The effects of delousing chemical, imidacloprid, on the copepod Acartia tonsa

The Norwegian aquaculture is critically important to the country’s industry and is the world largest exporter of Atlantic salmon (Salmo salar). However, the industry’s scale has led to an increase of louse pressure on both farmed and wild populations of salmonid species, with salmon louse (Lepeophtheirus salmonis) and Scottish louse (Caligus elongatus) having the greatest impact. Therefore, there are strict regulations in place to prevent louse infestations. To adhere to these regulations the industry uses chemotherapeutants and non-medical treatments. The chemotherapeutant imidacloprid was first approved for use in Norwegian aquaculture in 2021. Imidacloprid has a moiety resembling nicotine, which acts as a neurotoxin selectively targeting the insect’s central nervous system nicotinic acetylcholine receptors. Toxicity of imidacloprid has been extensively studied for insects and aquatic species, but little data for toxicity on marine species. In this study the acute toxicity of imidacloprid on the marine crustacean Acartia tonsa, using a 96-hours constant exposure and two pulse exposures, with a 24-hours and 72-hours intermediate non-exposure periods. Additionally, the stability of imidacloprid in saltwater solution was assessed during the 96-hours exposure. Raw data was further used to calculate LC and EC and make TKTD-RED-GUTS model. Experiments showed imidacloprid to be stable during the 96-hours and Acartia tonsa experiencing both immobilization and mortality in the 96-hours exposure, with EC10 = 0.06 mg/L, EC50 = 1.2 mg/L and EC90 = 25 mg/L after 24-hours and LC10 = 0.0021 mg/L, LC50 = 0.10 mg/L and LC90 = 4.3 mg/L after 96-hours. Both TKTD-RED-GUTS were poor fits compared with obtained mortality. The result obtained indicates Acartia tonsa would experience little effects based on treated release concentration, 0.30 µg/L, from the aquaculture. However, acute exposure from untreated treatment water, 20 mg/L, of imidacloprid could potentially have a significant impact on wild Acartia tonsa and potentially other non-target copepods.Masteroppgave i kjemiKJEM399KMAMN-LÆREMAMN-KJE

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 labelfree 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 ABCtransporter 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

<p>Abstract</p> <p>Background</p> <p>Membrane- and membrane-associated proteins are important for the pathogenicity of bacteria. We have analysed the content of these proteins in virulent <it>Mycobacterium tuberculosis </it>H37Rv using Triton X-114 detergent-phase separation for extraction of lipophilic proteins, followed by their identification with high resolution mass spectrometry.</p> <p>Results</p> <p>In total, 1417 different proteins were identified. <it>In silico </it>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 <it>M. tuberculosis </it>but not in <it>M. bovis</it>, revealed that the homologous gene in <it>M. bovis </it>lack the signal peptide and lipobox motif, suggesting impaired export to the membrane.</p> <p>Conclusions</p> <p>Altogether, we have identified a substantial proportion of membrane- and membrane-associated proteins of <it>M. tuberculosis </it>H37Rv, compared the relative abundance of the identified proteins and also revealed subtle differences between the different members of the <it>M. tuberculosis </it>complex.</p

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 highaccuracy 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.publishedVersio

Combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem mass spectrometry as an effective strategy for analyzing potential membrane protein complexes of Mycobacterium bovis bacillus Calmette-Guérin

<p>Abstract</p> <p>Background</p> <p>Tuberculosis is an infectious bacterial disease in humans caused primarily by <it>Mycobacterium tuberculosis</it>, and infects one-third of the world's total population. <it>Mycobacterium bovis </it>bacillus Calmette-Guérin (BCG) vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated <it>M. bovis </it>BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS) to characterize potential protein-protein interactions in membrane fractions.</p> <p>Results</p> <p>Using this approach, we analyzed native molecular composition of protein complexes in BCG membrane fractions. As a result, 40 proteins (including 12 integral membrane proteins), which were organized in 9 different gel bands, were unambiguous identified. The proteins identified have been experimentally confirmed using 2-D SDS PAGE. We identified MmpL8 and four neighboring proteins that were involved in lipid transport complexes, and all subunits of ATP synthase complex in their monomeric states. Two phenolpthiocerol synthases and three arabinosyltransferases belonging to individual operons were obtained in different gel bands. Furthermore, two giant multifunctional enzymes, Pks7 and Pks8, and four mycobacterial Hsp family members were determined. Additionally, seven ribosomal proteins involved in polyribosome complex and two subunits of the succinate dehydrogenase complex were also found. Notablely, some proteins with high hydrophobicity or multiple transmembrane helixes were identified well in our work.</p> <p>Conclusions</p> <p>In this study, we utilized LC-MS/MS in combination with blue native PAGE to characterize modular components of multiprotein complexes in BCG membrane fractions. The results demonstrated that the proteomic strategy was a reliable and reproducible tool for analysis of BCG multiprotein complexes. The identification in our study may provide some evidence for further study of BCG protein interaction.</p

Evaluation of signal peptide prediction algorithms for identification of mycobacterial signal peptides using sequence data from proteomic methods

Secreted proteins play an important part in the pathogenicity of Mycobacterium tuberculosis, and are the primary source of vaccine and diagnostic candidates. A majority of these proteins are exported via the signal peptidase I-dependent pathway, and have a signal peptide that is cleaved off during the secretion process. Sequence similarities within signal peptides have spurred the development of several algorithms for predicting their presence as well as the respective cleavage sites. For proteins exported via this pathway, algorithms exist for eukaryotes, and for Gram-negative and Gram-positive bacteria. However, the unique structure of the mycobacterial membrane raises the question of whether the existing algorithms are suitable for predicting signal peptides within mycobacterial proteins. In this work, we have evaluated the performance of nine signal peptide prediction algorithms on a positive validation set, consisting of 57 proteins with a verified signal peptide and cleavage site, and a negative set, consisting of 61 proteins that have an N-terminal sequence that confirms the annotated translational start site. We found the hidden Markov model of SignalP v3.0 to be the best-performing algorithm for predicting the presence of a signal peptide in mycobacterial proteins. It predicted no false positives or false negatives, and predicted a correct cleavage site for 45 of the 57 proteins in the positive set. Based on these results, we used the hidden Markov model of SignalP v3.0 to analyse the 10 available annotated proteomes of mycobacterial species, including annotations of M. tuberculosis H37Rv from the Wellcome Trust Sanger Institute and the J. Craig Venter Institute (JCVI). When excluding proteins with transmembrane regions among the proteins predicted to harbour a signal peptide, we found between 7.8 and 10.5 % of the proteins in the proteomes to be putative secreted proteins. Interestingly, we observed a consistent difference in the percentage of predicted proteins between the Sanger Institute and JCVI. We have determined the most valuable algorithm for predicting signal peptidase I-processed proteins of M. tuberculosis, and used this algorithm to estimate the number of mycobacterial proteins with the potential to be exported via this pathway

Computational Comparative Study of Tuberculosis Proteomes Using a Model Learned from Signal Peptide Structures

Secretome analysis is important in pathogen studies. A fundamental and convenient way to identify secreted proteins is to first predict signal peptides, which are essential for protein secretion. However, signal peptides are highly complex functional sequences that are easily confused with transmembrane domains. Such confusion would obviously affect the discovery of secreted proteins. Transmembrane proteins are important drug targets, but very few transmembrane protein structures have been determined experimentally; hence, prediction of the structures is essential. In the field of structure prediction, researchers do not make assumptions about organisms, so there is a need for a general signal peptide predictor

MSMEG_2731, an Uncharacterized Nucleic Acid Binding Protein from Mycobacterium smegmatis, Physically Interacts with RPS1

While the M. smegmatis genome has been sequenced, only a small portion of the genes have been characterized experimentally. Here, we purify and characterize MSMEG_2731, a conserved hypothetical alanine and arginine rich M. smegmatis protein. Using ultracentrifugation, we show that MSMEG_2731 is a monomer in vitro. MSMEG_2731 exists at a steady level throughout the M. smegmatis life-cycle. Combining results from pull-down techniques and LS-MS/MS, we show that MSMEG_2731 interacts with ribosomal protein S1. The existence of this interaction was confirmed by co-immunoprecipitation. We also show that MSMEG_2731 can bind ssDNA, dsDNA and RNA in vitro. Based on the interactions of MSMEG_2731 with RPS1 and RNA, we propose that MSMEG_2731 is involved in the transcription-translation process in vivo

Comparative Genomics of Cell Envelope Components in Mycobacteria

Mycobacterial cell envelope components have been a major focus of research due to their unique features that confer intrinsic resistance to antibiotics and chemicals apart from serving as a low-permeability barrier. The complex lipids secreted by Mycobacteria are known to evoke/repress host-immune response and thus contribute to its pathogenicity. This study focuses on the comparative genomics of the biosynthetic machinery of cell wall components across 21-mycobacterial genomes available in GenBank release 179.0. An insight into survival in varied environments could be attributed to its variation in the biosynthetic machinery. Gene-specific motifs like ‘DLLAQPTPAW’ of ufaA1 gene, novel functional linkages such as involvement of Rv0227c in mycolate biosynthesis; Rv2613c in LAM biosynthesis and Rv1209 in arabinogalactan peptidoglycan biosynthesis were detected in this study. These predictions correlate well with the available mutant and coexpression data from TBDB. It also helped to arrive at a minimal functional gene set for these biosynthetic pathways that complements findings using TraSH