Influence of Moraxella sp. colonization on the kidney proteome of farmed gilthead sea breams (Sparus aurata, L.)

Background: Currently, presence of Moraxella sp. in internal organs of fish is not considered detrimental for fish farming. However, bacterial colonization of internal organs can affect fish wellness and decrease growth rate, stress resistance, and immune response. Recently, there have been reports by farmers concerning slow growth, poor feed conversion, and low average weight increase of fish farmed in offshore floating sea cages, often associated with internal organ colonization by Moraxella sp. Therefore, presence of these opportunistic bacteria deserves further investigations for elucidating incidence and impact on fish metabolism. Results: A total of 960 gilthead sea breams (Sparus aurata, L.), collected along 17 months from four offshore sea cage plants and two natural lagoons in Sardinia, were subjected to routine microbiological examination of internal organs throughout the production cycle. Thirteen subjects (1.35%) were found positive for Moraxella sp. in the kidney (7), brain (3), eye (1), spleen (1), and perivisceral fat (1). In order to investigate the influence of Moraxella sp. colonization, positive and negative kidney samples were subjected to a differential proteomics study by means of 2-D PAGE and mass spectrometry. Interestingly, Moraxella sp. infected kidneys displayed a concerted upregulation of several mitochondrial enzymes compared to negative tissues, reinforcing previous observations following lipopolysaccharide (LPS) challenge in fish. Conclusions: Presence of Moraxella sp. in farmed sea bream kidney is able to induce proteome alterations similar to those described following LPS challenge in other fish species. This study revealed that Moraxella sp. might be causing metabolic alterations in fish, and provided indications on proteins that could be investigated as markers of infection by Gram-negative bacteria within farming plants.Pubblicat

Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples

Introduction and objectives. The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Worldwide archival tissue banks hold a significant number and variety of tissue samples, as well as a wealth of retrospective information regarding diagnosis, prognosis, and response to therapy. This makes them an important resource for protein biomarker discovery and validation. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun LC-MS/MS analysis of FFPE samples, but a critical comparison of the different methods is currently lacking. Methods DT was preceded by homogenization in ammonium bicarbonate, while ISD and FASP comprised protein extraction in SDS based-buffer, followed by SDS depletion with Detergent Removal Spin Columns and Microcon Ultracel YM-30 filtration devices, respectively. The three workflows were applied to consecutive tissue sections cut from an FFPE liver tissue block, and peptide mixtures were finally analyzed according to a label-free quantitative MS approach. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity. Results and Discussion. DT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility. Conclusion. These results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth.2014-10-06Madrid, Spain13th Human Proteome Organization World Congres

Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue

Background: The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking. Experimental design: DT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity. Results: DT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility. Conclusions: These results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth.Pubblicat

An Easy and efficient method for native and immunoreactive <i>Echinococcus granulosus</i> antigen 5 enrichment from hydatid cyst fluid

Background:Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis. Methodology/Principal Findings: Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure is very easy, efficient and reproducible, since different hydatid cyst fluid (HCF) sources produced very similar chromatograms, notwithstanding the clearly evident and extreme heterogeneity of the starting material. In addition, the performance of the antigen preparation in immunological assays was preliminarily assessed by western immunoblotting and ELISA on a limited panel of cystic echinococcosis patients and healthy controls. Following western immunoblotting and ELISA experiments, a high reactivity of patient sera was seen, with unambiguous and highly specific results. Conclusions/Significance: The methods and results reported open interesting perspectives for the development of sensitive diagnostic tools to enable the timely and unambiguous detection of cystic echinococcosis antibodies in patient sera

high throughput genomic and proteomic technologies in the fight against infectious diseases

New technologies have shown significant promise in the fight against infectious diseases, with the discovery of novel molecular targets for in vitro diagnostics and the improved design of vaccines. In developing countries, especially in areas of neglected diseases and resources-poor settings, a number of technological innovations are further needed, such as the integration of old and new biomarkers in suitable analysis platforms, the simplification of existing analysis systems, and the improvement of sample preservation and management. However, in these areas, identification of new biomarkers for infectious diseases is still a core issue in the diagnostic quest. Similarly, new technologies will allow scientists to design vaccines with improved immunogenicity, efficacy and safety in the local area, according to the circulating pathogenic strains and the genetic background of the population to be immunized. In this work we review the current omics-based technologies and their potential for accelerating the development of next generation vaccines and the identification of biomarkers suitable for point-of-care (POC) diagnostic applications

The impact of sequence database choice on metaproteomic results in gut microbiota studies

Background: Elucidating the role of gut microbiota in physiological and pathological processes has recently emerged as a key research aim in life sciences. In this respect, metaproteomics, the study of the whole protein complement of a microbial community, can provide a unique contribution by revealing which functions are actually being expressed by specific microbial taxa. However, its wide application to gut microbiota research has been hindered by challenges in data analysis, especially related to the choice of the proper sequence databases for protein identification. Results: Here, we present a systematic investigation of variables concerning database construction and annotation and evaluate their impact on human and mouse gut metaproteomic results. We found that both publicly available and experimental metagenomic databases lead to the identification of unique peptide assortments, suggesting parallel database searches as a mean to gain more complete information. In particular, the contribution of experimental metagenomic databases was revealed to be mandatory when dealing with mouse samples. Moreover, the use of a "merged" database, containing all metagenomic sequences from the population under study, was found to be generally preferable over the use of sample-matched databases. We also observed that taxonomic and functional results are strongly database-dependent, in particular when analyzing the mouse gut microbiota. As a striking example, the Firmicutes/Bacteroidetes ratio varied up to tenfold depending on the database used. Finally, assembling reads into longer contigs provided significant advantages in terms of functional annotation yields. Conclusions: This study contributes to identify host- and database-specific biases which need to be taken into account in a metaproteomic experiment, providing meaningful insights on how to design gut microbiota studies and to perform metaproteomic data analysis. In particular, the use of multiple databases and annotation tools has to be encouraged, even though this requires appropriate bioinformatic resources

Comparative evaluation of multiple protein extraction procedures from three species of the genus Caulerpa

AbstractThe aim of this study was to define the simplest and least expensive protocol for total protein extraction for three different macroalgae of the genus Caulerpa (the invasive C. taxifolia and C. cylindracea and the autochthonous C. prolifera). Five multi-step protein extraction procedures, set up for other macroalgal species, were tested. For each of them, different pre-treatment and extraction conditions were simultaneously examined, according to a factorial design, considering the starting material, the solvent-to-biomass ratio, and the incubation temperature. Protein yield in the obtained extracts was estimated with the Bradford method. Further, polyacrylamide gel electrophoresis (SDS-PAGE) was used to resolve proteins, assessing their quality and integrity. Significant differences in protein yield were observed among the extraction protocols and the conditions tested, also in relation to the considered species. Profiles having an acceptable quality were obtained for C. prolifera and C. cylindracea, and from the obtained results, the best method to obtain high yield and quality protein extracts for the two above-mentioned species appears to require the use of a primary TCA/acetone extraction buffer followed by a lysis buffer with NaCl, KCl, urea, Triton, SDS and a protease inhibitor. The best results, in particular, were obtained starting from fresh pulped material with a buffer-to-biomass ratio of 10:1 and an incubation temperature of 4°C. For C. taxifolia, instead, none of the tested protocols produced satisfactory results and further studies will be required

Identification of <i>Trichomonas vaginalis</i> alpha-actinin as the most common immunogen recognized by sera of women exposed to the parasite

A study on presence of antibodies to Trichomonis vaginalis in serum was done on a group of 500 pregnant, asymptomatic Angolan women. A serologic screening, done by ELISA, revealed that 41% of the women had IgG and IgM against the parasite. Analysis of sera by immunoblotting revealed that 94.4% of sera with anti-T. vaginalis IgG class antibodies were reactive against a common immunogenic protein of 115 kDa. The common immunogen was identified as the protozoan α-actinin. All sera recognizing the 115-kDa antigen were reactive against both native and recombinant T. vaginalis α-actinin and nonreactive against human α-actinin. The findings presented in this work offer a new tool for epidemiologic studies and open new perspectives for vaccination

Influence of <i>Moraxella</i> sp. colonization on the kidney proteome of farmed gilthead sea breams (<i>Sparus aurata</i>, L.)

Background: Currently, presence of Moraxella sp. in internal organs of fish is not considered detrimental for fish farming. However, bacterial colonization of internal organs can affect fish wellness and decrease growth rate, stress resistance, and immune response. Recently, there have been reports by farmers concerning slow growth, poor feed conversion, and low average weight increase of fish farmed in offshore floating sea cages, often associated with internal organ colonization by Moraxella sp. Therefore, presence of these opportunistic bacteria deserves further investigations for elucidating incidence and impact on fish metabolism. Results: A total of 960 gilthead sea breams (Sparus aurata, L.), collected along 17 months from four offshore sea cage plants and two natural lagoons in Sardinia, were subjected to routine microbiological examination of internal organs throughout the production cycle. Thirteen subjects (1.35%) were found positive for Moraxella sp. in the kidney (7), brain (3), eye (1), spleen (1), and perivisceral fat (1). In order to investigate the influence of Moraxella sp. colonization, positive and negative kidney samples were subjected to a differential proteomics study by means of 2-D PAGE and mass spectrometry. Interestingly, Moraxella sp. infected kidneys displayed a concerted upregulation of several mitochondrial enzymes compared to negative tissues, reinforcing previous observations following lipopolysaccharide (LPS) challenge in fish. Conclusions: Presence of Moraxella sp. in farmed sea bream kidney is able to induce proteome alterations similar to those described following LPS challenge in other fish species. This study revealed that Moraxella sp. might becausing metabolic alterations in fish, and provided indications on proteins that could be investigated as markers of infection by Gram-negative bacteria within farming plants

The liposoluble proteome of Mycoplasma agalactiae: an insight into the minimal protein complement of a bacterial membrane

<p>Abstract</p> <p>Background</p> <p>Mycoplasmas are the simplest bacteria capable of autonomous replication. Their evolution proceeded from gram-positive bacteria, with the loss of many biosynthetic pathways and of the cell wall. In this work, the liposoluble protein complement of <it>Mycoplasma agalactiae</it>, a minimal bacterial pathogen causing mastitis, polyarthritis, keratoconjunctivitis, and abortion in small ruminants, was subjected to systematic characterization in order to gain insights into its membrane proteome composition.</p> <p>Results</p> <p>The selective enrichment for <it>M. agalactiae </it>PG2^Tliposoluble proteins was accomplished by means of Triton X-114 fractionation. Liposoluble proteins were subjected to 2-D PAGE-MS, leading to the identification of 40 unique proteins and to the generation of a reference 2D map of the <it>M. agalactiae </it>liposoluble proteome. Liposoluble proteins from the type strain PG2 and two field isolates were then compared by means of 2D DIGE, revealing reproducible differences in protein expression among isolates. An in-depth analysis was then performed by GeLC-MS/MS in order to achieve a higher coverage of the liposoluble proteome. Using this approach, a total of 194 unique proteins were identified, corresponding to 26% of all <it>M. agalactiae </it>PG2^Tgenes. A gene ontology analysis and classification for localization and function was also carried out on all protein identifications. Interestingly, the 11.5% of expressed membrane proteins derived from putative horizontal gene transfer events.</p> <p>Conclusions</p> <p>This study led to the in-depth systematic characterization of the <it>M. agalactiae </it>liposoluble protein component, providing useful insights into its membrane organization.</p