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Shotgun Metaproteomics of the Human Distal Gut Microbiota

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Shotgun Metaproteomics of the Human Distal Gut Microbiota The ISME Journal (2009) 3, 179–189 & 2009 International Society for Microbial Ecology All rights reserved 1751-7362/09 $32.00 www.nature.com/ismej ORIGINAL ARTICLE Shotgun metaproteomics of the human distal gut microbiota Nathan C Verberkmoes1,7, Alison L Russell1,7, Manesh Shah1, Adam Godzik2, Magnus Rosenquist3,8, Jonas Halfvarson4, Mark G Lefsrud1,9, Juha Apajalahti5, Curt Tysk4, Robert L Hettich1 and Janet K Jansson3,6 1Oak Ridge National Laboratory, Chemical and Life Sciences Divisions, Oak Ridge, TN, USA; 2Department of Bioinformatics and Systems Biology, Burnham Institute for Medical Research, La Jolla, CA, USA; 3Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden; 4Department of Internal Medicine, Division of Gastroenterology, O¨rebro University Hospital, O¨rebro, Sweden; 5Alimetrics Ltd, Helsinki, Finland and 6Department of Ecology, Earth Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA The human gut contains a dense, complex and diverse microbial community, comprising the gut microbiome. Metagenomics has recently revealed the composition of genes in the gut microbiome, but provides no direct information about which genes are expressed or functioning. Therefore, our goal was to develop a novel approach to directly identify microbial proteins in fecal samples to gain information about the genes expressed and about key microbial functions in the human gut. We used a non-targeted, shotgun mass spectrometry-based whole community proteomics, or metaproteomics, approach for the first deep proteome measurements of thousands of proteins in human fecal samples, thus demonstrating this approach on the most complex sample type to date. The resulting metaproteomes had a skewed distribution relative to the metagenome, with more proteins for translation, energy production and carbohydrate metabolism when compared to what was earlier predicted from metagenomics. Human proteins, including antimicrobial peptides, were also identified, providing a non-targeted glimpse of the host response to the microbiota. Several unknown proteins represented previously undescribed microbial pathways or host immune responses, revealing a novel complex interplay between the human host and its associated microbes. The ISME Journal (2009) 3, 179–189; doi:10.1038/ismej.2008.108; published online 30 October 2008 Subject Category: microbe–microbe and microbe–host interactions Keywords: microbiome; human gut; metaproteome; shotgun proteomics; metagenomics; antimicrobial peptide Introduction has a unique and relatively stable gut microbiota, unless disrupted by external factors such as anti- The human gastrointestinal (GI) tract is host for 11 biotic treatment (Jernberg et al., 2007). Increasing myriads of microorganisms (approximately 10 evidence suggests that the composition of the GI per g feces) that carry out vital processes for normal microbiota is linked to inflammatory bowel diseases digestive functions of the host and play an impor- (Peterson et al., 2008), such as Crohn’s disease tant, although not yet not fully understood, role in (Dicksved et al., 2008), and can even influence the maturation of human immunity and defense against propensity for obesity (Ley et al., 2006). Current pathogens. Recent findings suggest that each human estimates, based on sequencing of 16S rRNA genes in DNA extracted from feces, are that 800–1000 different Correspondence: JK Jansson, Earth Science Division, Lawrence microbial species and 47000 different strains inhabit Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, the GI tract (Ba¨ckhead et al., 2005) and that the CA 94720, USA. majority of these (480%) have not yet been isolated E-mail: [email protected] 7These authors contributed equally to this work. or characterized (Eckburg et al., 2005). Therefore, 8Current address: Department of Oncology, Radiology and there is a vast microbial diversity with largely Clinical Immunology, Uppsala University Hospital, Uppsala, unknown function that is waiting to be explored. Sweden. Recently, metagenomic sequencing has revealed 9 Current address: Bioresource Engineering, McGill University, information about the complement of genes in the Ste-Anne-de-Bellevue, Quebec, Canada. Received 2 September 2008; revised 7 October 2008; accepted 8 gut microbiota of two healthy individuals (Gill et al., October 2008; published online 30 October 2008 2006). Although this dataset did not represent the Human gut metaproteome NC Verberkmoes et al 180 entire GI microbiota, analysis of identified genes can be confidently and reproducibly measured (that revealed that the GI microbiome has significantly is, with high specificity and low false-positive rates; enriched capacities for glycan, amino-acid and 1–5% maximum) from gut microorganisms by xenobiotic metabolism, methanogenesis, and synth- comparing with available metagenome databases esis of vitamins and isoprenoids. This indirect (Gill et al., 2006) and available gut isolate genomes evidence suggested that there are unique microbial and to determine whether unmatched datasets functions carried out in the gut environment. could suffice for accurate protein identifications. A major limitation of DNA-based approaches is An additional goal was to apply a novel bioinfor- that they predict potential functions, but it is not matics approach to assign putative functions to known whether the predicted genes are expressed at unknown proteins not covered by standard analysis all or if so, under what conditions and to what of clusters of orthologous groups (COGs). Ultimately, extent. In addition, it is not possible to determine our aim was to use the protein data to provide direct whether the DNA is from cells that are active and evidence of dominant and key microbial functions in viable, dormant or even dead. These limitations can the human gut for the first time, some of which could be overcome by directly assessing proteins, because serve as indicators of a healthy or diseased state. In the genes must have been transcribed and translated addition, this non-targeted approach enables identi- to produce a protein product. However, to date only fication of human proteins associated with the gut a couple of microbial proteins have been identified microbiota, thus illustrating potential interactions from the human gut and these were obtained by two- between the human microbiome and host. dimensional (2D) PAGE (Klaassens et al., 2007), followed by excision and de novo sequencing of targeted spots on the gel. Materials and methods Here, our aim was to develop a novel high- Fecal sample collection throughput, non-targeted mass spectrometry (MS) A female healthy monozygotic twin pair born in 1951 approach to determine the identities of thousands of was invited to take part in a larger double-blinded microbial proteins in the most complex sample type study, and details of these individuals with respect to to date (that is, feces) and to test the feasibility of diet, antibiotic usage, and so on are described earlier: using a non-matched metagenome dataset for individuals numbered 6a and 6b (Dicksved et al., protein identification. This MS-based shotgun 2008), who provided samples 7 and 8, respectively, proteomics approach relies on detection and identi- were the focus of this study. The only differences fication of all proteins in a lysed cell mixture between the individuals according to the submitted without the need for gel-based separation or de questionnaire data were that individual 6a had novo sequencing. Instead, the resulting peptides gastroenteritis and individual 6b had taken non- from an enzymatic digest of the entire proteome are steroidal anti-inflammatory drugs during the past 12 separated by liquid chromatography and infused months. Fecal samples were collected in 20 ml directly into rapidly scanning tandem mass spectro- colonic tubes by the twins and immediately sent to meters (2D-LC-MS/MS) through electrospray ioniza- O¨ rebro University Hospital on the day of collection, tion. The resulting peptide mass information and where they were placed at À70 1C and stored. The tandem mass spectra are used to search against Uppsala County Ethics Committee and the Oak Ridge protein databases generated from genome se- National Laboratory (ORNL) human study review quences. To date, the shotgun metaproteomics panel approved the study. approach has only been demonstrated in a limited number of studies and only for microbial commu- nities with low diversity, such as acid mine drainage Microbial cell extraction from fecal samples systems (Ram et al., 2005; Lo et al., 2007), Fecal samples were thawed at þ 4 1C and microbial endosymbionts (Markert et al., 2007) and sewage cells were extracted from the bulk fecal material by sludge water (Wilmes et al., 2008). It remains a differential centrifugation, as described earlier technical challenge to apply this shotgun approach (Apajalahti et al., 1998). This cell extraction method to more complex microbial communities, such as has previously been found to result in a highly those inhabiting the human gut. enriched bacterial fraction from complex samples, For this study, it was first necessary to develop the such as soil and chicken feces, with negligible shotgun proteomics approach to work with fecal bacterial cell loss and a good representation of fecal samples containing large amounts of particulate microbiota (Apajalahti et al., 1998). The resulting matter and undigested food and a large diversity of bacterial cell pellets were immediately
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