Escherichia coli are major bacterial pathogens causing bovine mastitis, a disease of great economic impact on dairy production worldwide. This work aimed to study the virulence determinants of mammary pathogenic E. coli (MPEC). By whole-genome sequencing analysis of 40 MPEC and 22 environmental (“dairy-farm” E. coli [DFEC]) strains, we found that only the fec locus (fecIRABCDE) for ferric dicitrate uptake was present in the core genome of MPEC and that it was absent in DFEC genomes (P < 0.05). Expression of the FecA receptor in the outer membrane was shown to be citrate dependent by mass spectrometry. FecA was overexpressed when bacteria were grown in milk. Transcription of the fecA gene and of the inner membrane transport component fecB gene was upregulated in bacteria recovered from experimental intramammary infection. The presence of the fec system was shown to affect the ability of E. coli to grow in milk. While the rate of growth in milk of fec-positive (fec+) DFEC was similar to that of MPEC, it was significantly lower in DFEC lacking fec. Furthermore, deletion of fec reduced the rate of growth in milk of MPEC strain P4, whereas fec-transformed non-mammary gland-pathogenic DFEC strain K71 gained the phenotype of the level of growth in milk observed in MPEC. The role of fec in E. coli intramammary pathogenicity was investigated in vivo in cows, with results showing that an MPEC P4 mutant lacking fec lost its ability to induce mastitis, whereas the fec+ DFEC K71 mutant was able to trigger intramammary inflammation. For the first time, a single molecular locus was shown to be crucial in MPEC pathogenicity.
We have examined HP1-chromatin interactions in different molecular contexts in vitro and in vivo. Employing purified components we show that HP1 exhibits selective, stoichiometric, and salt-resistant binding to recombinant histone H3, associating primarily with the helical "histone fold" domain. Furthermore, using "bulk" nucleosomes released by MNase digestion, S-phase extracts, and fragments of peripheral heterochromatin, we demonstrate that HP1 associates more tightly with destabilized or disrupted nucleosomes (H3/H4 subcomplexes) than with intact particles. Western blotting and mass spectrometry data indicate that HP1-selected H3/H4 particles and subparticles possess a complex pattern of posttranslational modifications but are not particularly enriched in me 3 K9-H3. Consistent with these results, mapping of HP1 and me 3 K9-H3 sites in vivo reveals overlapping, yet spatially distinct patterns, while transient transfection assays with synchronized cells show that stable incorporation of HP1-gfp into heterochromatin requires passage through the S-phase. The data amassed challenge the dogma that me 3 K9H3 is necessary and sufficient for HP1 binding and unveil a new mode of HP1-chromatin interactions.Histone modifications are thought to provide specific readouts that are selectively utilized in DNA transactions or chromatin state transitions (1). Given the multiplicity of modification sites and the diverse chemistries of post-translational modifications, the combinatorial repertoire of this putative "histone code" might have enormous dimensions; for instance, methylation of the five lysine residues that are located at the amino-terminal tail of histone H3 could yield alone over 15 ϫ 10 3 distinct patterns, while "saturation marking" of all lysines, arginines, serines, and threonines that are found in the same region would result in ϳ256 ϫ 10 6 combinations. Clearly then, even if 1% of the predicted patterns were materialized in vivo, this voluminous "instruction manual" could not be functionally interpreted without the aid of specific de-coding factors. Consistent with this idea, recent studies have identified a set of chromatin-associated proteins that bind specifically modified histones and could, at least in theory, fulfil such a de-coding role. As it turns out, these "effector" molecules are often components of large enzymatic assemblies and possess specialized modules known as bromo-, tudor-, or chromodomains (2).A classic example of a chromodomain-containing protein is HP1, a conserved constituent of eukaryotic cells, which, in metazoans, comprises three distinct variants: ␣, , and ␥ (3). HP1␣ and HP1 are localized in compact heterochromatic regions, while HP1␥ is more abundant in euchromatic territories (reviewed in Refs. 4 and 5). All HP1 variants have the same molecular architecture: they contain an amino-terminal chromodomain (CD), 5 an intervening region ("hinge") and a carboxylterminal chromoshadow domain (CSD). The CD is thought to be responsible for chromatin association, whereas the CSD rep...
BackgroundThe salmon louse, Lepeophtheirus salmonis, is an ectoparasitic copepod which feeds on the mucus, skin and blood of salmonid fish species. The parasite can persist on the surface of the fish without any effective control being exerted by the host immune system. Other ectoparasitic invertebrates produce compounds in their saliva, excretions and/or secretions which modulate the host immune responses allowing them to remain on or in the host during development. Similarly, compounds are produced in secretions of L. salmonis which are thought to be responsible for immunomodulation of the host responses as well as other aspects of crucial host-parasite interactions.MethodsIn this study we have identified and characterised the proteins in the excretory/secretory (E/S) products of L. salmonis using LC-ESI-MS/MS.ResultsIn total 187 individual proteins were identified in the E/S collected from adult lice and pre-adult sea lice. Fifty-three proteins, including 13 serine-type endopeptidases, 1 peroxidase and 5 vitellogenin-like proteins were common to both adult and pre-adult E/S products. One hundred and seven proteins were identified in the adult E/S but not in the pre-adult E/S and these included serine and cysteine-type endopeptidases, vitellogenins, sphingomyelinase and calreticulin. A total of 27 proteins were identified in pre-adult E/S products but not in adult E/S.ConclusionsThe assigned functions of these E/S products and the potential roles they play in host-parasite interaction is discussed.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2885-6) contains supplementary material, which is available to authorized users.
Lymph node cannulation allows the collection of lymph draining from a defined anatomical region. Proteomic analysis of that lymph offers a potentially valuable insight into the immunoinflammatory response of that particular region. In this study, ovine gastric lymph has been used to monitor the proteomic changes occurring in the tissue fluid of the abomasum, in response to infection with the parasitic nematode, Teladorsagia circumcincta. Lymph, collected temporally over an experimental infection period, was analysed by means of 2-DE and subsequent gel analysis using densitometry software. In addition, the composition of the lymphatic proteome was further explored by means of MALDI-TOF and MS/MS analyses. The concentration of gelsolin, alpha-1 beta glycoprotein and haemopexin were altered significantly (p<0.05) with infection.
Mycobacterium avium subsp. paratuberculosis causes paratuberculosis, a chronic granulomatous enteritis. Detecting animals with paratuberculosis infections is difficult because the currently available tools have low sensitivity and lack specificity; these tools are prone to generating spurious positive test results caused by exposure to environmental M. avium complex organisms. To generate candidate antigens for incorporation into a specific test for paratuberculosis, subspecies-specific proteins were determined by proteomic comparison of M. avium subsp. paratuberculosis and M. avium subsp. avium. Analysis was aimed at revealing proteins only expressed (or predominant) in the protein profile of M. avium subspecies paratuberculosis. Two-dimensional gel electrophoresis resolved approximately 1,000 protein spots from each subspecies. Proteome analysis identified protein spots whose expression profile appeared markedly increased in M. avium subsp. paratuberculosis, and 32 were identified by analysis of their tryptic peptide profile by matrix-assisted laser desorption ionizationtime of flight analysis. Thirty of these proteins were cloned, and their recombinant proteins were expressed. Ovine paratuberculosis sera were used to assess their immunoreactivity by enzyme-linked immunosorbent assay (ELISA), Western blotting, and dot blot analysis. Seventeen proteins were detected in at least one of the immunoassays, and eleven proteins were detected by ELISA with an optical density in excess of the cutoff of 0.1 in four of six sera tested. The immunoreactivity of these proteins indicates their potential as unique diagnostic antigens for the development of a specific serological detection of paratuberculosis.
Summary: This R package helps to implement a robust approach to deal with mass spectrometry (MS) data. It is aimed at alleviating reproducibility issues and pernicious effects of deviating signals on both data pre-processing and downstream data analysis. Based on robust statistical methods, it facilitates the identification and filtering of low-quality mass spectra and atypical peak profiles as well as monitoring and data handling through pre-processing, which extends existing computational tools for high-throughput data.
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