Effect of two constant light regimens on antibody profiles and immune gene expression in Atlantic salmon following vaccination and experimental challenge with salmonid alphavirus.

Before seawater transfer, farmed Atlantic salmon are subjected to treatments that may affect the immune system and susceptibility to pathogens. E.g., exposure to constant light (CL) stimulates smoltification, which prepares salmon to life in sea water, but endocrine changes in this period are associated with suppression of immune genes. Salmon are vaccinated towards end of the freshwater period to safeguard that adequate vaccine efficacy is achieved by the time the fish is transferred to sea. In the present study, we investigated how the responses to vaccination and viral infection varied depending on the time of CL onset relative to vaccination. The salmon were either exposed to CL two weeks prior to vaccination (2-PRI) or exposed to CL at the time of vaccination (0-PRI). A cohabitant challenge with salmonid alphavirus, the causative agent of pancreatic disease, was performed 9 weeks post vaccination. The immunological effects of the different light manipulation were examined at 0- and 6-weeks post vaccination, and 6 weeks post challenge. Antibody levels in serum were measured using a serological bead-based multiplex panel as well as ELISA, and 92 immune genes in heart and spleen were measured using an integrated fluidic circuit-based qPCR array for multiple gene expression. The 2-PRI group showed a moderate transcript down-regulation of genes in the heart at the time of vaccination, which were restored 6 weeks after vaccination (WPV). Conversely, at 6WPV a down-regulation was seen for the 0-PRI fish. Moreover, the 2-PRI group had significantly higher levels of antibodies binding to three of the vaccine components at 6WPV, compared to 0-PRI. In response to SAV challenge, transcription of immune genes between 2-PRI and 0-PRI was markedly dissimilar in the heart and spleen of control fish, but no difference was found between vaccinated salmon from the two CL regimens. Thus, by using labor-saving high throughput detection methods, we demonstrated that light regimens affected antibody production and transcription of immune genes in non-vaccinated and virus challenged salmon, but the differences between the light treatment groups appeared eliminated by vaccination.

Consequences of Haemorrhagic Smolt Syndrome (HSS) for the Immune Status of Atlantic salmon (Salmo salar L.) (Case Study).

Haemorrhagic smolt syndrome (HSS) is a disorder of unknown aetiology causing losses in the fresh water phase of Atlantic salmon farming. Normally, the mortality is limited and symptoms disappear upon seawater exposure. In this case study, classical HSS pathology with internal organ haemorrhages and nephrocalcinosis was diagnosed, and the losses were substantial. Microarray analyses of head kidney revealed association between HSS and enhanced expression of stress genes and proteins reducing bioavailability of iron, heme, and retinol. In parallel, suppression of multiple metabolic pathways was observed. Up-regulation of genes encoding acute phase proteins, complement, and lectins indicated mild inflammation but without characteristic features of viral or bacterial infections. Microarray analyses highlighted several members of tumor necrosis factor receptor superfamily that may control development of B-cell immunity. Examination of IgM at the mRNA and protein levels showed the impact of HSS on vaccine responses. In fish without HSS symptoms (non-HSS), titres of vaccine specific antibodies to A-layer of Aeromonas salmonicida subsp. salmonicida and Moritella viscosa and antibodies binding to DNP-keyhole limpet hemocyanin (DNP-KLH), which are presumably polyreactive, were respectively four- and 14-fold higher than in HSS-diseased fish. Parallel sequencing of variable regions of immunoglobulin Mrevealed a larger size of most abundant clonotypes shared by multiple individuals in the non-HSS group. The results of the current case study indicated that, in addition to direct damage, HSS suppresses humoral immune responses including the production of specific and polyreactive antibodies.

Comparison of membrane proteins of Mycobacterium tuberculosis H37Rv and H37Ra strains.

BACKGROUND: The potential causes for variation in virulence between distinct M. tuberculosis strains are still not fully known. However, differences in protein expression are probably an important factor. In this study we used a label-free quantitative proteomic approach to estimate differences in protein abundance between two closely related M. tuberculosis strains; the virulent H37Rv strain and its attenuated counterpart H37Ra. RESULTS: We were able to identify more than 1700 proteins from both strains. As expected, the majority of the identified proteins had similar relative abundance in the two strains. However, 29 membrane-associated proteins were observed with a 5 or more fold difference in their relative abundance in one strain compared to the other. Of note, 19 membrane- and lipo-proteins had higher abundance in H37Rv, while another 10 proteins had a higher abundance in H37Ra. Interestingly, the possible protein-export membrane protein SecF (Rv2586c), and three ABC-transporter proteins (Rv0933, Rv1273c and Rv1819c) were among the more abundant proteins in M. tuberculosis H37Rv. CONCLUSION: Our data suggests that the bacterial secretion system and the transmembrane transport system may be important determinants of the ability of distinct M. tuberculosis strains to cause disease.

Definition of novel cell envelope associated proteins in Triton X-114 extracts of Mycobacterium tuberculosis H37Rv.

BACKGROUND: Membrane- and membrane-associated proteins are important for the pathogenicity of bacteria. We have analysed the content of these proteins in virulent Mycobacterium tuberculosis H37Rv using Triton X-114 detergent-phase separation for extraction of lipophilic proteins, followed by their identification with high resolution mass spectrometry. RESULTS: In total, 1417 different proteins were identified. In silico analysis of the identified proteins revealed that 248 proteins had at least one predicted trans-membrane region. Also, 64 of the identified proteins were predicted lipoproteins, and 54 proteins were predicted as outer membrane proteins. Three-hundred-and-ninety-five of the observed proteins, including 91 integral membrane proteins were described for the first time. Comparison of abundance levels of the identified proteins was performed using the exponentially modified protein abundance index (emPAI) which takes into account the number of the observable peptides to the number of experimentally observed peptide ions for a given protein. The outcome showed that among the membrane-and membrane-associated proteins several proteins are present with high relative abundance. Further, a close examination of the lipoprotein LpqG (Rv3623) which is only detected in the membrane fractions of M. tuberculosis but not in M. bovis, revealed that the homologous gene in M. bovis lack the signal peptide and lipobox motif, suggesting impaired export to the membrane. CONCLUSIONS: Altogether, we have identified a substantial proportion of membrane- and membrane-associated proteins of M. tuberculosis H37Rv, compared the relative abundance of the identified proteins and also revealed subtle differences between the different members of the M. tuberculosis complex.

Validating divergent ORF annotation of the Mycobacterium leprae genome through a full translation data set and peptide identification by tandem mass spectrometry.

Mycobacterium leprae has undergone extensive degenerative evolution, with a large number of pseudogenes. It is also the organism with the greatest divergence between gene annotations from independent institutes. Therefore, M. leprae is a good model to verify the currently predicted coding sequence regions between different annotations, to identify new ones and to investigate the expression of pseudogenes. We submitted a total extract of the bacteria isolated from Armadillo to Gel-LC-MS/MS using a linear quadrupole ion trap-Orbitrap mass spectrometer. Spectra were analyzed using the Leproma (1614 genes and 1133 pseudogenes) and TIGR (5446 genes) databases and a database containing the full genome translation. We identified a total of 1046 proteins, including five proteins encoded by previously predicted pseudogenes, which upon closer inspection appeared to be proper genes. Only 11 of the additional annotations by TIGR were verified. We also identified six tryptic peptides from five proteins from regions not considered to be coding sequences, in addition to peptides from two unannotated gene candidates that overlap with other genes. Our data show that the Leproma annotation of M. leprae is quite accurate, and there were no peptide observations corresponding to true pseudogenes, except for a new gene candidate, overlapping with an essential enolase on the complementary strand.

High accuracy mass spectrometry analysis as a tool to verify and improve gene annotation using Mycobacterium tuberculosis as an example.

BACKGROUND: While the genomic annotations of diverse lineages of the Mycobacterium tuberculosis complex are available, divergences between gene prediction methods are still a challenge for unbiased protein dataset generation. M. tuberculosis gene annotation is an example, where the most used datasets from two independent institutions (Sanger Institute and Institute of Genomic Research-TIGR) differ up to 12% in the number of annotated open reading frames, and 46% of the genes contained in both annotations have different start codons. Such differences emphasize the importance of the identification of the sequence of protein products to validate each gene annotation including its sequence coding area. RESULTS: With this objective, we submitted a culture filtrate sample from M. tuberculosis to a high-accuracy LTQ-Orbitrap mass spectrometer analysis and applied refined N-terminal prediction to perform comparison of two gene annotations. From a total of 449 proteins identified from the MS data, we validated 35 tryptic peptides that were specific to one of the two datasets, representing 24 different proteins. From those, 5 proteins were only annotated in the Sanger database. In the remaining proteins, the observed differences were due to differences in annotation of transcriptional start sites. CONCLUSION: Our results indicate that, even in a less complex sample likely to represent only 10% of the bacterial proteome, we were still able to detect major differences between different gene annotation approaches. This gives hope that high-throughput proteomics techniques can be used to improve and validate gene annotations, and in particular for verification of high-throughput, automatic gene annotations.

Membrane and membrane-associated proteins in Triton X-114 extracts of Mycobacterium bovis BCG identified using a combination of gel-based and gel-free fractionation strategies.

Tuberculosis is an ancient disease that remains a significant global health problem. Because many membrane and membrane-associated proteins of this pathogen represent potential targets for drugs, diagnostic probes or vaccine components, we have analysed Mycobacterium bovis, bacillus Calmette-Guérin (BCG) substrain Moreau, using Triton X-114 for extraction of lipophilic proteins, followed by identification with LC coupled MS/MS. We identified 351 different proteins in total, and 103 (29%) were predicted as integral membrane proteins with at least one predicted transmembrane region and another 84 (23.9%) proteins had a positive grand average of hydropathicity (GRAVY) value, indicating increased probability for membrane association. Altogether 43 predicted lipoproteins (Lpps) were identified which is close to 50% of the total number of Lpps in the genome. Fifty-four proteins, including twenty-four predicted integral membrane proteins and seven predicted Lpps are described for the first time. The proportion of hydrophobic membrane and membrane-associated proteins shows that Triton X-114 is a highly efficient method for extraction of membrane proteins from bacteria, without the need for preisolation of membranes. ATP synthase, NAD(P) transhydrogenase, ubiquinone oxidoreductase and ubiquinol-cytochrome C reductase appear to represent major enzyme complexes in the membrane of Mycobacterium tuberculosis complex organisms.