Dynamic Properties of the Intestinal Ecosystem Call for Combination Therapies, Targeting Inflammation and Microbiota, in Ulcerative Colitis.

BACKGROUND & AIMS: Intestinal microbiota-host interactions play a major role in health and disease. This has been documented at the microbiota level ("dysbiosis" in chronic immune-mediated diseases) and through the study of specific bacteria-host interactions but rarely at the level of intestinal ecosystem dynamics. However, understanding the behavior of this ecosystem may be key to the successful treatment of disease. We recently postulated that health and disease represent alternative stable states of the intestinal ecosystem (different configurations that can exist under identical external conditions), which would require adapted strategies in disease treatment. Here, we examine if alternative stable states indeed exist in this ecosystem and if they could affect remission from ulcerative colitis (UC). METHODS: We analyzed data from a study on pediatric UC. The data reflect current treatment practice following the recruitment of treatment-naive patients with new-onset disease. Patients received personalized anti-inflammatory treatments over a period of 1 year. Stool samples at 0, 4, 12, and 52 weeks allowed an estimation of microbiota status (through 16S ribosomal RNA gene sequencing) and host inflammatory status (through the measurement of fecal calprotectin levels). RESULTS: We identify 4 microbiota states and 4 host states. Longitudinal data show that the improvement of inflammatory status is accompanied by an improvement of microbiota status. However, they also provide strong indications that both improvements are retarded or blocked by alternative states barriers. CONCLUSIONS: Our observations strongly suggest that inflammation suppression should be combined with microbiota management where possible to improve the efficacy of UC treatment.

Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus: a chronicle of evolution in action.

BACKGROUND: Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus are lactic acid producing bacteria that are largely used in dairy industries, notably in cheese-making and yogurt production. An earlier in-depth study of the first completely sequenced ssp. bulgaricus genome revealed the characteristics of a genome in an active phase of rapid evolution, in what appears to be an adaptation to the milk environment. Here we examine for the first time if the same conclusions apply to the ssp. lactis, and discuss intra- and inter-subspecies genomic diversity in the context of evolutionary adaptation. RESULTS: Both L. delbrueckii ssp. show the signs of reductive evolution through the elimination of superfluous genes, thereby limiting their carbohydrate metabolic capacities and amino acid biosynthesis potential. In the ssp. lactis this reductive evolution has gone less far than in the ssp. bulgaricus. Consequently, the ssp. lactis retained more extended carbohydrate metabolizing capabilities than the ssp. bulgaricus but, due to high intra-subspecies diversity, very few carbohydrate substrates, if any, allow a reliable distinction of the two ssp. We further show that one of the most important traits, lactose fermentation, of one of the economically most important dairy bacteria, L. delbruecki ssp. bulgaricus, relies on horizontally acquired rather than deep ancestral genes. In this sense this bacterium may thus be regarded as a natural GMO avant la lettre. CONCLUSIONS: The dairy lactic acid producing bacteria L. delbrueckii ssp. lactis and ssp. bulgaricus appear to represent different points on the same evolutionary track of adaptation to the milk environment through the loss of superfluous functions and the acquisition of functions that allow an optimized utilization of milk resources, where the ssp. bulgaricus has progressed further away from the common ancestor.

BioNLP Shared Task--The Bacteria Track.

BACKGROUND: We present the BioNLP 2011 Shared Task Bacteria Track, the first Information Extraction challenge entirely dedicated to bacteria. It includes three tasks that cover different levels of biological knowledge. The Bacteria Gene Renaming supporting task is aimed at extracting gene renaming and gene name synonymy in PubMed abstracts. The Bacteria Gene Interaction is a gene/protein interaction extraction task from individual sentences. The interactions have been categorized into ten different sub-types, thus giving a detailed account of genetic regulations at the molecular level. Finally, the Bacteria Biotopes task focuses on the localization and environment of bacteria mentioned in textbook articles. We describe the process of creation for the three corpora, including document acquisition and manual annotation, as well as the metrics used to evaluate the participants' submissions. RESULTS: Three teams submitted to the Bacteria Gene Renaming task; the best team achieved an F-score of 87%. For the Bacteria Gene Interaction task, the only participant's score had reached a global F-score of 77%, although the system efficiency varies significantly from one sub-type to another. Three teams submitted to the Bacteria Biotopes task with very different approaches; the best team achieved an F-score of 45%. However, the detailed study of the participating systems efficiency reveals the strengths and weaknesses of each participating system. CONCLUSIONS: The three tasks of the Bacteria Track offer participants a chance to address a wide range of issues in Information Extraction, including entity recognition, semantic typing and coreference resolution. We found common trends in the most efficient systems: the systematic use of syntactic dependencies and machine learning. Nevertheless, the originality of the Bacteria Biotopes task encouraged the use of interesting novel methods and techniques, such as term compositionality, scopes wider than the sentence.

Development of an efficient in vivo system (Pjunc-TpaseIS1223) for random transposon mutagenesis of Lactobacillus casei.

The random transposon mutagenesis system P(junc)-TpaseIS(1223) is composed of plasmids pVI129, expressing IS1223 transposase, and pVI110, a suicide transposon plasmid carrying the P(junc) sequence, the substrate of the IS1223 transposase. This system is particularly efficient in Lactobacillus casei, as more than 10,000 stable, random mutants were routinely obtained via electroporation.

Complete genome sequence of the commensal Streptococcus salivarius strain JIM8777.

The commensal bacterium Streptococcus salivarius is a prevalent species of the human oropharyngeal tract with an important role in oral ecology. Here, we report the complete 2.2-Mb genome sequence and annotation of strain JIM8777, which was recently isolated from the oral cavity of a healthy, dentate infant.

Characterization of the insoluble proteome of Lactococcus lactis by SDS-PAGE LC-MS/MS leads to the identification of new markers of adaptation of the bacteria to the mouse digestive tract.

We characterized the insoluble proteome of Lactococcus lactis using 1D electrophoresis-LC-MS/MS and identified 313 proteins with at least two different peptides. The identified proteins include 89 proteins having a predicted signal peptide and 25 predicted to be membrane-located. In addition, 67 proteins had alkaline isoelectric point values. Using spectra and peptide counts, we compared protein abundances in two different conditions: growth in rich medium, and after transit in the mouse digestive tract. We identified the large mechanosensitive channel and a putative cation transporter as membrane markers of bacterial adaptation to the digestive tract.

Alternative stable states in the intestinal ecosystem: proof of concept in a rat model and a perspective of therapeutic implications.

BACKGROUND: Chronic immune-mediated diseases are rapidly expanding and notoriously difficult to cure. Altered relatively stable intestinal microbiota configurations are associated with several of these diseases, and with a possible pre-disease condition (more susceptible to disease development) of the host-microbiota ecosystem. These observations are reminiscent of the behavior of an ecosystem with alternative stable states (different stable configurations that can exist under identical external conditions), and we recently postulated that health, pre-disease and disease represent such alternative states. Here, our aim was to examine if alternative stable states indeed exist in the intestinal ecosystem. RESULTS: Rats were exposed to varying concentrations of DSS in order to create a wide range of mildly inflammatory conditions, in a context of diet-induced low microbiota diversity. The consequences for the intestinal microbiota were traced by 16S rRNA gene profiling over time, and inflammation of the distal colon was evaluated at sacrifice, 45 days after the last DSS treatment. The results provide the first formal experimental proof for the existence of alternative stable states in the rat intestinal ecosystem, taking both microbiota and host inflammatory status into consideration. The alternative states are host-microbiota ecosystem states rather than independent and dissociated microbiota and host states, and inflammation can prompt stable state-transition. Based on these results, we propose a conceptual model providing new insights in the interplay between host inflammatory status and microbiota status. These new insights call for innovative therapeutic strategies to cure (pre-)disease. CONCLUSIONS: We provide proof of concept showing the existence of alternative stable states in the rat intestinal ecosystem. We further propose a model which, if validated in humans, will support innovative diagnosis, therapeutic strategy, and monitoring in the treatment of chronic inflammatory conditions. This model provides a strong rationale for the application of combinatorial therapeutic strategies, targeting host and microbiota rather than only one of the two in chronic immune-mediated diseases. Video Abstract.

Prediction of surface exposed proteins in Streptococcus pyogenes, with a potential application to other Gram-positive bacteria.

The in silico prediction of bacterial surface exposed proteins is of growing interest for the rational development of vaccines and in the study of bacteria-host relationships, whether pathogenic or host beneficial. This interest is driven by the increase in the use of DNA sequencing as a major tool in the early characterization of pathogenic bacteria and, more recently, even of complex ecosystems at the host-environment interface in metagenomics approaches. Current protein localization protocols are not suited to this prediction task as they ignore the potential surface exposition of many membrane-associated proteins. Therefore, we developed a new flow scheme, SurfG+, for the processing of protein sequence data with the particular aim of identification of potentially surface exposed (PSE) proteins from Gram-positive bacteria, which was validated for Streptococcus pyogenes. The results of an exploratory case study on closely related lactobacilli of the acidophilus group suggest that the yogurt bacterium Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) dedicates a relatively important fraction of its coding capacity to secreted proteins, while the probiotic gastrointestinal (GI) tract bacteria L. johnsonii and L. gasseri appear to encode a larger variety of PSE proteins, that may play a role in the interaction with the host.

Rerouting of pyruvate metabolism during acid adaptation in Lactobacillus bulgaricus.

Lactic acid bacteria (LAB) gradually acidify their environment through the conversion of pyruvate to lactate, an essential process to regenerate NAD(+) used during glycolysis. A clear demonstration of acidification can be found in yogurt, the product of milk fermentation by the LAB Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) and Streptococcus thermophilus, where the pH falls to 4.2. Acid adaptation therefore plays an important role in the physiology of LAB. Here we present the results of a proteomic approach to reveal cellular changes associated with acid adaptation in L. bulgaricus. These results were complemented with transcription data for selected genes to show three major effects: (i) induction of the chaperones GroES, GroEL, HrcA, GrpE, DnaK, DnaJ, ClpE, ClpP, and ClpL, and the repression of ClpC; (ii) induction of genes involved in the biosynthesis of fatty acids (fabH, accC, fabI); (iii) repression of genes involved in the mevalonate pathway of isoprenoid synthesis (mvaC, mvaS). Together with changes in the expression of other genes from the local metabolic network, these results for the first time show a coherent picture of changes in gene expression expected to result in a rerouting of pyruvate metabolism to favor fatty acid biosynthesis, and thereby affect membrane fluidity.

Humans as holobionts: implications for prevention and therapy.

The human gut microbiota is increasingly recognized for its important or even decisive role in health. As it becomes clear that microbiota and host mutually affect and depend on each other in an intimate relationship, a holistic view of the gut microbiota-host association imposes itself. Ideally, a stable state of equilibrium, homeostasis, is maintained and serves health, but signs are that perturbation of this equilibrium beyond the limits of resilience can propel the system into an alternative stable state, a pre-disease state, more susceptible to the development of chronic diseases. The microbiota-host equilibrium of a large and growing proportion of individuals in Western society may represent such a pre-disease state and explain the explosive development of chronic diseases such as inflammatory bowel disease, obesity, and other inflammatory diseases. These diseases themselves represent other alternative stable states again and are therefore hard to cure. The holistic view of the microbiota-host association where feedback loops between microbiota and host are thought to maintain the system in a stable state-be it a healthy, pre-disease, or disease state-implies that integrated approaches, addressing host processes and microbiota, should be used to treat or prevent (pre-)disease.

Extensive horizontal transfer of core genome genes between two Lactobacillus species found in the gastrointestinal tract.

BACKGROUND: While genes that are conserved between related bacterial species are usually thought to have evolved along with the species, phylogenetic trees reconstructed for individual genes may contradict this picture and indicate horizontal gene transfer. Individual trees are often not resolved with high confidence, however, and in that case alternative trees are generally not considered as contradicting the species tree, although not confirming it either. Here we conduct an in-depth analysis of 401 protein phylogenetic trees inferred with varying levels of confidence for three lactobacilli from the acidophilus complex. At present the relationship between these bacteria, isolated from environments as diverse as the gastrointestinal tract (Lactobacillus acidophilus and Lactobacillus johnsonii) and yogurt (Lactobacillus delbrueckii ssp. bulgaricus), is ambiguous due to contradictory phenotypical and 16S rRNA based classifications. RESULTS: Among the 401 phylogenetic trees, those that could be reconstructed with high confidence support the 16S-rRNA tree or one alternative topology in an astonishing 3:2 ratio, while the third possible topology is practically absent. Lowering the confidence threshold for trees to be taken into consideration does not significantly affect this ratio, and therefore suggests that gene transfer may have affected as much as 40% of the core genome genes. Gene function bias suggests that the 16S rRNA phylogeny of the acidophilus complex, which indicates that L. acidophilus and L. delbrueckii ssp. bulgaricus are the closest related of these three species, is correct. A novel approach of comparison of interspecies protein divergence data employed in this study allowed to determine that gene transfer most likely took place between the lineages of the two species found in the gastrointestinal tract. CONCLUSION: This case-study reports an unprecedented level of phylogenetic incongruence, presumably resulting from extensive horizontal gene transfer. The data give a first indication of the large extent of gene transfer that may take place in the gastrointestinal tract and its accumulated effect. For future studies, our results should encourage a careful weighing of data on phylogenetic tree topology, confidence and distribution to conclude on the absence or presence and extent of horizontal gene transfer.

Horizontal Gene Transfer and Ecosystem Function Dynamics.

Horizontal gene transfer can provide bacteria with new functions that confer an important competitive advantage, and is therefore likely to affect the dynamics of bacterial ecosystems. Two studies by Wolfe et al. and Bonham et al. prepare the way to study this hypothesis in a model ecosystem with reproducible properties.

Unprecedented large inverted repeats at the replication terminus of circular bacterial chromosomes suggest a novel mode of chromosome rescue.

The first Lactobacillus delbrueckii ssp. bulgaricus genome sequence revealed the presence of a very large inverted repeat (IR), a DNA sequence arrangement which thus far seemed inconceivable in a non-manipulated circular bacterial chromosome, at the replication terminus. This intriguing observation prompted us to investigate if similar IRs could be found in other bacteria. IRs with sizes varying from 38 to 76 kbp were found at the replication terminus of all 5 L. delbrueckii ssp. bulgaricus chromosomes analysed, but in none of 1373 other chromosomes. They represent the first naturally occurring very large IRs detected in circular bacterial genomes. A comparison of the L. bulgaricus replication terminus regions and the corresponding regions without IR in 5 L. delbrueckii ssp. lactis genomes leads us to propose a model for the formation and evolution of the IRs. The DNA sequence data are consistent with a novel model of chromosome rescue after premature replication termination or irreversible chromosome damage near the replication terminus, involving mechanisms analogous to those proposed in the formation of very large IRs in human cancer cells. We postulate that the L. delbrueckii ssp. bulgaricus-specific IRs in different strains derive from a single ancestral IR of at least 93 kbp.

Functional Comparison of Bacteria from the Human Gut and Closely Related Non-Gut Bacteria Reveals the Importance of Conjugation and a Paucity of Motility and Chemotaxis Functions in the Gut Environment.

The human GI tract is a complex and still poorly understood environment, inhabited by one of the densest microbial communities on earth. The gut microbiota is shaped by millennia of evolution to co-exist with the host in commensal or symbiotic relationships. Members of the gut microbiota perform specific molecular functions important in the human gut environment. This can be illustrated by the presence of a highly expanded repertoire of proteins involved in carbohydrate metabolism, in phase with the large diversity of polysaccharides originating from the diet or from the host itself that can be encountered in this environment. In order to identify other bacterial functions that are important in the human gut environment, we investigated the distribution of functional groups of proteins in a group of human gut bacteria and their close non-gut relatives. Complementary to earlier global comparisons between different ecosystems, this approach should allow a closer focus on a group of functions directly related to the gut environment while avoiding functions related to taxonomically divergent microbiota composition, which may or may not be relevant for gut homeostasis. We identified several functions that are overrepresented in the human gut bacteria which had not been recognized in a global approach. The observed under-representation of certain other functions may be equally important for gut homeostasis. Together, these analyses provide us with new information about this environment so critical to our health and well-being.

Combining selected immunomodulatory Propionibacterium freudenreichii and Lactobacillus delbrueckii strains: Reverse engineering development of an anti-inflammatory cheese.

SCOPE: Inflammatory bowel disease (IBD) constitutes a growing public health concern in western countries. Bacteria with anti-inflammatory properties are lacking in the dysbiosis accompanying IBD. Selected strains of probiotic bacteria with anti-inflammatory properties accordingly alleviate symptoms and enhance treatment of ulcerative colitis in clinical trials. Such properties are also found in selected strains of dairy starters such as Propionibacterium freudenreichii and Lactobacillus delbrueckii (Ld). We thus investigated the possibility to develop a fermented dairy product, combining both starter and probiotic abilities of both lactic acid and propionic acid bacteria, designed to extend remissions in IBD patients. METHODS AND RESULTS: We developed a single-strain Ld-fermented milk and a two-strain P. freudenreichii and Ld-fermented experimental pressed cheese using strains previously selected for their anti-inflammatory properties. Consumption of these experimental fermented dairy products protected mice against trinitrobenzenesulfonic acid induced colitis, alleviating severity of symptoms, modulating local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages. As a control, the corresponding sterile dairy matrix failed to afford such protection. CONCLUSION: This work reveals the probiotic potential of this bacterial mixture, in the context of fermented dairy products. It opens new perspectives for the reverse engineering development of anti-inflammatory fermented foods designed for target populations with IBD, and has provided evidences leading to an ongoing pilot clinical study in ulcerative colitis patients.

Genome Sequence of Lactobacillus delbrueckii subsp. lactis CNRZ327, a Dairy Bacterium with Anti-Inflammatory Properties.

Lactobacillus delbrueckii subsp. lactis CNRZ327 is a dairy bacterium with anti-inflammatory properties both in vitro and in vivo. Here, we report the genome sequence of this bacterium, which appears to contain no less than 215 insertion sequence (IS) elements, an exceptionally high number regarding the small genome size of the strain.

Local and systemic immune mechanisms underlying the anti-colitis effects of the dairy bacterium Lactobacillus delbrueckii.

Several probiotic bacteria have been proposed for treatment or prevention of inflammatory bowel diseases (IBD), showing a protective effect in animal models of experimental colitis and for some of them also in human clinical trials. While most of these probiotic bacteria are isolated from the digestive tract, we recently reported that a Lactobacillus strain isolated from cheese, L. delbrueckii subsp. lactis CNRZ327 (Lb CNRZ327), also possesses anti-inflammatory effects in vitro and in vivo, demonstrating that common dairy bacteria may be useful in the treatment or prevention of IBD. Here, we studied the mechanisms underlying the protective effects of Lb CNRZ327 in vivo, in a mouse dextran sodium sulfate (DSS) colitis model. During colitis, Lb CNRZ327 modulated the production of TGF-ß, IL-6, and IL-12 in colonic tissue and of TGF-ß and IL-6 in the spleen, and caused an expansion of CD4+Foxp3+ regulatory T cells in the cecal lymph nodes. Moreover, a strong tendency to CD4+Foxp3+ expansion was also observed in the spleen. The results of this study for the first time show that orally administered dairy lactobacilli can not only modulate mucosal but also systemic immune responses and constitute an effective treatment of IBD.

Genome Sequence of "Candidatus Arthromitus" sp. Strain SFB-Mouse-NL, a Commensal Bacterium with a Key Role in Postnatal Maturation of Gut Immune Functions.

"Candidatus Arthromitus" sp. strain SFB-mouse-NL (SFB, segmented filamentous bacteria) is a commensal bacterium necessary for inducing the postnatal maturation of homeostatic innate and adaptive immune responses in the mouse gut. Here, we report the genome sequence of this bacterium, which sets it apart from earlier sequenced mouse SFB isolates.

High-throughput system for the presentation of secreted and surface-exposed proteins from Gram-positive bacteria in functional metagenomics studies.

Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli-B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.

Properties of probiotic bacteria explored by proteomic approaches.

The study of health-beneficial effects that probiotic bacteria can exert on humans and animals is at its beginning. Pending scientific questions include the identification of molecular markers of the health-promoting activity of specific strains, which may be used to select novel probiotic strains and to gain understanding of the mechanisms underlying their effects. In that perspective, the role of bacterial proteins must be evaluated, placing proteomics-based approaches at the core of the field. Until now, most proteomic analyses focused on the dynamics of abundant cytoplasmic proteins during adaptation of bacteria to conditions mimicking the gastro-intestinal tract environment. The development of in silico and experimental procedures allowing identification and quantification of surface-exposed and secreted proteins should boost our understanding of bacteria-host crosstalk.