Functional Metagenomic Investigations of the Human Intestinal Microbiota

The human intestinal microbiota encode multiple critical functions impacting human health, including metabolism of dietary substrate, prevention of pathogen invasion, immune system modulation, and provision of a reservoir of antibiotic resistance genes accessible to pathogens. The complexity of this microbial community, its recalcitrance to standard cultivation, and the immense diversity of its encoded genes has necessitated the development of novel molecular, microbiological, and genomic tools. Functional metagenomics is one such culture-independent technique, used for decades to study environmental microorganisms, but relatively recently applied to the study of the human commensal microbiota. Metagenomic functional screens characterize the functional capacity of a microbial community, independent of identity to known genes, by subjecting the metagenome to functional assays in a genetically tractable host. Here we highlight recent work applying this technique to study the functional diversity of the intestinal microbiota, and discuss how an approach combining high-throughput sequencing, cultivation, and metagenomic functional screens can improve our understanding of interactions between this complex community and its human host

From screen to structure with a harvestable microfluidic device

Microfluidic crystallization using the Crystal Former improves the identification of initial crystallization conditions relative to screening via vapour diffusion

Oral supplementation of healthy adults with 2'-O-fucosyllactose and lacto-N-neotetraose is well tolerated and shifts the intestinal microbiota

The gut microbiota has been established as an important player influencing many aspects of human physiology. Breast milk, the first diet for an infant, contains human milk oligosaccharides (HMO) that shape the infant's gut microbiota by selectively stimulating the growth of specific bacteria, especially bifidobacteria. In addition to their bifidogenic activity, the ability of HMO to modulate immune function and the gut barrier makes them prime candidates to restore a beneficial microbiota in dysbiotic adults and provide health benefits. We conducted a parallel, double-blind, randomised, placebo-controlled, HMO-supplementation study in 100 healthy, adult volunteers, consuming chemically produced 2'-O-fucosyllactose (2'FL) and/or lacto-N-neotetraose (LNnT) at various daily doses and mixes or placebo for 2 weeks. All participants completed the study without premature discontinuation. Supplementation of 2'FL and LNnT at daily doses up to 20 g was shown to be safe and well tolerated, as assessed using the gastrointestinal symptoms rating scale. 16S rRNA sequencing analysis showed that HMO supplementation specifically modified the adult gut microbiota with the primary impact being substantial increases in relative abundance of Actinobacteria and Bifidobacterium in particular and a reduction in relative abundance of Firmicutes and Proteobacteria. This study provides the first set of data on safety, tolerance and impact of HMO on the adult gut microbiota. Collectively, the results from this study show that supplementing the diet with HMO is a valuable strategy to shape the human gut microbiota and specifically promote the growth of beneficial bifidobacteria

Simulating Serial-Target Antibacterial Drug Synergies Using Flux Balance Analysis

Flux balance analysis (FBA) is an increasingly useful approach for modeling the behavior of metabolic systems. However, standard FBA modeling of genetic knockouts cannot predict drug combination synergies observed between serial metabolic targets, even though such synergies give rise to some of the most widely used antibiotic treatments. Here we extend FBA modeling to simulate responses to chemical inhibitors at varying concentrations, by diverting enzymatic flux to a waste reaction. This flux diversion yields very similar qualitative predictions to prior methods for single target activity. However, we find very different predictions for combinations, where flux diversion, which mimics the kinetics of competitive metabolic inhibitors, can explain serial target synergies between metabolic enzyme inhibitors that we confirmed in Escherichia coli cultures. FBA flux diversion opens the possibility for more accurate genome-scale predictions of drug synergies, which can be used to suggest treatments for infections and other diseases

Human Intestinal Cells Modulate Conjugational Transfer of Multidrug Resistance Plasmids between Clinical Escherichia coli Isolates.

Bacterial conjugation in the human gut microbiota is believed to play a major role in the dissemination of antibiotic resistance genes and virulence plasmids. However, the modulation of bacterial conjugation by the human host remains poorly understood and there is a need for controlled systems to study this process. We established an in vitro co-culture system to study the interaction between human intestinal cells and bacteria. We show that the conjugation efficiency of a plasmid encoding an extended spectrum beta-lactamase is reduced when clinical isolates of Escherichia coli are co-cultured with human intestinal cells. We show that filtered media from co-cultures contain a factor that reduces conjugation efficiency. Protease treatment of the filtered media eliminates this inhibition of conjugation. This data suggests that a peptide or protein based factor is secreted on the apical side of the intestinal cells exposed to bacteria leading to a two-fold reduction in conjugation efficiency. These results show that human gut epithelial cells can modulate bacterial conjugation and may have relevance to gene exchange in the gut

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery