Unique Sugar Metabolic Pathways of Bifidobacteria

Bifidobacteria have many beneficial effects for human health. The gastrointestinal tract, where natural colonization of bifidobacteria occurs, is an environment poor in nutrition and oxygen. Therefore, bifidobacteria have many unique glycosidases, transporters, and metabolic enzymes for sugar fermentation to utilize diverse carbohydrates that are not absorbed by host humans and animals. They have a unique, effective central fermentative pathway called bifid shunt. Recently, a novel metabolic pathway that utilizes both human milk oligosaccharides and host glycoconjugates was found. The galacto-N-biose/lacto-N-biose I metabolic pathway plays a key role in colonization in the infant gastrointestinal tract. These pathways involve many unique enzymes and proteins. This review focuses on their molecular mechanisms, as revealed by biochemical and crystallographic studies.     

Carbohydrate metabolism in Bifidobacteria

Members of the genus Bifidobacterium can be found as components of the gastrointestinal microbiota, and are believed to play an important role in maintaining and promoting human health by eliciting a number of beneficial properties. Bifidobacteria can utilize a diverse range of dietary carbohydrates that escape degradation in the upper parts of the intestine, many of which are plant-derived oligo- and polysaccharides. The gene content of a bifidobacterial genome reflects this apparent metabolic adaptation to a complex carbohydrate-rich gastrointestinal tract environment as it encodes a large number of predicted carbohydrate-modifying enzymes. Different bifidobacterial strains may possess different carbohydrate utilizing abilities, as established by a number of studies reviewed here. Carbohydrate-degrading activities described for bifidobacteria and their relevance to the deliberate enhancement of number and/or activity of bifidobacteria in the gut are also discussed in this review.     

Diversity, vitality and activities of intestinal lactic acid bacteria and bifidobacteria assessed by molecular approaches

While lactic acid bacteria and bifidobacteria have been scientifically important for over a century, many of these are marketed today as probiotics and have become a valuable and rapidly expanding sector of the food market that is leading functional foods in many countries. The human gastro-intestinal tract with its various compartments and complex microbiota is the primary target of most of these functional foods containing lactic acid bacteria and bifidobacteria (LAB&B). In addition, their use as vectors for delivery of molecules with therapeutic value to the host via the intestinal tract is being studied. This review focuses on molecular approaches for the investigation of the diversity of lactic acid bacteria and bifidobacteria in the human intestine, as well as tracking of probiotic bacteria within this complex ecosystem. Moreover, methodologies to determine the viability of the lactic acid bacteria and bifidobacteria and molecular approaches to study the mechanisms by which they adapt, establish and interact with the human host via the digestive tract, are described.     

Live/dead state is not the factor influencing adhesion ability of Bifidobacterium animalis KLDS2.0603

Two essential requirements for probiotic bifidobacteria are that they be “live” and have “colonization” ability, following FAO/WHO guideline recommendations. The amount of research on the adhesion ability of bifidobacteria compares poorly with that of other probiotic bacteria, such as lactobacilli. The aim of the present study was to determine how gastrointestinal conditions affect the adhesion ability of bifidobacteria, and to investigate the relationship between the adhesion ability and the live/dead state of bifidobacteria. The adhesion ability of Bifidobacterium animalis KLDS2.0603 that had been subjected to the digestive enzymes, pepsin, trypsin, and proteinase K, was decreased significantly, but these treatments did not significantly change the strain’s survival rates, which were 98.78%, 97.60%, and 97.63% respectively. B. animalis KLDS2.0603 subjected to LiCl retained its adhesion ability but had a lower survival rate (59.28%) than the control group (P<0.01). B. animalis KLDS 2.0603 subjected to sodium metaperiodate exhibited higher adhesion ability than the control group (P<0.01), but the bacterial cells were killed totally. The results of transmission electron microscopy and laser scanning confocal microscopy showed that live/dead state of bifidobacteria was not one of the main factors that affected the adhesion ability of bifidobacteira, and that the substances affecting the adhesion ability of bifidobacteria were on the outer surface layer of the bifidobacterial cells. Our results also indicated that the substances related to the adhesion ability of bifidobacteria are proteinaceous. The above results will help us to understand the adhesion and colonization processes of bifidobacteria in the human gastrointestinal tract.     

Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens

The gastrointestinal tract is a complex ecosystem that associates a resident microbiota and cells of various phenotypes lining the epithelial wall expressing complex metabolic activities. The resident microbiota in the digestive tract is a heterogeneous microbial ecosystem containing up to 1 x 10(14) colony-forming units (CFUs) of bacteria. The intestinal microbiota plays an important role in normal gut function and maintaining host health. The host is protected from attack by potentially harmful microbial microorganisms by the physical and chemical barriers created by the gastrointestinal epithelium. The cells lining the gastrointestinal epithelium and the resident microbiota are two partners that properly and/or synergistically function to promote an efficient host system of defence. The gastrointestinal cells that make up the epithelium, provide a physical barrier that protects the host against the unwanted intrusion of microorganisms into the gastrointestinal microbiota, and against the penetration of harmful microorganisms which usurp the cellular molecules and signalling pathways of the host to become pathogenic. One of the basic physiological functions of the resident microbiota is that it functions as a microbial barrier against microbial pathogens. The mechanisms by which the species of the microbiota exert this barrier effect remain largely to be determined. There is increasing evidence that lactobacilli and bifidobacteria, which inhabit the gastrointestinal microbiota, develop antimicrobial activities that participate in the host's gastrointestinal system of defence. The objective of this review is to analyze the in vitro and in vivo experimental and clinical studies in which the antimicrobial activities of selected lactobacilli and bifidobacteria strains have been documented.     

Diversity,ecology and intestinal function of bifidobacteria

The human gastrointestinal tract represents an environment which is a densely populated home for a microbiota that has evolved to positively contribute to host health. At birth the essentially sterile gastrointestinal tract (GIT) is rapidly colonized by microorganisms that originate from the mother and the surrounding environment. Within a short timeframe a microbiota establishes within the (breastfed) infant's GIT where bifidobacteria are among the dominant members, although their numerical dominance disappears following weaning. The numerous health benefits associated with bifidobacteria, and the consequent commercial relevance resulting from their incorporation into functional foods, has led to intensified research aimed at the molecular understanding of claimed probiotic attributes of this genus. In this review we provide the current status on the diversity and ecology of bifidobacteria. In addition, we will discuss the molecular mechanisms that allow this intriguing group of bacteria to colonize and persist in the GIT, so as to facilitate interaction with its host.     

Identification of N-acetylhexosamine 1-kinase in the complete lacto-N-biose I/galacto-N-biose metabolic pathway in Bifidobacterium longum

We have determined the functions of the enzymes encoded by the lnpB, lnpC, and lnpD genes, located downstream of the lacto-N-biose phosphorylase gene (lnpA), in Bifidobacterium longum JCM1217. The lnpB gene encodes a novel kinase, N-acetylhexosamine 1-kinase, which produces N-acetylhexosamine 1-phosphate; the lnpC gene encodes UDP-glucose hexose 1-phosphate uridylyltransferase, which is also active on N-acetylhexosamine 1-phosphate; and the lnpD gene encodes a UDP-glucose 4-epimerase, which is active on both UDP-galactose and UDP-N-acetylgalactosamine. These results suggest that the gene operon lnpABCD encodes a previously undescribed lacto-N-biose I/galacto-N-biose metabolic pathway that is involved in the intestinal colonization of bifidobacteria and that utilizes lacto-N-biose I from human milk oligosaccharides or galacto-N-biose from mucin sugars.     

Studies on Streptococci

The population levels of bacteria in the contents and the walls of the gastrointestinal tract of gnotobiotic rats inoculated with lactic acid bacteria (streptococci, lactobacilli, and bifidobacteria) from humans and rats were determined. Lactobacilli as well as streptococci isolated from rats colonized in the digestive tracts of the gnotobiotic rats at a high population level, characteristically highest in the stomach. On the other hand, in the rats inoculated with human lactic acid bacteria, streptococci were dominant in the lower tract. The human lactobacilli or bifidobacteria did not colonize when the organisms in each genus were inoculated together with streptococci. However, when all three genera were inoculated together, lactobacilli and bifidobacteria colonized. Observations on the species of streptococci showed that the intestinal type of streptococci was found to colonize at a high population level, but the oral type was not. Strains of the same genus of lactic acid bacteria from humans and from rats showed different colonization patterns.     

Inflammation and gastrointestinal Candida colonization

Candida organisms commonly colonize the human gastrointestinal tract as a component of the resident microbiota. Their presence is generally benign. Recent studies, however, show that high level Candida colonization is associated with several diseases of the gastrointestinal tract. Further, results from animal models argue that Candida colonization delays healing of inflammatory lesions and that inflammation promotes colonization. These effects may create a vicious cycle in which low-level inflammation promotes fungal colonization and fungal colonization promotes further inflammation. Both inflammatory bowel disease and gastrointestinal Candida colonization are associated with elevated levels of the pro-inflammatory cytokine IL-17. Therefore, effects on IL-17 levels may underlie the ability of Candida colonization to enhance inflammation. Because Candida is a frequent colonizer, these effects have the potential to impact many people.     

Bacteria and mucosal inflammation of the gut: lessons from Helicobacter pylori

The human gastrointestinal tract is colonized by an abundance of bacteria, which are in constant interaction with the epithelial lining usually leading to an intricate balance between tolerance and immunological response. There is ample evidence that the abundant presence of bacteria thus plays a role in the maintenance of human health, as well as in the induction of chronic inflammatory diseases of the gastrointestinal tract. Research in this field is, however, considerably hampered by the abundance of bacterial species, many of which have not even been characterized, and are difficult to culture specifically. These important limitations may to some extent be overcome by recent molecular biologic methods. Furthermore however, the adherent mucosal flora may differ largely from the luminal flora and that in excreta. These characteristics do not pertain to Helicobacter pylori, which generally colonizes the human stomach as a single strain with stable characteristics. Such colonization is stable throughout life, but can be treated. Furthermore, the association with chronic gastritis is very strong. For these reasons, H. pylori serves as an excellent model for the understanding of the processes involved in bacterial colonization and host response including mediation of immunoregulation, and the mechanisms by which this response can lead to disease.     

Lessons from the genomes of bifidobacteria

The gut microbiota is a complex ecosystem composed of hundreds of different bacterial species that altogether play an important role in the physiology of their host. In the past few years the complete genome sequence of a number of bacterial strains isolated from the human gastrointestinal tract has been established including that of Bifidobacterium longum NCC2705 isolated from the feces of a healthy infant. Bifidobacteria are among the first species to colonise the human gastrointestinal tract and as such are believed to play an important role in gut homeostasis and normal development. The genome sequence of NCC2705 has revealed a number of features that suggest how this bacterium has adapted to its environment and that could help understanding how it interacts with its host. Here, we review general features of bifidobacteria and illustrate how genome-based approaches can help us better understand the biology of these organisms.     

DNA damage and reactive nitrogen species are barriers to Vibrio cholerae colonization of the infant mouse intestine

Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. Here, we show that V. cholerae requires at least two types of DNA repair systems to efficiently compete for colonization of the infant mouse intestine. These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract. Agreeing with this, we show that passage through the murine gut increases the mutation frequency of V. cholerae compared to liquid culture passage. Our genetic analysis identifies known and novel defense enzymes required for detoxifying reactive nitrogen species (but not reactive oxygen species) that are also required for V. cholerae to efficiently colonize the infant mouse intestine, pointing to reactive nitrogen species as the potential cause of DNA damage. We demonstrate that potential reactive nitrogen species deleterious for V. cholerae are not generated by host inducible nitric oxide synthase (iNOS) activity and instead may be derived from acidified nitrite in the stomach. Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media. Moreover, we demonstrate that neutralizing stomach acid rescues the colonization defect of the DNA repair and reactive nitrogen species defense defective mutants suggesting a common defense pathway for these mutants.     

Barcoded pyrosequencing reveals that consumption of galactooligosaccharides results in a highly specific bifidogenic response in humans

Prebiotics are selectively fermented ingredients that allow specific changes in the gastrointestinal microbiota that confer health benefits to the host. However, the effects of prebiotics on the human gut microbiota are incomplete as most studies have relied on methods that fail to cover the breadth of the bacterial community. The goal of this research was to use high throughput multiplex community sequencing of 16S rDNA tags to gain a community wide perspective of the impact of prebiotic galactooligosaccharide (GOS) on the fecal microbiota of healthy human subjects. Fecal samples from eighteen healthy adults were previously obtained during a feeding trial in which each subject consumed a GOS-containing product for twelve weeks, with four increasing dosages (0, 2.5, 5, and 10 gram) of GOS. Multiplex sequencing of the 16S rDNA tags revealed that GOS induced significant compositional alterations in the fecal microbiota, principally by increasing the abundance of organisms within the Actinobacteria. Specifically, several distinct lineages of Bifidobacterium were enriched. Consumption of GOS led to five- to ten-fold increases in bifidobacteria in half of the subjects. Increases in Firmicutes were also observed, however, these changes were detectable in only a few individuals. The enrichment of bifidobacteria was generally at the expense of one group of bacteria, the Bacteroides. The responses to GOS and the magnitude of the response varied between individuals, were reversible, and were in accordance with dosage. The bifidobacteria were the only bacteria that were consistently and significantly enriched by GOS, although this substrate supported the growth of diverse colonic bacteria in mono-culture experiments. These results suggest that GOS can be used to enrich bifidobacteria in the human gastrointestinal tract with remarkable specificity, and that the bifidogenic properties of GOS that occur in vivo are caused by selective fermentation as well as by competitive interactions within the intestinal environment.     

Nutritional influences on the gut microbiota and the consequences for gastrointestinal health

The human colonic microbiota degrades dietary substrates that are indigestible in the upper GIT (gastrointestinal tract), releasing bacterial metabolites, some of which are important for gut health. Advances in molecular biology techniques have facilitated detailed analyses of the composition of the bacterial community resident in the lower GIT. Such analyses have indicated that more than 500 different bacterial species colonize an individual, and that, although there is much functional consistency in the resident bacterial groups, there is considerable inter-individual variation at the species/strain level. The bacterial community develops during early childhood until it reaches an adult-like composition. Whereas colonization and host factors influence the species composition, dietary factors also have an important impact, with specific bacterial groups changing in response to specific dietary interventions. Since bacterial species have different metabolic activities, specific diets have various consequences for health, dependent on the effect exerted on the bacterial population.     

Genomic Overview and Biological Functions of Exopolysaccharide Biosynthesis in Bifidobacterium spp.

For many years, bacterial exopolysaccharides (EPS) have received considerable scientific attention, mainly due to their contribution to biofilm formation and, above all, because EPS are potential virulence factors. In recent times, interest in EPS research has enjoyed a welcome boost thanks to the discovery of their ability to mediate communication processes with their surrounding environment and to their contribution to host health maintenance. In this review, we provide a fresh perspective on the genetics and activity of these polymers in members of the Bifidobacterium genus, a common gut inhabitant of humans and animals that has been associated with several health-promoting effects. Bifidobacteria can use EPS to protect themselves against the harsh conditions of the gastrointestinal tract, thus improving their persistence in the host. Indeed, the relevant function of EPS for bifidobacteria is underlined by the fact that most genomes sequenced until now contain genes related to EPS biosynthesis. A high interspecies variability in the number of genes and structural organization is denoted among species/subspecies; thus, eps clusters in this genus do not display a consensus genetic architecture. Their different G+C content compared to that of the whole genome suggests that eps genes have been acquired by horizontal transfer. From the host perspective, EPS-producing bifidobacteria are able to trigger both innate and adaptive immune responses, and they are able to modulate the composition and activity of the gut microbiota. Thus, these polymers seem to be critical in understanding the physiology of bifidobacteria and their interaction with the host.     

in silico analyses of metabolic pathway and protein interaction network for identification of next gen therapeutic targets in Chlamydophila pneumoniae

Chlamydophila pneumoniae, the causative agent of chronic obstructive pulmonary disease (COPD), is presently the fifth mortality causing chronic disease in the world. The understanding of disease and treatment options are limited represents a severe concern and a need for better therapeutics. With the advancements in the field of complete genome sequencing and computational approaches development have lead to metabolic pathway analysis and protein-protein interaction network which provides vital evidence to the protein function and has been appropriate to the fields such as systems biology and drug discovery. Protein interaction network analysis allows us to predict the most potential drug targets among large number of the non-homologous proteins involved in the unique metabolic pathway. A computational comparative metabolic pathway analysis of the host H. sapiens and the pathogen C pneumoniae AR39 has been carried out at three level analyses. Firstly, metabolic pathway analysis was performed to identify unique metabolic pathways and non-homologous proteins were identified. Secondly, essentiality of the proteins was checked, where these proteins contribute to the growth and survival of the organism. Finally these proteins were further subjected to predict protein interaction networks. Among the total 65 pathways in the C pneumoniae AR39 genome 10 were identified as the unique metabolic pathways which were not found in the human host, 32 enzymes were predicted as essential and these proteins were considered for protein interaction analysis, later using various criteria''s we have narrowed down to prioritize ribonucleotide-diphosphate reductase subunit beta as a potential drug target which facilitate for the successful entry into drug designing.     

Gastrointestinal Colonization of Fungi

The human gastrointestinal tract is colonized with trillions of commensal microbes, and disturbances in the equilibrium of the gut microbiota have now been shown to be associated with a number of human diseases. Fungi, particularly Candida spp., are normal, harmless residents of the human gut, but in certain instances can cause invasive infections and inflammatory disorders. This paper will review the fungal diversity in the human gut, host and fungal factors that regulate GI colonization, and how these factors play into the pathogenesis of human disease.     

The HOG Pathway Is Critical for the Colonization of the Mouse Gastrointestinal Tract by Candida albicans

The opportunistic pathogen Candida albicans is a frequent inhabitant of the human gastrointestinal tract where it usually behaves as a harmless commensal. In this particular niche, it needs to adapt to the different micro environments that challenge its survival within the host. In order to determine those factors involved in gut adaptation, we have used a gastrointestinal model of colonization in mouse to trace the behaviour of fungal cells. We have developed a genetic labelling system based on the complementary spectral properties of the fluorescent proteins GFP and a new C. albicans codon-adapted RFP (dTOM2) that allow a precise quantification of the fungal population in the gut via standard in vitro cultures or flow cytometry. This methodology has allowed us to determine the role of the three MAP kinase pathways of C. albicans (mediated by the MAPK Mkc1, Cek1 or Hog1) in mouse gut colonization via competitive assays with MAPK pathway mutants and their isogenic wild type strain. This approach reveals the signalling through HOG pathway as a critical factor influencing the establishment of C. albicans in the mouse gut. Less pronounced effects for mkc1 or cek1 mutants were found, only evident after 2–3 weeks of colonization. We have also seen that hog1 mutants is defective in adhesion to the gut mucosa and sensitive to bile salts. Finally, we have developed a genetic strategy for the in vivo excision (tetracycline-dependent) of any specific gene during the course of colonization in this particular niche, allowing the analysis of its role during gut colonization.     

Cj1386 is an ankyrin-containing protein involved in heme trafficking to catalase in Campylobacter jejuni

Campylobacter jejuni, a microaerophilic bacterium, is the most frequent cause of human bacterial gastroenteritis. C. jejuni is exposed to harmful reactive oxygen species (ROS) produced during its own normal metabolic processes and during infection from the host immune system and from host intestinal microbiota. These ROS will damage DNA and proteins and cause peroxidation of lipids. Consequently, identifying ROS defense mechanisms is important for understanding how Campylobacter survives this environmental stress during infection. Construction of a ΔCj1386 isogenic deletion mutant and phenotypic assays led to its discovery as a novel oxidative stress defense gene. The ΔCj1386 mutant has an increased sensitivity toward hydrogen peroxide. The Cj1386 gene is located directly downstream from katA (catalase) in the C. jejuni genome. A ΔkatAΔ Cj1386 double deletion mutant was constructed and exhibited a sensitivity to hydrogen peroxide similar to that seen in the ΔCj1386 and ΔkatA single deletion mutants. This observation suggests that Cj1386 may be involved in the same detoxification pathway as catalase. Despite identical KatA abundances, catalase activity assays showed that the ΔCj1386 mutant had a reduced catalase activity relative to that of wild-type C. jejuni. Heme quantification of KatA protein from the ΔCj1386 mutant revealed a significant decrease in heme concentration. This indicates an important role for Cj1386 in heme trafficking to KatA within C. jejuni. Interestingly, the ΔCj1386 mutant had a reduced ability to colonize the ceca of chicks and was outcompeted by the wild-type strain for colonization of the gastrointestinal tract of neonate piglets. These results indicate an important role for Cj1386 in Campylobacter colonization and pathogenesis.