Host-centric Proteomics of Stool: A Novel Strategy Focused on intestinal Responses to the Gut Microbiota

The diverse community of microbes that inhabits the human bowel is vitally important to human health. Host-expressed proteins are essential for maintaining this mutualistic relationship and serve as reporters on the status of host-microbiota interaction. Therefore, unbiased and sensitive methods focused on host proteome characterization are needed. Herein we describe a novel method for applying shotgun proteomics to the analysis of feces, focusing on the secreted host proteome. We have conducted the most complete analysis of the extracellular mouse gut proteome to date by employing a gnotobiotic mouse model. Using mice colonized with defined microbial communities of increasing complexity or a complete human microbiota (‘humanized‘), we show that the complexity of the host stool proteome mirrors the complexity of microbiota composition. We further show that host responses exhibit signatures specific to the different colonization states. We demonstrate feasibility of this approach in human stool samples and provide evidence for a “core” stool proteome as well as personalized host response features. Our method provides a new avenue for noninvasive monitoring of host-microbiota interaction dynamics via host-produced proteins in stool. The diverse community of microbes that inhabits the human bowel is vitally important to human health. Host-expressed proteins are essential for maintaining this mutualistic relationship and serve as reporters on the status of host-microbiota interaction. Therefore, unbiased and sensitive methods focused on host proteome characterization are needed. Herein we describe a novel method for applying shotgun proteomics to the analysis of feces, focusing on the secreted host proteome. We have conducted the most complete analysis of the extracellular mouse gut proteome to date by employing a gnotobiotic mouse model. Using mice colonized with defined microbial communities of increasing complexity or a complete human microbiota (‘humanized‘), we show that the complexity of the host stool proteome mirrors the complexity of microbiota composition. We further show that host responses exhibit signatures specific to the different colonization states. We demonstrate feasibility of this approach in human stool samples and provide evidence for a “core” stool proteome as well as personalized host response features. Our method provides a new avenue for noninvasive monitoring of host-microbiota interaction dynamics via host-produced proteins in stool. Hundreds to thousands of microbial “species” and ∼1013 individual organisms make up any one person's gut microbiota (1Bäckhed F. Ley R.E. Sonnenburg J.L. Peterson D.A. Gordon J.I. Host-bacterial mutualism in the human intestine.Science. 2005; 307: 1915-1920Crossref PubMed Scopus (3614) Google Scholar), making the gastrointestinal (GI) 1The abbreviations used are: GIgastrointestinalLC-MS/MSliquid chromatography-tandem MS. 1The abbreviations used are: GIgastrointestinalLC-MS/MSliquid chromatography-tandem MS. tract one of the most complex biological ecosystems ever studied. The dynamic interaction between these communities and the host organism is linked to many aspects of health and disease in humans including inflammatory bowel diseases (2Round J.L. Mazmanian S.K. The gut microbiota shapes intestinal immune responses during health and disease.Nature Rev. 2006; 9: 313-323Google Scholar), obesity (3Sanderson S. Boardman W. Ciofi C. Gibson R. Human gut microbes associated with obesity.Nature. 2006; 444: 1022-1023Crossref PubMed Scopus (6151) Google Scholar), allergies (4Maslowski K.M. Mackay C.R. Diet, gut microbiota and immune responses.Nat. Immunol. 2011; 12: 5-9Crossref PubMed Scopus (882) Google Scholar), and autoimmunity (5Reid G. Younes J.A. Van der Mei H.C. Gloor G.B. Knight R. Busscher H.J. Microbiota restoration: natural and supplemented recovery of human microbial communities.Nat. Rev. Microbiol. 2011; 9: 27-38Crossref PubMed Scopus (386) Google Scholar). Sequence-based approaches (e.g. metagenomics and 16S community profiling) have effectively elucidated the gene and species composition of several microbial communities that influence health and disease (3Sanderson S. Boardman W. Ciofi C. Gibson R. Human gut microbes associated with obesity.Nature. 2006; 444: 1022-1023Crossref PubMed Scopus (6151) Google Scholar, 6Gill S.R. Pop M. Deboy R.T. Eckburg P.B. Turnbaugh P.J. Samuel B.S. Gordon J.I. Relman D.A. Fraser-Liggett C.M. Nelson K.E. Metagenomic analysis of the human distal gut microbiome.Science. 2006; 312: 1355-1359Crossref PubMed Scopus (3285) Google Scholar, 7Eckburg P.B. Bik E.M. Bernstein C.N. Purdom E. Dethlefsen L. Sargent M. Gill S.R. Nelson K.E. Relman D.A. Diversity of the human intestinal microbial flora.Science. 2005; 308: 1635-1638Crossref PubMed Scopus (5532) Google Scholar). However, sequencing alone is limited to defining microbial community constituents, providing little insight into the myriad ways hosts can respond to their resident microbes. Despite an individualized “fingerprint” (7Eckburg P.B. Bik E.M. Bernstein C.N. Purdom E. Dethlefsen L. Sargent M. Gill S.R. Nelson K.E. Relman D.A. Diversity of the human intestinal microbial flora.Science. 2005; 308: 1635-1638Crossref PubMed Scopus (5532) Google Scholar) of microbiota composition, a major gap separates our understanding of how differently composed microbial communities specifically impact host responses in the gut. Enhanced methods that sensitively probe the microbial impact on host biology will be critical to expanding insight into the host-microbiota super-organism. Stool presents an easily sampled biological material that offers a window into complex host–microbe relationships. gastrointestinal liquid chromatography-tandem MS. gastrointestinal liquid chromatography-tandem MS. Early studies of the host response to microbiota utilized laser-capture micro dissection (LCM) (8Stappenbeck T.S. Hooper L.V. Manchester J.K. Wong M.H. Gordon J.I. Laser capture microdissection of mouse intestine: characterizing mRNA and protein expression, and profiling intermediary metabolism in specified cell populations.Methods Enzymol. 2002; 356: 167-196Crossref PubMed Scopus (50) Google Scholar), followed by gene expression analysis of particular cell types in the GI epithelium. Although providing an unprecedented view into the ways microbiota can impact host biology, this approach is technically difficult, provides only a semiquantitative estimate of biologically pertinent protein expression, and requires the collection of intestinal tissue. Therefore, LCM and subsequent transcriptional profiling of host tissue prevents time-course experimentation in animal models and is not readily translated to patient studies. The combination of liquid chromatography and tandem mass spectrometry (LC-MS/MS) provides a flexible, dynamic platform for the simultaneous identification and quantification of thousands of proteins in fecal samples. Implementing this technology to study gut biology has been inhibited by technical limitations stemming from the overwhelming complexity of the resident microbiota metagenome: it greatly overshadows the host's genome, its composition varies between individuals, and it encodes only a sparsely defined proteome. Pioneering studies of this complex system focused on the metaproteome, attempting to identify as many host and bacterial proteins as possible using matched metagenomic sequencing and shotgun proteomics (9Verberkmoes, N. C., Russell, A. L., Shah, M., Godzik, A., Rosenquist, M., Halfvarson, J., Lefsrud, M. G., Apajalahti, J., Tysk, C., Hettich, R. L., Jansson, J. K., Shotgun metaproteomics of the human distal gut microbiota. ISME J. 3, 179–89.Google Scholar, 10Erickson A.R. Cantarel B.L. Lamendella R. Darzi Y. Mongodin E.F. Pan C. Shah M. Halfvarson J. Tysk C. Henrissat B. Raes J. Verberkmoes N.C. Fraser C.M. Hettich R.L. Jansson J.K. Integrated metagenomics/metaproteomics reveals human host-microbiota signatures of Crohn's disease.PloS one. 2012; 7: e49138Crossref PubMed Scopus (325) Google Scholar). Although matched sequencing data can improve bacterial protein identifications, drawing biological conclusions from data that is composed predominantly of proteins with ill-defined functions and origins remains difficult (10Erickson A.R. Cantarel B.L. Lamendella R. Darzi Y. Mongodin E.F. Pan C. Shah M. Halfvarson J. Tysk C. Henrissat B. Raes J. Verberkmoes N.C. Fraser C.M. Hettich R.L. Jansson J.K. Integrated metagenomics/metaproteomics reveals human host-microbiota signatures of Crohn's disease.PloS one. 2012; 7: e49138Crossref PubMed Scopus (325) Google Scholar). Our approach acknowledges the contrast between the technical challenges posed by measuring bacterial proteins in the context of complex microbial communities and the importance of elucidating the host response to microbial dynamics. By combining technical improvements in sample preparation before LC-MS/MS and subsequent data analysis, we have developed a workflow in which abundance changes of >3000 host proteins shed into the GI tract can be sensitively assayed. Applying these techniques to defined perturbations of the gnotobiotic mouse model establishes a pathway for discovering functional relationships between microbiota and host response. Furthermore, extending this approach to archived or freshly collected human stool samples makes possible the elucidation of specific host responses to microbiota for which extensive characterization is already complete or planned. Gnotobiotic and conventional (RF, Taconic, Inc.) Swiss-Webster mice were maintained as previously described (11Sonnenburg E.D. Zheng H. Joglekar P. Higginbottom S.K. Firbank S.J. Bolam D.N. Sonnenburg J.L. Specificity of polysaccharide use in intestinal bacteroides species determines diet-induced microbiota alterations.Cell. 2010; 141: 1241-1252Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). Humanization was performed using human fecal samples from one healthy human donor (male, western diet) as previously described (12Kashyap P. Marcobal A. Ursell L. Complex interactions among diet, gastrointestinal transit, and gut microbiota in humanized mice.Gastroenterology. 2013; (doi:10.1053/j.gastro.2013.01.047)Abstract Full Text Full Text PDF Scopus (305) Google Scholar). Mice were fed with standard diet (Purine LabDiet 5K67). Cultures of bacteria were prepared as following. Tryptone-yeast extract-glucose (TYG) medium was used for growing Bacteroides species (BacteroidesthetaiotaomicronVPI-5482 and B. caccae ATCC 43185T) as described previously (11Sonnenburg E.D. Zheng H. Joglekar P. Higginbottom S.K. Firbank S.J. Bolam D.N. Sonnenburg J.L. Specificity of polysaccharide use in intestinal bacteroides species determines diet-induced microbiota alterations.Cell. 2010; 141: 1241-1252Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). Bifidobacterium species (Bif. longum NCC2705 and Bif. bifidum DSM 20456) were grown in Man-Rogosa-Sharpe (MRS) medium (Oxoid LTD., Basingstoke, Hampshire, England).Reinforced clostridium medium (RCM; Becton Dickinson, Franklin Lakes, NJ) was used for Clostridium spiroforme DSM 1552and Clostridium scindensATCC35704growth.Luria broth (LB; Becton Dickinson) media was used for the growth of Edwardsiella tarda ATCC 23685 and Provedencia stuarii ATCC 25827. Peptone yeast glucose broth (Anaerobe Systems, Morgan Hill, CA) was used for Roseburia inulinivorans DSM 16841, Prevotella copri CB7, and Prevotella stercorea CB35. Enterococcus faecalis ATCC 27276 were grown in broth bacteria were in using an mice were with (8Stappenbeck T.S. Hooper L.V. Manchester J.K. Wong M.H. Gordon J.I. Laser capture microdissection of mouse intestine: characterizing mRNA and protein expression, and profiling intermediary metabolism in specified cell populations.Methods Enzymol. 2002; 356: 167-196Crossref PubMed Scopus (50) Google Scholar) of as previously described (11Sonnenburg E.D. Zheng H. Joglekar P. Higginbottom S.K. Firbank S.J. Bolam D.N. Sonnenburg J.L. Specificity of polysaccharide use in intestinal bacteroides species determines diet-induced microbiota alterations.Cell. 2010; 141: 1241-1252Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). The for the mice Bif. bifidum DSM Enterococcus faecalis ATCC Prevotella copri CB7, Roseburia inulinivorans DSM 16841, and Prevotella The for mice Bacteroides B. caccae ATCC Bif. Clostridium Clostridium spiroforme DSM and Edwardsiella tarda ATCC to we of mouse was by with of the bacterial community was using were conducted the mouse proteome and the proteome of the microbes. data to a and for that to only one microbial were for organism organism was it were to one of which was a and that were from mice for before mice in a colonization and were and sampled the were to the the fecal samples were collected from in liquid and before were on in with were with the of a and were to The was to The was and with proteins were using a and were to and in were into and using and via liquid chromatography tandem mass spectrometry were in sample of sample a in an were from this a using an The was to a and mass Full were collected in the in followed by a standard with in which were from of were for protein were from the protein the 2012; PubMed Scopus Google Scholar) and bacterial were from the Integrated a E. B. A. M. 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The and associated status in 2013; PubMed Scopus Google Scholar) with the data analysis, of was to the for a particular protein samples. were using and with the of with further analysis was conducted on using the in functional were conducted using the functional analysis of gene PubMed Scopus Google Scholar, and analysis of gene using PubMed Scopus Google Scholar) functional platform with the specified protein are were in of gut were inhibited by major of host proteins to stool a and ill-defined associated with the microbiota and that during gut of their in the complex the proteins most biologically to host responses are to be to between a mass to sensitively proteins and analysis to and in a of protein with these we a gnotobiotic mouse model to microbial complexity and to study host Mice colonized with microbial communities of increasing complexity with defined the and of our of colonization of conventional conventional microbiota. in a new a defined of the intestinal we a previously described recovery of bacterial and analysis of community in the gastrointestinal tract of Microbiol. 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E. J.K. M. M. J. Y. R. microbial colonization of mice reveals the dynamics of immune 2010; PubMed Scopus Google We several proteins in microbial colonization with expression a relationship between the microbes and these particular aspects of the host we aspects of the host responses that were specific to colonization by demonstrate that increasing and complexity in host responses are linked to community complexity and to colonization microbiota composition varies between in mice dynamics in the mouse gut a possible for in community one. 2010; PubMed Scopus Google Scholar) and humans M. N. Gordon J.I. Knight R. community in human and PubMed Scopus Google Scholar). is the host proteome is or We this by from of and conventional mice for The fecal of the mice were with or between conventional mice in their responses to the microbiota to this was between mice secreted protein in and data that and in the host fecal proteome is on the gut microbiota composition, and a sensitive of host responses to the microbiota. Furthermore, our data that mice provide an for monitoring host the of the gnotobiotic mouse model as a platform for using communities to the between particular and of to specific host We our and method be to the analysis of human fecal samples. We stool samples from healthy human with gut microbiota Using a stool sample from we a of human proteins with from with protein and of and Although the of proteins from these samples was in mouse samples proteins in analysis of these proteins were for the types of functions we to in the gut Our data reveals a of proteins from a analysis of the fecal to our on secreted host proteins (10Erickson A.R. Cantarel B.L. Lamendella R. Darzi Y. Mongodin E.F. Pan C. Shah M. Halfvarson J. Tysk C. Henrissat B. Raes J. Verberkmoes N.C. Fraser C.M. Hettich R.L. Jansson J.K. Integrated metagenomics/metaproteomics reveals human host-microbiota signatures of Crohn's disease.PloS one. 2012; 7: e49138Crossref PubMed Scopus (325) Google Scholar). We further a proteome that of proteins between these Despite the of this the abundance of most proteins between the with signatures The proteins that were to functional the the proteins were for secreted and extracellular of the proteins in the human proteome have that in the mouse proteome with a of the response to resident microbes Samuel B.S. Gordon J.I. Gnotobiotic responses to the gut S. A. PubMed Scopus Google Scholar). fecal sample from one human was used as the for the humanized mouse analysis to individualized aspects of the host fecal proteome be in the by analysis of proteins in the mouse and human data the for the fecal donor was most to of the mice of the human microbiota on into the mouse gut P. The of diet on the human gut a metagenomic analysis in humanized gnotobiotic Scopus Google Scholar) and of host that are P.J. Knight R. Gordon J.I. The of diet on the human gut a metagenomic analysis in humanized gnotobiotic PubMed Scopus Google Scholar). Our study these a of host responses in humanized including personalized aspects of these our method is with analysis of human fecal it be easily to archived or samples. our method to samples will the of the secreted host proteome and that specific interaction in health and We a novel method for proteins from mouse and human fecal samples. these methods in with a gnotobiotic mouse we have the of microbial community complexity on the secreted host proteome. by this of our methods several associated with gut microbiota proteomics including the complexity of feces, the of the microbial metaproteome, and in the GI as described will metagenomic the of host with health method the of host responses from the GI tract in an unbiased and noninvasive will fecal samples previously used for microbial studies to be for host protein it can be used to critical between the gene and species composition of microbial and the have on host studies have microbiota in healthy disease states. many of these have that microbiota is linked to specific host that to states. stool proteomics will between microbiota and host response and provide an avenue for discovering novel By the in which these interactions it is that these proteins will be linked with status with in or method several for and of by we proteins from the secreted host proteins on which our analysis microbial associated proteins secreted by the host B. J. J. B. F. L. R. H. H. The human and and are by specific and PubMed Google Scholar), which to be to health and protein signatures from microbial and host proteins will further of sample preparation and from the distal of the gastrointestinal tract provides an of host interactions that the of the GI we fecal proteome analysis host responses in the of the gut. of a mouse model to as used will focused study of these by and specific of the it be to host responses in stool to specific the GI the between with the semiquantitative of that of and the of A. J. S. J. G. R. C. mass a novel quantification for analysis of complex protein by PubMed Scopus Google Scholar, B. S. N. S. S. S. S. P. S. M. F. A. protein in using Full Text Full Text PDF PubMed Scopus Google improve analysis of these this approach can be further and analysis using previously developed mass spectrometry providing and understanding this complex biological for mass spectrometry of complex microbial communities as the gut as in human (9Verberkmoes, N. C., Russell, A. L., Shah, M., Godzik, A., Rosenquist, M., Halfvarson, J., Lefsrud, M. G., Apajalahti, J., Tysk, C., Hettich, R. L., Jansson, J. K., Shotgun metaproteomics of the human distal gut microbiota. ISME J. 3, 179–89.Google Scholar, 10Erickson A.R. Cantarel B.L. Lamendella R. Darzi Y. Mongodin E.F. Pan C. Shah M. Halfvarson J. Tysk C. Henrissat B. Raes J. Verberkmoes N.C. Fraser C.M. Hettich R.L. Jansson J.K. Integrated metagenomics/metaproteomics reveals human host-microbiota signatures of Crohn's disease.PloS one. 2012; 7: e49138Crossref PubMed Scopus (325) Google Scholar) and mouse H. Turnbaugh P.J. A. Shah N. C. Cantarel B.L. Henrissat B. A. Verberkmoes N.C. Hettich R.L. Gordon J.I. a model human gut microbiota composed of of its bacterial S. A. PubMed Scopus Google Scholar). gnotobiotic mouse model the of our communities composed only of species it possible to host from microbial The of the gnotobiotic mouse model with analysis of stool has for and interactions the gut proteome (9Verberkmoes, N. C., Russell, A. L., Shah, M., Godzik, A., Rosenquist, M., Halfvarson, J., Lefsrud, M. G., Apajalahti, J., Tysk, C., Hettich, R. L., Jansson, J. K., Shotgun metaproteomics of the human distal gut microbiota. ISME J. 3, 179–89.Google Scholar, 10Erickson A.R. Cantarel B.L. Lamendella R. Darzi Y. Mongodin E.F. Pan C. Shah M. Halfvarson J. Tysk C. Henrissat B. Raes J. Verberkmoes N.C. Fraser C.M. Hettich R.L. Jansson J.K. Integrated metagenomics/metaproteomics reveals human host-microbiota signatures of Crohn's disease.PloS one. 2012; 7: e49138Crossref PubMed Scopus (325) Google Scholar, H. Turnbaugh P.J. A. Shah N. C. Cantarel B.L. Henrissat B. A. Verberkmoes N.C. Hettich R.L. Gordon J.I. a model human gut microbiota composed of of its bacterial S. A. PubMed Scopus Google identification were cell or in mice microbes we a of we from a standard proteome We this to proteins that a of (e.g. to and to that with of which in that are not readily We further and will these The complex interactions between the gut microbiota and their host unbiased of host and By the described proteomics method with bacterial metagenomics and and by these interactions in the gnotobiotic mouse the can to improve its understanding of this new of which host responses to any of and natural will improve our to and complex GI We for Higginbottom for with the gnotobiotic mice and many of the and Sonnenburg for and to the defined colonization with