method exceptionally suited for the proteomic analysis of rare cell states, including immune cell subsets and cell cycle subphases. To demonstrate the method, we characterized the proteome changes across 16 cell cycle states (CCSs) isolated from an asynchronous TK6 cells, avoiding synchronization. States included late mitotic cells present at extremely low frequency. We identified 119 pseudoperiodic proteins that vary across the cell cycle. Clustering of the pseudoperiodic proteins showed abundance patterns consistent with “waves” of protein degradation in late S, at the G2&M border, midmitosis, and at mitotic exit. These clusters were distinguished by significant differences in predicted nuclear localization and interaction with the anaphase-promoting complex/cyclosome. The dataset also identifies putative anaphase-promoting complex/cyclosome substrates in mitosis and the temporal order in which they are targeted for degradation. We demonstrate that a protein signature made of these 119 high-confidence cell cycle–regulated proteins can be used to perform unbiased classification of proteomes into CCSs. We applied this signature to 296 proteomes that encompass a range of quantitation methods, cell types, and experimental conditions. The analysis confidently assigns a CCS for 49 proteomes, including correct classification for proteomes from synchronized cells. We anticipate that this robust cell cycle protein signature will be crucial for classifying cell states in single-cell proteomes.     

Molecular weight assessment of proteins in total proteome profiles using 1D-PAGE and LC/MS/MS

Background  

The observed molecular weight of a protein on a 1D polyacrylamide gel can provide meaningful insight into its biological function. Differences between a protein's observed molecular weight and that predicted by its full length amino acid sequence can be the result of different types of post-translational events, such as alternative splicing (AS), endoproteolytic processing (EPP), and post-translational modifications (PTMs). The characterization of these events is one of the important goals of total proteome profiling (TPP). LC/MS/MS has emerged as one of the primary tools for TPP, but since this method identifies tryptic fragments of proteins, it has not generally been used for large-scale determination of the molecular weight of intact proteins in complex mixtures.     

Gel and gel-free proteomics to identify Saccharomyces cerevisiae cell surface proteins

The study of Saccharomyces cerevisiae cell surface proteins was performed because of their important role in cell wall biogenesis and in the physiology of the yeast. Two different proteomic approaches were carried out. First, proteins loosely associated or S–S linked to structural wall components were released by treatment of whole intact cells with dithiothreitol, separated by 2D-PAGE and identified by mass spectrometry. Second, cell surface-exposed proteins (surfome) were digested with trypsin and DTT from whole intact cells, and analyzed by LC–MS/MS. Ninety-nine different proteins were identified: 67 with DTT treatment and 52 with DTT and trypsin digestion. These proteins were classified in different cellular processes: control of cell wall organization, cell rescue, defence, and virulence, protein fate, protein synthesis and metabolism. Most of the proteins have already been reported as present on the cell surface showing that the yeast cell surface is composed not only by typical but also by atypical cell wall proteins. “Bona fide” cell wall proteins were identified by both protocols but a higher number with the non-gel strategy. However, only 20% of the proteins identified were common to both protocols, thus, for a complete knowledge of the cell surface proteome, several strategies have to be used.