Mary O’Connell Motherway

Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection

Bifidobacteria comprise a significant proportion of the human gut microbiota. Several bifidobacterial strains are currently used as therapeutic interventions, claiming various health benefits by acting as probiotics. However, the precise mechanisms by which they maintain habitation within their host and consequently provide these benefits are not fully understood. Here we show that Bifidobacterium breve UCC2003 produces a cell surface-associated exopolysaccharide (EPS), the biosynthesis of which is directed by either half of a bidirectional gene cluster, thus leading to production of one of two possible EPSs. Alternate transcription of the two opposing halves of this cluster appears to be the result of promoter reorientation. Surface EPS provided stress tolerance and promoted in vivo persistence, but not initial colonization. Marked differences were observed in host immune response: strains producing surface EPS (EPS+) failed to elicit a strong immune response compared with EPS-deficient variants. Specifically, EPS production was shown to be linked to the evasion of adaptive B-cell responses. Furthermore, presence of EPS+ B. breve reduced colonization levels of the gut pathogen Citrobacter rodentium. Our data thus assigns a pivotal and beneficial role for EPS in modulating various aspects of bifidobacterial–host interaction, including the ability of commensal bacteria to remain immunologically silent and in turn provide pathogen protection. This finding enforces the probiotic concept and provides mechanistic insights into health-promoting benefits for both animal and human hosts.

Functional genome analysis of Bifidobacterium breve UCC2003 reveals type IVb tight adherence (Tad) pili as an essential and conserved host-colonization factor

Development of the human gut microbiota commences at birth, with bifidobacteria being among the first colonizers of the sterile newborn gastrointestinal tract. To date, the genetic basis of Bifidobacterium colonization and persistence remains poorly understood. Transcriptome analysis of the Bifidobacterium breve UCC2003 2.42-Mb genome in a murine colonization model revealed differential expression of a type IVb tight adherence (Tad) pilus-encoding gene cluster designated “tad2003.” Mutational analysis demonstrated that the tad2003 gene cluster is essential for efficient in vivo murine gut colonization, and immunogold transmission electron microscopy confirmed the presence of Tad pili at the poles of B. breve UCC2003 cells. Conservation of the Tad pilus-encoding locus among other B. breve strains and among sequenced Bifidobacterium genomes supports the notion of a ubiquitous pili-mediated host colonization and persistence mechanism for bifidobacteria.

Molecular dialogue between the human gut microbiota and the host: a Lactobacillus and Bifidobacterium perspective

The human gut represents a highly complex ecosystem, which is densely colonized by a myriad of microorganisms that influence the physiology, immune function and health status of the host. Among the many members of the human gut microbiota, there are microorganisms that have co-evolved with their host and that are believed to exert health-promoting or probiotic effects. Probiotic bacteria isolated from the gut and other environments are commercially exploited, and although there is a growing list of health benefits provided by the consumption of such probiotics, their precise mechanisms of action have essentially remained elusive. Genomics approaches have provided exciting new opportunities for the identification of probiotic effector molecules that elicit specific responses to influence the physiology and immune function of their human host. In this review, we describe the current understanding of the intriguing relationships that exist between the human gut and key members of the gut microbiota such as bifidobacteria and lactobacilli, discussed here as prototypical groups of probiotic microorganisms.

Role of sortase-dependent pili of Bifidobacterium bifidum PRL2010 in modulating bacterium–host interactions

Bifidobacteria represent one of the dominant groups of microorganisms colonizing the human infant intestine. Commensal bacteria that interact with a eukaryotic host are believed to express adhesive molecules on their cell surface that bind to specific host cell receptors or soluble macromolecules. Whole-genome transcription profiling of Bifidobacterium bifidum PRL2010, a strain isolated from infant stool, revealed a small number of commonly expressed extracellular proteins, among which were genes that specify sortase-dependent pili. Expression of the coding sequences of these B. bifidum PRL2010 appendages in nonpiliated Lactococcus lactis enhanced adherence to human enterocytes through extracellular matrix protein and bacterial aggregation. Furthermore, such piliated L. lactis cells evoked a higher TNF-α response during murine colonization compared with their nonpiliated parent, suggesting that bifidobacterial sortase-dependent pili not only contribute to adherence but also display immunomodulatory activity.

Host-microbe interactions that facilitate gut colonization by commensal bifidobacteria.

Microorganisms live in a myriad of ecological niches. The human intestine is among the most densely populated environments; here, a multitude of bacteria appear to have co-evolved to impact beneficially upon the health of their human host. The precise molecular mechanisms and signaling pathways employed by commensal bacteria, including those that facilitate colonization and persistence, remain largely unknown despite the perceived positive effects of such host-microbe interactions. In this review we discuss several fascinating relationships between the gastrointestinal tract and commensal bacteria, with particular emphasis on bifidobacteria as a prototypical group of human enteric microorganisms.

Genetic analysis and morphological identification of pilus-like structures in members of the genus Bifidobacterium

BackgroundCell surface pili in Gram positive bacteria have been reported to orchestrate the colonization of host tissues, evasion of immunity and the development of biofilms. So far, little if any information is available on the presence of pilus-like structures in human gut commensals like bifidobacteria.Results and discussionIn this report, Atomic Force Microscopy (AFM) of various bifidobacterial strains belonging to Bifidobacterium bifidum, Bifidobacterium longum subsp. longum, Bifidobacterium dentium, Bifidobacterium adolescentis and Bifidobacterium animalis subsp. lactis revealed the existence of appendages resembling pilus-like structures. Interestingly, these microorganisms harbour two to six predicted pilus gene clusters in their genome, with each organized in an operon encompassing the major pilin subunit-encoding gene (designated fim A or fim P) together with one or two minor pilin subunit-encoding genes (designated as fim B and/or fim Q), and a gene encoding a sortase enzyme (str A). Quantitative Real Time (qRT)-PCR analysis and RT-PCR experiments revealed a polycistronic mRNA, encompassing the fim A/P and fim B/Q genes, which are differentially expressed upon cultivation of bifidobacteria on various glycans.

Overcoming the restriction barrier to plasmid transformation and targeted mutagenesis in Bifidobacterium breve UCC2003.

In silico analysis of the Bifidobacterium breve UCC2003 genome predicted two distinct loci, which encode three different restriction/modification systems, each comprising a modification methylase and a restriction endonuclease. Based on sequence homology and observed protection against restriction we conclude that the first restriction endonuclease, designated BbrI, is an isoschizomer of BbeI, the second, BbrII, is a neoschizomer of SalI, while the third, BbrIII, is an isoschizomer of PstI. Expression of each of the B. breve UCC2003 methylase‐encoding genes in B. breve JCM 7017 established that BbrII and BbrIII are active and restrict incoming DNA. By exploiting knowledge on restriction/modification in B. breve UCC2003 we successfully increased the transformation efficiency to a level that allows the reliable generation of mutants by homologous recombination using a non‐replicative plasmid.

Characterization of ApuB, an Extracellular Type II Amylopullulanase from Bifidobacterium breve UCC2003

ABSTRACT The apuB gene of Bifidobacterium breve UCC2003 was shown to encode an extracellular amylopullulanase. ApuB is composed of a distinct N-terminally located α-amylase-containing domain which hydrolyzes α-1,4-glucosidic linkages in starch and related polysaccharides and a C-terminally located pullulanase-containing domain which hydrolyzes α-1,6 linkages in pullulan, allowing the classification of this enzyme as a bifunctional class II pullulanase. A knockout mutation of the apuB gene in B. breve UCC2003 rendered the resulting mutant incapable of growth in medium containing starch, amylopectin, glycogen, or pullulan as the sole carbon and energy source, confirming the crucial physiological role of this gene in starch metabolism.

Comparative genomics of the Bifidobacterium breve taxon

BackgroundBifidobacteria are commonly found as part of the microbiota of the gastrointestinal tract (GIT) of a broad range of hosts, where their presence is positively correlated with the host’s health status. In this study, we assessed the genomes of thirteen representatives of Bifidobacterium breve, which is not only a frequently encountered component of the (adult and infant) human gut microbiota, but can also be isolated from human milk and vagina.ResultsIn silico analysis of genome sequences from thirteen B. breve strains isolated from different environments (infant and adult faeces, human milk, human vagina) shows that the genetic variability of this species principally consists of hypothetical genes and mobile elements, but, interestingly, also genes correlated with the adaptation to host environment and gut colonization. These latter genes specify the biosynthetic machinery for sortase-dependent pili and exopolysaccharide production, as well as genes that provide protection against invasion of foreign DNA (i.e. CRISPR loci and restriction/modification systems), and genes that encode enzymes responsible for carbohydrate fermentation. Gene-trait matching analysis showed clear correlations between known metabolic capabilities and characterized genes, and it also allowed the identification of a gene cluster involved in the utilization of the alcohol-sugar sorbitol.ConclusionsGenome analysis of thirteen representatives of the B. breve species revealed that the deduced pan-genome exhibits an essentially close trend. For this reason our analyses suggest that this number of B. breve representatives is sufficient to fully describe the pan-genome of this species. Comparative genomics also facilitated the genetic explanation for differential carbon source utilization phenotypes previously observed in different strains of B. breve.

Metabolism of sialic acid by Bifidobacterium breve UCC2003.

ABSTRACT Bifidobacteria constitute a specific group of commensal bacteria that inhabit the gastrointestinal tracts of humans and other mammals. Bifidobacterium breve UCC2003 has previously been shown to utilize several plant-derived carbohydrates that include cellodextrins, starch, and galactan. In the present study, we investigated the ability of this strain to utilize the mucin- and human milk oligosaccharide (HMO)-derived carbohydrate sialic acid. Using a combination of transcriptomic and functional genomic approaches, we identified a gene cluster dedicated to the uptake and metabolism of sialic acid. Furthermore, we demonstrate that B. breve UCC2003 can cross feed on sialic acid derived from the metabolism of 3′-sialyllactose, an abundant HMO, by another infant gut bifidobacterial strain, Bifidobacterium bifidum PRL2010.