A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis.
ABSTRACT: BACKGROUND: Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats. RESULTS: An optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins. CONCLUSIONS: Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far.
Project description:BACKGROUND: Pan-genomic studies aim, for instance, at defining the core, dispensable and unique genes within a species. A pan-genomics study for vaccine design tries to assess the best candidates for a vaccine against a specific pathogen. In this context, rather than studying genes predicted to be exported in a single genome, with pan-genomics it is possible to study genes present in different strains within the same species, such as virulence factors. The target organism of this pan-genomic work here presented is Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis (CLA) in goat and sheep, which causes significant economic losses in those herds around the world. Currently, only a few antigens against CLA are known as being the basis of commercial and still ineffective vaccines. In this regard, the here presented work analyses, in silico, five C. pseudotuberculosis genomes and gathers data to predict common exported proteins in all five genomes. These candidates were also compared to two recent C. pseudotuberculosis in vitro exoproteome results. RESULTS: The complete genome of five C. pseudotuberculosis strains (1002, C231, I19, FRC41 and PAT10) were submitted to pan-genomics analysis, yielding 306, 59 and 12 gene sets, respectively, representing the core, dispensable and unique in silico predicted exported pan-genomes. These sets bear 150 genes classified as secreted (SEC) and 227 as potentially surface exposed (PSE). Our findings suggest that the main C. pseudotuberculosis in vitro exoproteome could be greater, appended by a fraction of the 35 proteins formerly predicted as making part of the variant in vitro exoproteome. These genomes were manually curated for correct methionine initiation and redeposited with a total of 1885 homogenized genes. CONCLUSIONS: The in silico prediction of exported proteins has allowed to define a list of putative vaccine candidate genes present in all five complete C. pseudotuberculosis genomes. Moreover, it has also been possible to define the in silico predicted dispensable and unique C. pseudotuberculosis exported proteins. These results provide in silico evidence to further guide experiments in the areas of vaccines, diagnosis and drugs. The work here presented is the first whole C. pseudotuberculosis in silico predicted pan-exoproteome completed till today.
Project description:The exoproteome of parasitic protists constitutes extracellular proteins that play a fundamental role in host-parasite interactions. Lytic factors, especially secreted proteases, are capable of modulating tissue invasion, thereby aggravating host susceptibility. Despite the important role of exoproteins during infection, the exoproteomic data on Histomonas meleagridis are non-existent. The present study employed traditional 1D-in-gel-zymography (1D-IGZ) and micro-LC-ESI-MS/MS (shotgun proteomics), to investigate H. meleagridis exoproteomes, obtained from a clonal virulent and an attenuated strain. Both strains were maintained as mono-eukaryotic monoxenic cultures with Escherichia coli. We demonstrated active in vitro secretion kinetics of proteases by both parasite strains, with a widespread proteolytic activity ranging from 17 kDa to 120 kDa. Based on protease inhibitor susceptibility assay, the majority of proteases present in both exoproteomes belonged to the family of cysteine proteases and showed stronger activity in the exoproteome of a virulent H. meleagridis. Shotgun proteomics, aided by customized database search, identified 176 proteins including actin, potential moonlighting glycolytic enzymes, lytic molecules such as pore-forming proteins (PFPs) and proteases like cathepsin-L like cysteine protease. To quantify the exoproteomic differences between the virulent and the attenuated H. meleagridis cultures, a sequential window acquisition of all theoretical spectra mass spectrometric (SWATH-MS) approach was applied. Surprisingly, results showed most of the exoproteomic differences to be of bacterial origin, especially targeting metabolism and locomotion. By deciphering such molecular signatures, novel insights into a complex in vitro protozoan- bacteria relationship were elucidated.
Project description:The interest in examining the subset of proteins present in the extracellular milieu, the exoproteome, has been growing due to novel insights highlighting their role on extracellular matrix organization and biofilm formation, but also on homeostasis and development. The cyanobacterial exoproteome is poorly studied, and the role of cyanobacterial exoproteins on cell wall biogenesis, morphology and even physiology is largely unknown. Here, we present a comprehensive examination of the Anabaena sp. PCC 7120 exoproteome under various growth conditions. Altogether, 139 proteins belonging to 16 different functional categories have been identified. A large fraction (48%) of the identified proteins is classified as "hypothetical", falls into the "other categories" set or presents no similarity to other proteins. The evidence presented here shows that Anabaena sp. PCC 7120 is capable of outer membrane vesicle formation and that these vesicles are likely to contribute to the exoproteome profile. Furthermore, the activity of selected exoproteins associated with oxidative stress has been assessed, suggesting their involvement in redox homeostasis mechanisms in the extracellular space. Finally, we discuss our results in light of other cyanobacterial exoproteome studies and focus on the potential of exploring cyanobacteria as cell factories to produce and secrete selected proteins.
Project description:The opportunistic pathogen Pseudomonas aeruginosa uses secretion systems to deliver exoproteins into the environment. These exoproteins contribute to bacterial survival, adaptation, and virulence. The Twin arginine translocation (Tat) export system enables the export of folded proteins into the periplasm, some of which can then be further secreted outside the cell. However, the full range of proteins that are conveyed by Tat is unknown, despite the importance of Tat for the adaptability and full virulence of P. aeruginosa. In this work, we explored the P. aeruginosa Tat-dependent exoproteome under phosphate starvation by two-dimensional gel analysis. We identified the major secreted proteins and new Tat-dependent exoproteins. These exoproteins were further analyzed by a combination of in silico analysis, regulation studies, and protein localization. Altogether we reveal that the absence of the Tat system significantly affects the composition of the exoproteome by impairing protein export and affecting gene expression. Notably we discovered three new Tat exoproteins and one novel type II secretion substrate. Our data also allowed the identification of two new start codons highlighting the importance of protein annotation for subcellular predictions. The new exoproteins that we identify may play a significant role in P. aeruginosa pathogenesis, host interaction and niche adaptation.
Project description:Wound-colonizing microorganisms can form complex and dynamic polymicrobial communities where pathogens and commensals may co-exist, cooperate or compete with each other. The present study was aimed at identifying possible interactions between different bacteria isolated from the same chronic wound of a patient with the genetic blistering disease epidermolysis bullosa (EB). Specifically, this involved two different isolates of the human pathogen Staphylococcus aureus, and isolates of Bacillus thuringiensis and Klebsiella oxytoca. Particular focus was attributed to interactions of S. aureus with the two other species, because of the high staphylococcal prevalence among chronic wounds. Intriguingly, upon co-cultivation, none of the wound isolates inhibited each other's growth. Since the extracellular proteome of bacterial pathogens is a reservoir of virulence factors, the exoproteomes of the staphylococcal isolates in monoculture and co-culture with B. thuringiensis and K. oxytoca were characterized by Mass Spectrometry to explore the inherent relationships between these co-exisiting bacteria. This revealed a massive reduction in the number of staphylococcal exoproteins upon co-culturing with K. oxytoca or B. thuringiensis. Interestingly, this decrease was particularly evident for extracellular proteins with a predicted cytoplasmic localization, which were recently implicated in staphylococcal virulence and epidemiology. Furthermore, our exoproteome analysis uncovered potential cooperativity between the two different S. aureus isolates. Altogether, the observed exoproteome variations upon co-culturing are indicative of unprecedented adaptive mechanisms that set limits to the production of secreted staphylococcal virulence factors.
Project description:At low density, Bacillus cereus cells release a large variety of proteins into the extracellular medium when cultivated in pH-regulated, glucose-containing minimal medium, either in the presence or absence of oxygen. The majority of these exoproteins are putative virulence factors, including toxin-related proteins. Here, B. cereus exoproteome time courses were monitored by nanoLC-MS/MS under low-oxidoreduction potential (ORP) anaerobiosis, high-ORP anaerobiosis, and aerobiosis, with a specific focus on oxidative-induced post-translational modifications of methionine residues. Principal component analysis (PCA) of the exoproteome dynamics indicated that toxin-related proteins were the most representative of the exoproteome changes, both in terms of protein abundance and their methionine sulfoxide (Met(O)) content. PCA also revealed an interesting interconnection between toxin-, metabolism-, and oxidative stress-related proteins, suggesting that the abundance level of toxin-related proteins, and their Met(O) content in the B. cereus exoproteome, reflected the cellular oxidation under both aerobiosis and anaerobiosis.
Project description:Analysis of the culture supernatant exoproteins produced by two PFGE clusters of high-level gentamicin and ciprofloxacin-resistant clinical isolates of Enterococcus faecalis from the UK and Ireland revealed two distinct protein profiles. This grouping distinguished OG1RF and GelE metalloprotease-expressing isolates from JH2-2 and other GelE-negative isolates. The integrity of the fsrABDC operon was found to determine the exoproteome composition, since an fsrB mutant of strain OG1RF appeared very similar to that of strain JH2-2, and complementation of the latter with the fsrABDC operon produced an OG1RF-like exoproteome. The proteins present in the supernatant fraction of OG1RF were separated using 2D gels and identified by mass spectrometry and comprised many mass and pI variants of the GelE and SprE proteases. In addition cell wall synthesis and cell division proteins were identified. An OG1RF fsrB mutant had a distinct exoprotein fraction with an absence of the Fsr-regulated proteases and was characterised by general stress and glycolytic proteins. The exoproteome of the OG1RF fsrB mutant resembles that of a divIVA mutant of E. faecalis, suggestive of a stress phenotype.
Project description:Probiotics must not only exert a health-promoting effect but also be capable of adapting to the harsh environment of the gastrointestinal (GI) tract. Probiotics in the GI tract must survive the cell wall-disrupting effect of bile acids. We investigated the exoproteome of <i>Lactobacillus johnsonii</i> PF01 and C1-10 under bile stress. A comparative analysis revealed the similarities between the two <i>L</i>. <i>johnsonii</i> exoproteomes, as well as their different responses to bile. The large number of metabolic proteins in <i>L</i>. <i>johnsonii</i> revealed its metabolic adaptation to meet protein synthesis requirements under bile stress. In addition, cell wall modifications occurred in response to bile. Furthermore, some extracellular proteins of <i>L</i>. <i>johnsonii</i> may have moonlighting function in the presence of bile. Enolase, L-lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, 50s ribosomal protein L7/L12, and cellobiose-specific phosphotransferase system (PTS) sugar transporter were significantly upregulated under bile stress, suggesting a leading role in the collective bile stress response of <i>L</i>. <i>johnsonii</i> from its exoproteome perspective.
Project description:The exported protein fraction of an organism may reflect its life strategy and, ultimately, the way it is perceived by the outside world. Bioinformatic prediction of the exported pan-proteome of Prochlorococcus and Synechococcus lineages demonstrated that (i) this fraction of the encoded proteome had a much higher incidence of lineage-specific proteins than the cytosolic fraction (57% and 73% homologue incidence respectively) and (ii) exported proteins are largely uncharacterized to date (54%) compared with proteins from the cytosolic fraction (35%). This suggests that the genomic and functional diversity of these organisms lies largely in the diverse pool of novel functions these organisms export to/through their membranes playing a key role in community diversification, e.g. for niche partitioning or evading predation. Experimental exoproteome analysis of marine Synechococcus showed transport systems for inorganic nutrients, an interesting array of strain-specific exoproteins involved in mutualistic or hostile interactions (i.e. hemolysins, pilins, adhesins), and exoenzymes with a potential mixotrophic goal (i.e. exoproteases and chitinases). We also show how these organisms can remodel their exoproteome, i.e. by increasing the repertoire of interaction proteins when grown in the presence of a heterotroph or decrease exposure to prey when grown in the dark. Finally, our data indicate that heterotrophic bacteria can feed on the exoproteome of Synechococcus.
Project description:The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections.