Progress 09/01/07 to 08/31/12
Outputs
OUTPUTS: Our group has recently uncovered the first example of host specificity among a non-pathogenic member of the vertebrate gut microbiota: the gram-positive bacterium Lactobacillus reuter. These studies used a combination of population genetics and comparative genomics, and revealed host-specific phylogenetic clusters within the species that possess differences in genome content reflective of niche characteristics in the gastrointestinal tract (GIT) of respective host. We further conducted animal experiments in gnotobiotic mice with the aim to experimentally demonstrate host specialization by measuring competitive indices of strains originating from different hosts. In addition, we have identified genes of Lactobacillus reuteri that contribute to host adaptation, and begun and study the mechanisms by which host specialization is facilitated. The results of this research have been disseminated by publications in several peer-reviewed journals and presented at several scientific meetings. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We used a combination of population genetics and comparative genomics to identify signatures of host adaptation in the phylogeny and genomes of this gut symbiont. These studies showed that strains of L. reuteri from global sources comprised distinct phylogenetic clusters that can be detected with Multilocus Sequence Analysis (MLSA) and Amplified Fragment Length Polymorphism (AFLP) and that were tightly reflective of host origin. The population structure suggests a stable association of L. reuteri with particular vertebrates and groups of host species (e.g. mice and rats, and chicken and turkey) over evolutionary time and the emergence of host adapted subpopulations. We further employed animal experiments in ex germ-free and Lactobacillus-free mice and empirically demonstrated host specialization by measuring competitive indices of strains originating from different hosts. This research revealed that only rodent strains display high ecological performance in mice and, most importantly, efficiently colonized Lactobacillus-free mice. To identify genomic events marking adaptations to the murine host, comparative genome hybridizations were performed to query 57 L. reuteri strains originating from six different vertebrate hosts in combination with genome sequence comparisons of nine strains encompassing five phylogenetic lineages of the species. This approach revealed that rodent strains, although showing a high degree of genomic plasticity, possessed a specific genome inventory that was rare or absent in strains associated with other vertebrate hosts. Most importantly, although topologies of dendrograms inferred from UPGMA analysis of gene polymorphisms and MLSA sequences were different, both methods resulted in trees with essentially the same host-specific phylogenetic clusters. This indicates that L. reuteri has diverged into genetically and ecologically cohesive subpopulations (ecotypes) restricted to particular hosts. The analysis of the bacterial genomes of representative strains revealed that gene content of L. reuteri strains reflected the niche characteristics in the GITs of their respective hosts, and inactivation of seven out of eight rodent-specific genes in L. reuteri 100-23 resulted in impaired ecological performance in the gut of mice. We have begun to determine the mechanisms by which host specificity is mediated and maintained between Lactobacillus reuteri and the murine host, focusing on the role of host-specific bacterial adherence and biofilm formation. In addition, the experimental approaches that we have established have recently been used to infer the evolutionary relationship of Lactobacillus reuteri strains from fermented foods.
Publications
- Su MS, Oh PL, Walter J, and Ganzle, MG. 2012. Intestinal origin of sourdough Lactobacillus reuteri isolates as revealed by phylogenetic, genetic, and physiological analysis. Applied and Environmental Microbiology. 78: 6777-6780.
- Frese SA, and Walter J. 2012. Host-specific adherence of Lactobacillus reuteri contributes to ecological fitness in vivo. Invited Talk. 1 October 2012. International Scientific Association for Probiotics and Prebiotics - Annual Meeting. Cork, Ireland. Frese SA, and Walter J. 2012. Host-specific adherence of Lactobacillus reuteri contributes to ecological fitness in vivo. Poster Presentation. October 2012. International Scientific Association for Probiotics and Prebiotics - Annual Meeting. Cork, Ireland.
|
Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: In order to contribute to our understanding of the co-evolution between vertebrate gut symbionts and their hosts, we studied the host-L. reuteri interrelationship from an evolutionary standpoint, using a combination of population genetics, evolutionary genomics, and gnotobiotic animal work. This work has demonstrated host specificity of this vertebrate gut synbiont, and we used a comparative genomic analysis to identify host specific genome content and genomic events marking adaptations to the murine host. The ecological significance of representative rodent specific genes in animal experiments were tested using Lactobacillus-free mice, and we have started to characterize the ecological functionality of of these genes and the mechanisms by which they contribute to ecological performance. PARTICIPANTS: Collaboration has been established with Nathalie Juge (Institute of Food Research, Norwich Research Park, Norwich, UK) to study symbiotic interactions between Lactobacillus reuteri and the host. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have begun to study the mechanistic role of host-specific genes of Lactobacillus reuteri, both in animal and in vitro experiments. For example, we performed a structural and functional analysis of exopolysacchrides of L. reuteri that are produced by a rodent-specific fructosyltransferase (Ftf). This analysis revealed that Ftf contribute to ecological performance in the mouse gastrointestinal tract, probably through a mechanistic role in immunomodulation and an induction of regulatory T cells (Foxp3+ cells). The ecological role of other host specific genes is currently under investigation.
Publications
- Walter, J., Britton, R. A., and Roos, S. Microbes and Health Sackler Colloquium: Host-microbial symbiosis in the vertebrate gastrointestinal tract and the Lactobacillus reuteri paradigm. Proceedings of the National Academics Sciences USA. 108: Suppl. 1:4645-4652 (2011)
- Frese SA, Benson AK, Tannock GW, Loach, DM, Kim J, Zhang M, Oh PL, Heng NCK, Patil PB, Juge N, MacKenzie DA, Pearson BM, Lapidus A, Dalin E, Tice H, Goltsman E, Land M, Hauser L, Ivanova N, Kyrpides NC, and Walter J. The evolution of host specialization in the vertebrate gut symbiont Lactobacillus reuteri. PLoS Genetics. 7(2): e1001314 (2011).
- Sims I.M., Frese S.A., Walter J., Loach D., Wilson M., Appleyard K., Eason J., Livingston M., Baird M., Cook G., and Tannock G.W. Structure and functions of exopolysaccharide produced by gut commensal Lactobacillus reuteri 100-23. ISME Journal. 5:1115-1124 (2011).
- MacKenzie, D.A., Jeffers, F., Parker, M.L., Vibert-Vallet, A., Bongaerts, R.J., Roos, S., Walter, J., and Juge, N. Strain-specific diversity of mucus-binding proteins in the adhesion and aggregation properties of Lactobacillus reuteri. Microbiology. 156:3368-3378 (2010).
|
Progress 10/01/10 to 01/30/11
Outputs
OUTPUTS: We have examined the evolutionary relationships of 165 Lactobacillus reuteri strains isolated from humans, rodents, pigs, and poultry originating from a wide variety of geographic locations. We further used a comparative genomic analysis to identify genomic events marking adaptations to the murine host, and confirmed ecological significance of representative rodent specific genes in animal experiments using Lactobacillus-free mice. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
To gain insight into the evolution of a gut symbiont, we have characterized the population genetic structure and phylogeny of Lactobacillus reuteri strains isolated from six different host species (human, mouse, rat, pig, chicken, and turkey) using Amplified Fragment Length Polymorphism (AFLP) and Multi Locus Sequence Analysis (MLSA). The results revealed considerable genetic heterogeneity within the L. reuteri population and distinct monophyletic clades reflecting host origin but not provenance. The evolutionary patterns detected indicate a long-term association of L. reuteri lineages with particular vertebrate species and host-driven diversification. Results from a competition experiment in a gnotobiotic mouse model revealed that rodent isolates showed elevated ecological performance, indicating that evolution of L. reuteri lineages was adaptive. Further experiments with Lactobacillus-free mice revealed that isolates L. reuteri evolved a high degree of specialization and host restriction. These findings provide evidence that some vertebrate gut microbes are not promiscuous but have diversified into host-adapted lineages by a long-term evolutionary process, allowing the development of a highly specialized symbiosis. To identify genomic events marking adaptations to the murine host, we compared the genome of the rodent isolate L. reuteri 100-23 with that of the human isolate L. reuteri F275, and identified hundreds of genes that were specific to each strain. In order to differentiate true host-specific genome content from strain-level differences, comparative genome hybridizations were performed to query 57 L. reuteri strains originating from six different vertebrate hosts in combination with genome sequence comparisons of nine strains encompassing five phylogenetic lineages of the species. This approach revealed that rodent strains, although showing a high degree of genomic plasticity, possessed a specific genome inventory that was rare or absent in strains from other vertebrate hosts. The distinct genome content of L. reuteri lineages reflected the niche characteristics in the gastrointestinal tracts of their respective hosts, and inactivation of seven out of eight representative rodent-specific genes in L. reuteri 100-23 resulted in impaired ecological performance in the gut of mice. The comparative genomic analyses suggested fundamentally different trends of genome evolution in rodent and human L. reuteri populations, with the former possessing a large and adaptable pan-genome while the latter being subjected to a process of reductive evolution. In conclusion, this study provided experimental evidence and a molecular basis for the evolution of host specificity in a vertebrate gut symbiont, and identified genomic events that have shaped this process.
Publications
- Frese SA, Benson AK, Tannock GW, Loach DM, Kim J, Zhang M, Oh PL, Heng NCK, Patil PB, Juge N, MacKenzie, DA, Pearson BM, Lapidus A, Dalin E, Tice H, Goltsman E, Land M, Hauser L, Ivanova N, Kyrpides NC, and Walter J. 2011. The Evolution of Host Specialization in the Vertebrate Gut Symbiont Lactobacillus reuteri. PLoS Genetics. In press.
- Frese SA, Benson AK, Tannock GW, Loach DM, Kim J, Zhang M, Oh PL, Patil PB, Lapidus A, Heng NCK, Dalin E, Tice H, Goltsman E, Land M, Hauser L, Ivanova N, Kyrpides NC, and Walter J. 2010. The Evolution of Host Specialization in the Vertebrate Gut Symbiont Lactobacillus reuteri. German-American Kavli Frontiers of Science Symposium. Potsdam, Germany. Poster and Poster Flash Presentation.
- Oh, P.L., Benson, A.K., Peterson, D.A., Patil, P.B., Moriyama, E.N., Roos, S., and Walter, J. 2010. Diversification of the gut symbiont Lactobacillus reuteri as a result of host-driven evolution. ISME Journal. 4:377-387.
|
Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: We have examined the evolutionary relationships of 165 Lactobacillus reuteri strains isolated from humans, rodents, pigs, and poultry originating from a wide variety of geographic locations. DNA microarray analysis was used to identify lineage-specific differences in genome content. 58 strains of L. reuteri representing all major phylogenetic lineages were compared with the genomes of Lactobacillus reuteri 100-23 (rodent isolate) and DSM20016T (human isolate) to identify putative colonization factors of Lactobacillus reuteri. Several genes were inactivated in strain 100-23 and tested for their ecological performance in the gastrointestinal tract of Lactobacillus-free mice. PARTICIPANTS: This is a collaboration between investigators of the University of Nebraska, Lincoln, the University of Otago, Dunedin, New Zealand, and the Joint Genome Institute (JGI). The project provides training for two PhD students at the University of Nebraska. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
To gain insight into the evolution of a gut symbiont, we have characterized the population genetic structure and phylogeny of Lactobacillus reuteri strains isolated from six different host species (human, mouse, rat, pig, chicken, and turkey) using Amplified Fragment Length Polymorphism (AFLP) and Multi Locus Sequence Analysis (MLSA). The results revealed considerable genetic heterogeneity within the L. reuteri population and distinct monophyletic clades reflecting host origin but not provenance. The evolutionary patterns detected indicate a long-term association of L. reuteri lineages with particular vertebrate species and host-driven diversification. Further experiments with Lactobacillus-free mice revealed that isolates L. reuteri evolved a high degree of specialization and host restriction. These findings provide evidence that some vertebrate gut microbes are not promiscuous but have diversified into host-adapted lineages by a long-term evolutionary process. To identify candidate genes contributing to host adaptation within the lineages, DNA microarray analysis was used to identify lineage-specific differences in genome content. 54 strains of L. reuteri representing all major phylogenetic lineages were compared with the genomes of Lactobacillus reuteri 100-23 (rodent isolate) and DSM20016T (human isolate). Interestingly, a dendrogram derived from UPGMA analysis of microarray binary data revealed virtually the same phylogenetic lineages of L. reuteri strains as obtained with AFLP and MLST. It is therefore reasonable to conclude that the gene distribution is the result of evolutionary events in the development of host confined phylogenetic lineages. Lineage specific genome content was identified using MARKFIND (Zhang et al., 2003, J. Bacteriol. 185: 5573-5584) and revealed polymorphisms that are conserved within all members or most members of a lineage. The rodent clade of strains maintained or acquired a wide range of genes that were lost in the human clade I throughout evolution. These genes comprise mainly 6 functional categories: Surface proteins, capsular polysaccharide formation, environmental sensing, protein secretion, and genes associated with prophages. Interestingly, these are groups of genes one would expect to contribute to the biofilm formation of L. reuteri in the murine forestomach. We hypothesized that genes maintained by strains in the rodent clade but not in human specific clade I have been maintained or acquired during evolution of L. reuteri as they increase ecological fitness in the rodent GIT. To test this hypothesis, we did inactivate representative rodent specific genes in L. reuteri 100-23 and showed their contribution to colonization of the murine GIT. In conclusion, our ongoing work revealed important insight into the evolution of a vertebrate gut symbiont and identified colonization factors of L. reuteri for life in the murine digestive tract.
Publications
- Steven A. Frese, Andrew K. Benson, Gerald W. Tannock, Prabhu B. Patil, Phaik Lyn Oh, Min Zhang, Jaehyoung Kim, Diane M. Loach, Nicholas C.K. Heng, Alla Lapidus, Eileen Dalin, Hope Tice, Eugene Goltsman, Miriam Land, Loren Hauser, Natalia Ivanova, Nikos C. Kyrpides, and Jens Walter, 2009. Genomic changes underlying the evolution of host-restriction in the vertebrate gut symbiont Lactobacillus reuteri. Poster presentation (selected for oral presentation), Sackler Microbes and Health Colloquia. Anaheim, CA, Nov 2-3, 2009.
- Oh, P.L., Benson, A.K., Peterson, D.A., Patil, P.B., Roos, S., and Walter, J. 2009. Diversification of the gut symbiont Lactobacillus reuteri as a result of host-driven bacterial evolution. German-American Kavli Frontiers of Science Symposium. Irvine, California. Poster and Poster Flash Presentation.
- Oh, P.L., Benson, A.K., Peterson, D.A., Patil, P.B., Moriyama, E.N., Roos, S., and Walter, J. Diversification of the gut symbiont Lactobacillus reuteri as a result of host-driven evolution. ISME Journal. Epub ahead of print. 2010.
- Phaik Lyn Oh, Andrew K. Benson, Daniel A. Peterson, Prabhu B. Patil, Etsuko N. Moriyama, Stefan Roos, and Jens Walter. 2009. Diversification of the gut symbiont Lactobacillus reuteri as a result of host-driven evolution. Poster presentation (selected for oral presentation), Sackler Microbes and Health Colloquia. Anaheim, CA, Nov 2-3, 2009.
|
Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: We have examined the evolutionary relationships of Lactobacillus reuteri strains isolated from humans, rodents, pigs, and poultry originating from a wide variety of geographic locations. DNA microarray analysis was used to identify lineage-specific differences in genome content. 54 strains of L. reuteri representing all major phylogenetic lineages were compared with the genomes of Lactobacillus reuteri 100-23 (rodent isolate) and DSM20016T (human isolate) to identify putative colonization factors of Lactobacillus reuteri. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Reconstructed phylogenetic trees obtained with Amplified Fragment Length Polymorphism (AFLP) and Multi Locus Sequence Typing (MLST) analysis revealed clusters of strains that showed high specificity to particular hosts with only limited confounding effects of geography. The relative branch lengths observed in both AFLP and MLST dendrograms is indicative of the fact that these are deep phylogenetic divisions and that independent L. reuteri lineages have likely been associated with respective animal species throughout long evolutionary time spans, leading to a host-driven bacterial selection and diversification. To identify candidate genes contributing to these deep phylogenetic divisions (and ultimately to host adaptation within the lineages) DNA microarray analysis was used to identify lineage-specific differences in genome content. 54 strains of L. reuteri representing all major phylogenetic lineages were compared with the genomes of Lactobacillus reuteri 100-23 (rodent isolate) and DSM20016T (human isolate). Interestingly, a dendrogram derived from UPGMA analysis of microarray binary data revealed virtually the same phylogenetic lineages of L. reuteri strains as obtained with AFLP and MLST. Lineage specific genome content was identified using MARKFIND (Zhang et al., 2003, J. Bacteriol. 185: 5573-5584) and revealed polymorphisms that are conserved within all members or most members of a lineage. The rodent clade of strains maintained or acquired a wide range of genes that were lost in the human clade I throughout evolution. Since the phylogenetic clustering was virtual identical with AFLP, MLST, and microarray analysis, it is reasonable to conclude that the gene distribution is the result of evolutionary events in the development of host confined phylogenetic lineages. Furthermore, since L. reuteri DSM 20016T, which groups in human clade I, does not colonize the gut of mice, it is likely that genes present in the rodent clade but not in human clade I encode traits important for life in the rodent gut. These genes comprise mainly 6 functional categories: Surface proteins, capsular polysaccharide formation, environmental sensing, bacterocin production, protein secretion, and genes associated with prophages. Interestingly, these are groups of genes one would expect of an organism colonizing the gut of mammals by forming biofilms. We hypothesize that genes maintained by strains in the rodent confined phylogenetic clade but not in human specific clade I have been maintained or acquired during evolution of L. reuteri as they increase ecological fitness in the rodent GIT. The aim of this ongoing project is to characterize these genes in order to confirm their ecological significance, and to determine evolutionary event that shaped this gut symbiont.
Publications
- Phaik Lyn Oh, Andrew K. Benson, Stefan Roos, and Jens Walter. 2008. Genotypic characterization of Lactobacillus reuteri strains isolated from different host animals reveals host confined phylogenetic lineages. Poster presentation of the 9th Symposium on Lactic Acid Bacteria, Egmond aan Zee, The Netherlands.
|
|