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

ProteoClade: A taxonomic toolkit for multi-species and metaproteomic analysis

We present ProteoClade, a Python toolkit that performs taxa-specific peptide assignment, protein inference, and quantitation for multi-species proteomics experiments. ProteoClade scales to hundreds of millions of protein sequences, requires minimal computational resources, and is open source, multi-platform, and accessible to non-programmers. We demonstrate its utility for processing quantitative proteomic data derived from patient-derived xenografts and its speed and scalability enable a novel de novo proteomic workflow for complex microbiota samples

A shotgun metaproteomics approach to study the microbiome of cystic fibrosis (CF) patients

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Metaproteomic evidence of changes in protein expression following a change in electrode potential in a robust biocathode microbiome

Microorganisms that respire electrodes may be exploited for biotechnology applications if key pathways for extracellular electron transfer (EET) can be identified and manipulated through bioengineering. To determine whether expression of proposed Biocathode-MCL EET proteins are changed by modulating electrode potential without disrupting the relative distribution of microbial constituents, metaproteomic and 16S rRNA gene expression analyses were performed after switching from an optimal to suboptimal potential based on an expected decrease in electrode respiration. Five hundred and seventy-nine unique proteins were identified across both potentials, the majority of which were assigned to three previously defined Biocathode-MCL metagenomic clusters: a Marinobacter sp., a member of the family Chromatiaceae, and a Labrenzia sp. Statistical analysis of spectral counts using the Fisher's exact test identified 16 proteins associated with the optimal potential, five of which are predicted electron transfer proteins. The majority of proteins associated with the suboptimal potential were involved in protein turnover/turnover, motility, and membrane transport. Unipept and 16S rRNA gene expression analyses indicated that the taxonomic profile of the microbiome did not change after 52 hours at the suboptimal potential. These findings show that protein expression is sensitive to the electrode potential without inducing shifts in community composition, a feature that may be exploited for engineering Biocathode-MCL

The effect of imipenem and diffusible signaling factors on the secretion of outer membrane vesicles and associated Ax21 proteins in Stenotrophomonas maltophilia

Outer membrane vesicles (OMVs) are small nanoscale structures that are secreted by bacteria and that can carry nucleic acids, proteins, and small metabolites. They can mediate intracellular communication and play a role in virulence. In this study, we show that treatment with the -lactam antibiotic imipenem leads to a dramatic increase in the secretion of outer membrane vesicles in the nosocomial pathogen Stenotrophomonas maltophilia. Proteomic analysis of their protein content demonstrated that the OMVs contain the chromosomal encoded Li metallo-Hactamase and L2 serine-Hactamase. Moreover, the secreted OMVs contain large amounts of two Ax21 homologs, i.e., outer membrane proteins known to be involved in virulence and biofilm formation. We show that OMV secretion and the levels of Ax21 in the OMVs are dependent on the quorum sensing diffusible signal system (DSF). More specific, we demonstrate that the S. maltophilia DSF cis-42-11 -methyl-dodecenoic acid and, to a lesser extent, the Burkholderia cenocepacia DSF cis-42-dodecenoic acid, stimulate OMV secretion. By a targeted proteomic analysis, we confirmed that DSF-induced OMVs contain large amounts of the Ax21 homologs, but not the Hactamases. This work illustrates that both quorum sensing and disturbance of the peptidoglycan biosynthesis provoke the release of OMVs and that OMV content is context dependent

Microfluidics-based liquid chromatography/mass spectrometry multiple reaction monitoring approach for the relative quantification of Burkholderia cenocepacia secreted virulence factors

Rationale: Burkholderia cenocepacia is an opportunistic pathogen that is commonly isolated from patients with cystic fibrosis (CF). Quorum sensing has been suggested to play a role in the activity of type II and type VI secretion systems and the release of virulence factors. Apart from the classical acyl homoserine lactone quorum sensing, B. cenocepacia also uses the diffusible signal factor system (DSF). Quantitative information on the true impact of DSF molecules on the release of ZmpA and other virulence factors is lacking. Methods: Based on results of a label-free proteomics analysis addressing changes in the secretome in response to DSFs, a panel of peptides was selected to develop a microfluidics liquid chromatography/mass spectrometry (LC/MS) method implementing single reaction monitoring (SRM) to quantify B. cenocepacia virulence factors. Results: Increase in secretion of virulence factors upon treatment with BDSF was observed for ZmpA and Aida, but not for ZmpB. Type VI secretion system dependent Hcp1 and TecA were decreased. However, non-physiological amounts of BDSF were needed to provoke the effect. DSFs from P. aeruginosa and S. maltophilia were also affecting virulence factor secretion, but the effect was smaller than for the endogenous BDSF. Conclusions: Microfluidics-based SRM is a useful tool to quantitatively assess the impact of quorum sensing on the release of virulence factors by (opportunistic) pathogens

A fast Peptide Match service for UniProt Knowledgebase

Summary: We have developed a new web application for peptide matching using Apache Lucene-based search engine. The Peptide Match service is designed to quickly retrieve all occurrences of a given query peptide from UniProt Knowledgebase (UniProtKB) with isoforms. The matched proteins are shown in summary tables with rich annotations, including matched sequence region(s) and links to corresponding proteins in a number of proteomic/peptide spectral databases. The results are grouped by taxonomy and can be browsed by organism, taxonomic group or taxonomy tree. The service supports queries where isobaric leucine and isoleucine are treated equivalent, and an option for searching UniRef100 representative sequences, as well as dynamic queries to major proteomic databases. In addition to the web interface, we also provide RESTful web services. The underlying data are updated every 4 weeks in accordance with the UniProt releases. Availability: http://proteininformationresource.org/peptide.shtml Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

A Robust and Universal Metaproteomics Workflow for Research Studies and Routine Diagnostics Within 24 h Using Phenol Extraction, FASP Digest, and the MetaProteomeAnalyzer

The investigation of microbial proteins by mass spectrometry (metaproteomics) is a key technology for simultaneously assessing the taxonomic composition and the functionality of microbial communities in medical, environmental, and biotechnological applications. We present an improved metaproteomics workflow using an updated sample preparation and a new version of the MetaProteomeAnalyzer software for data analysis. High resolution by multidimensional separation (GeLC, MudPIT) was sacrificed to aim at fast analysis of a broad range of different samples in less than 24 h. The improved workflow generated at least two times as many protein identifications than our previous workflow, and a drastic increase of taxonomic and functional annotations. Improvements of all aspects of the workflow, particularly the speed, are first steps toward potential routine clinical diagnostics (i.e., fecal samples) and analysis of technical and environmental samples. The MetaProteomeAnalyzer is provided to the scientific community as a central remote server solution at www.mpa.ovgu.de.Peer Reviewe

Faecal (meta)proteomics : a tool to investigate dysbiosis and inflammation in patients with cystic fibrosis

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Nitrous oxide emission by the non-denitrifying, nitrate ammonifier Bacillus licheniformis

Background: Firmicutes have the capacity to remove excess nitrate from the environment via either denitrification, dissimilatory nitrate reduction to ammonium or both. The recent renewed interest in their nitrogen metabolism has revealed many interesting features, the most striking being their wide variety of dissimilatory nitrate reduction pathways. In the present study, nitrous oxide production from Bacillus licheniformis, a ubiquitous Gram-positive, spore-forming species with many industrial applications, is investigated. Results: B. licheniformis has long been considered a denitrifier but physiological experiments on three different strains demonstrated that nitrous oxide is not produced from nitrate in stoichiometric amounts, rather ammonium is the most important end-product, produced during fermentation. Significant strain dependency in end-product ratios, attributed to nitrite and ammonium, and medium dependency in nitrous oxide production were also observed. Genome analyses confirmed the lack of a nitrite reductase to nitric oxide, the key enzyme of denitrification. Based on the gene inventory and building on knowledge from other non-denitrifying nitrous oxide emitters, hypothetical pathways for nitrous oxide production, involving NarG, NirB, qNor and Hmp, are proposed. In addition, all publically available genomes of B. licheniformis demonstrated similar gene inventories, with specific duplications of the nar operon, narK and hmp genes as well as NarG phylogeny supporting the evolutionary separation of previously described distinct BALI1 and BALI2 lineages. Conclusions: Using physiological and genomic data we have demonstrated that the common soil bacterium B. licheniformis does not denitrify but is capable of fermentative dissimilatory nitrate/nitrite reduction to ammonium (DNRA) with concomitant production of N2O. Considering its ubiquitous nature and non-fastidious growth in the lab, B. licheniformis is a suitable candidate for further exploration of the actual mechanism of N2O production in DNRA bacteria and its relevance in situ