Sialylated Milk Oligosaccharides Promote Microbiota-Dependent Growth in Models of Infant Undernutrition.

Identifying interventions that more effectively promote healthy growth of children with undernutrition is a pressing global health goal. Analysis of human milk oligosaccharides (HMOs) from 6-month-postpartum mothers in two Malawian birth cohorts revealed that sialylated HMOs are significantly less abundant in those with severely stunted infants. To explore this association, we colonized young germ-free mice with a consortium of bacterial strains cultured from the fecal microbiota of a 6-month-old stunted Malawian infant and fed recipient animals a prototypic Malawian diet with or without purified sialylated bovine milk oligosaccharides (S-BMO). S-BMO produced a microbiota-dependent augmentation of lean body mass gain, changed bone morphology, and altered liver, muscle, and brain metabolism in ways indicative of a greater ability to utilize nutrients for anabolism. These effects were also documented in gnotobiotic piglets using the same consortium and Malawian diet. These preclinical models indicate a causal, microbiota-dependent relationship between S-BMO and growth promotion.

Cultivating healthy growth and nutrition through the gut microbiota.

Microbiota assembly is perturbed in children with undernutrition, resulting in persistent microbiota immaturity that is not rescued by current nutritional interventions. Evidence is accumulating that this immaturity is causally related to the pathogenesis of undernutrition and its lingering sequelae. Preclinical models in which human gut communities are replicated in gnotobiotic mice have provided an opportunity to identify and predict the effects of different dietary ingredients on microbiota structure, expressed functions, and host biology. This capacity sets the stage for proof-of-concept tests designed to deliberately shape the developmental trajectory and configurations of microbiota in children representing different geographies, cultural traditions, and states of health. Developing these capabilities for microbial stewardship is timely given the global health burden of childhood undernutrition, the effects of changing eating practices brought about by globalization, and the realization that affordable nutritious foods need to be developed to enhance our capacity to cultivate healthier microbiota in populations at risk for poor nutrition.

Regulators of gut motility revealed by a gnotobiotic model of diet-microbiome interactions related to travel.

To understand how different diets, the consumers' gut microbiota, and the enteric nervous system (ENS) interact to regulate gut motility, we developed a gnotobiotic mouse model that mimics short-term dietary changes that happen when humans are traveling to places with different culinary traditions. Studying animals transplanted with the microbiota from humans representing diverse culinary traditions and fed a sequence of diets representing those of all donors, we found that correlations between bacterial species abundances and transit times are diet dependent. However, the levels of unconjugated bile acids-generated by bacterial bile salt hydrolases (BSH)-correlated with faster transit, including during consumption of a Bangladeshi diet. Mice harboring a consortium of sequenced cultured bacterial strains from the Bangladeshi donor's microbiota and fed a Bangladeshi diet revealed that the commonly used cholekinetic spice, turmeric, affects gut motility through a mechanism that reflects bacterial BSH activity and Ret signaling in the ENS. These results demonstrate how a single food ingredient interacts with a functional microbiota trait to regulate host physiology.

Development of the gut microbiota and mucosal IgA responses in twins and gnotobiotic mice.

Immunoglobulin A (IgA), the major class of antibody secreted by the gut mucosa, is an important contributor to gut barrier function. The repertoire of IgA bound to gut bacteria reflects both T-cell-dependent and -independent pathways, plus glycans present on the antibody's secretory component. Human gut bacterial taxa targeted by IgA in the setting of barrier dysfunction are capable of producing intestinal pathology when isolated and transferred to gnotobiotic mice. A complex reorientation of gut immunity occurs as infants transition from passively acquired IgA present in breast milk to host-derived IgA. How IgA responses co-develop with assembly of the microbiota during this period remains poorly understood. Here, we (1) identify a set of age-discriminatory bacterial taxa whose representations define a program of microbiota assembly and maturation during the first 2 postnatal years that is shared across 40 healthy twin pairs in the USA; (2) describe a pattern of progression of gut mucosal IgA responses to bacterial members of the microbiota that is highly distinctive for family members (twin pairs) during the first several postnatal months then generalizes across pairs in the second year; and (3) assess the effects of zygosity, birth mode, and breast feeding. Age-associated differences in these IgA responses can be recapitulated in young germ-free mice, colonized with faecal microbiota obtained from two twin pairs at 6 and 18 months of age, and fed a sequence of human diets that simulate the transition from milk feeding to complementary foods. Most of these responses were robust to diet, suggesting that 'intrinsic' properties of community members play a dominant role in dictating IgA responses. The approach described can be used to define gut mucosal immune development in health and disease states and to help discover ways of repairing or preventing perturbations in this facet of host immunity.

A microbial perspective of human developmental biology.

When most people think of human development, they tend to consider only human cells and organs. Yet there is another facet that involves human-associated microbial communities. A microbial perspective of human development provides opportunities to refine our definitions of healthy prenatal and postnatal growth and to develop innovative strategies for disease prevention and treatment. Given the dramatic changes in lifestyles and disease patterns that are occurring with globalization, we issue a call for the establishment of 'human microbial observatories' designed to examine microbial community development in birth cohorts representing populations with diverse anthropological characteristics, including those undergoing rapid change.

Gut DNA viromes of Malawian twins discordant for severe acute malnutrition.

The bacterial component of the human gut microbiota undergoes a definable program of postnatal development. Evidence is accumulating that this program is disrupted in children with severe acute malnutrition (SAM) and that their persistent gut microbiota immaturity, which is not durably repaired with current ready-to-use therapeutic food (RUTF) interventions, is causally related to disease pathogenesis. To further characterize gut microbial community development in healthy versus malnourished infants/children, we performed a time-series metagenomic study of DNA isolated from virus-like particles (VLPs) recovered from fecal samples collected during the first 30 mo of postnatal life from eight pairs of mono- and dizygotic Malawian twins concordant for healthy growth and 12 twin pairs discordant for SAM. Both members of discordant pairs were sampled just before, during, and after treatment with a peanut-based RUTF. Using Random Forests and a dataset of 17,676 viral contigs assembled from shotgun sequencing reads of VLP DNAs, we identified viruses that distinguish different stages in the assembly of the gut microbiota in the concordant healthy twin pairs. This developmental program is impaired in both members of SAM discordant pairs and not repaired with RUTF. Phage plus members of the Anelloviridae and Circoviridae families of eukaryotic viruses discriminate discordant from concordant healthy pairs. These results disclose that apparently healthy cotwins in discordant pairs have viromes associated with, although not necessarily mediators, of SAM; as such, they provide a human model for delineating normal versus perturbed postnatal acquisition and retention of the gut microbiota's viral component in populations at risk for malnutrition.

Stable and robust Xi and Y transcriptomes drive cell-type-specific autosomal and Xa responses in vivo and in vitro in four human cell types.

Recent in vitro studies of human sex chromosome aneuploidy showed that the Xi ("inactive" X) and Y chromosomes broadly modulate autosomal and Xa ("active" X) gene expression in two cell types. We tested these findings in vivo in two additional cell types. Using linear modeling in CD4+ T cells and monocytes from individuals with one to three X chromosomes and zero to two Y chromosomes, we identified 82 sex-chromosomal and 344 autosomal genes whose expression changed significantly with Xi and/or Y dosage in vivo . Changes in sex-chromosomal expression were remarkably constant in vivo and in vitro across all four cell types examined. In contrast, autosomal responses to Xi and/or Y dosage were largely cell-type-specific, with up to 2.6-fold more variation than sex-chromosomal responses. Targets of the X- and Y-encoded transcription factors ZFX and ZFY accounted for a significant fraction of these autosomal responses both in vivo and in vitro . We conclude that the human Xi and Y transcriptomes are surprisingly robust and stable across the four cell types examined, yet they modulate autosomal and Xa genes - and cell function - in a cell-type-specific fashion. These emerging principles offer a foundation for exploring the wide-ranging regulatory roles of the sex chromosomes across the human body.

The human Y and inactive X chromosomes similarly modulate autosomal gene expression.

Somatic cells of human males and females have 45 chromosomes in common, including the "active" X chromosome. In males the 46th chromosome is a Y; in females it is an "inactive" X (Xi). Through linear modeling of autosomal gene expression in cells from individuals with zero to three Xi and zero to four Y chromosomes, we found that Xi and Y impact autosomal expression broadly and with remarkably similar effects. Studying sex chromosome structural anomalies, promoters of Xi- and Y-responsive genes, and CRISPR inhibition, we traced part of this shared effect to homologous transcription factors-ZFX and ZFY-encoded by Chr X and Y. This demonstrates sex-shared mechanisms by which Xi and Y modulate autosomal expression. Combined with earlier analyses of sex-linked gene expression, our studies show that 21% of all genes expressed in lymphoblastoid cells or fibroblasts change expression significantly in response to Xi or Y chromosomes.

The human inactive X chromosome modulates expression of the active X chromosome.

The "inactive" X chromosome (Xi) has been assumed to have little impact, in trans, on the "active" X (Xa). To test this, we quantified Xi and Xa gene expression in individuals with one Xa and zero to three Xis. Our linear modeling revealed modular Xi and Xa transcriptomes and significant Xi-driven expression changes for 38% (162/423) of expressed X chromosome genes. By integrating allele-specific analyses, we found that modulation of Xa transcript levels by Xi contributes to many of these Xi-driven changes (≥121 genes). By incorporating metrics of evolutionary constraint, we identified 10 X chromosome genes most likely to drive sex differences in common disease and sex chromosome aneuploidy syndromes. We conclude that human X chromosomes are regulated both in cis, through Xi-wide transcriptional attenuation, and in trans, through positive or negative modulation of individual Xa genes by Xi. The sum of these cis and trans effects differs widely among genes.

The human Y and inactive X chromosomes similarly modulate autosomal gene expression.

Somatic cells of human males and females have 45 chromosomes in common, including the "active" X chromosome. In males the 46th chromosome is a Y; in females it is an "inactive" X (Xi). Through linear modeling of autosomal gene expression in cells from individuals with zero to three Xi and zero to four Y chromosomes, we found that Xi and Y impact autosomal expression broadly and with remarkably similar effects. Studying sex-chromosome structural anomalies, promoters of Xi- and Y-responsive genes, and CRISPR inhibition, we traced part of this shared effect to homologous transcription factors - ZFX and ZFY - encoded by Chr X and Y. This demonstrates sex-shared mechanisms by which Xi and Y modulate autosomal expression. Combined with earlier analyses of sex-linked gene expression, our studies show that 21% of all genes expressed in lymphoblastoid cells or fibroblasts change expression significantly in response to Xi or Y chromosomes.

Aggregative Adherence and Intestinal Colonization by Enteroaggregative Escherichia coli Are Produced by Interactions among Multiple Surface Factors.

Enteroaggregative Escherichia coli (EAEC) bacteria are exceptional colonizers that are associated with diarrhea. The genome of EAEC strain 042, a diarrheal pathogen validated in a human challenge study, encodes multiple colonization factors. Notable among them are aggregative adherence fimbriae (AAF/II) and a secreted antiaggregation protein (Aap). Deletion of aap is known to increase adherence, autoaggregation, and biofilm formation, so it was proposed that Aap counteracts AAF/II-mediated interactions. We hypothesized that Aap sterically masks heat-resistant agglutinin 1 (Hra1), an integral outer membrane protein recently identified as an accessory colonization factor. We propose that this masking accounts for reduced in vivo colonization upon hra1 deletion and yet no colonization-associated phenotypes when hra1 is deleted in vitro. Using single and double mutants of hra1, aap, and the AAF/II structural protein gene aafA, we demonstrated that increased adherence in aap mutants occurs even when AAF/II proteins are genetically or chemically removed. Deletion of hra1 together with aap abolishes the hyperadherence phenotype, demonstrating that Aap indeed masks Hra1. The presence of all three colonization factors, however, is necessary for optimal colonization and for rapidly building stacked-brick patterns on slides and cultured monolayers, the signature EAEC phenotype. Altogether, our data demonstrate that Aap serves to mask nonstructural adhesins such as Hra1 and that optimal colonization by EAEC is mediated through interactions among multiple surface factors. IMPORTANCE Enteroaggregative Escherichia coli (EAEC) bacteria are exceptional colonizers of the human intestine and can cause diarrhea. Compared to other E. coli pathogens, little is known about the genes and pathogenic mechanisms that differentiate EAEC from harmless commensal E. coli. EAEC bacteria attach via multiple proteins and structures, including long appendages produced by assembling molecules of AafA and a short surface protein called Hra1. EAEC also secretes an antiadherence protein (Aap; also known as dispersin) which remains loosely attached to the cell surface. This report shows that dispersin covers Hra1 such that the adhesive properties of EAEC seen in the laboratory are largely produced by AafA structures. When the bacteria colonize worms, dispersin is sloughed off, or otherwise removed, such that Hra1-mediated adherence occurs. All three factors are required for optimal colonization, as well as to produce the signature EAEC stacked-brick adherence pattern. Interplay among multiple colonization factors may be an essential feature of exceptional colonizers.

Childhood undernutrition, the gut microbiota, and microbiota-directed therapeutics.

Childhood undernutrition is a major global health challenge. Although current therapeutic approaches have reduced mortality in individuals with severe disease, they have had limited efficacy in ameliorating long-term sequelae, notably stunting, immune dysfunction, and neurocognitive deficits. Recent work is providing insights about the role of impaired development of the human gut microbiota in disease pathogenesis, leading to new concepts for treatment and prevention. These findings raise intriguing basic questions about the mechanisms that direct normal gut microbial community assembly and functional maturation. Designing and implementing new microbiota-directed therapeutics for undernutrition highlights the need to simultaneously consider a variety of features of human biology as well as broader societal issues.

Effects of a gut pathobiont in a gnotobiotic mouse model of childhood undernutrition.

To model how interactions among enteropathogens and gut microbial community members contribute to undernutrition, we colonized gnotobiotic mice fed representative Bangladeshi diets with sequenced bacterial strains cultured from the fecal microbiota of two 24-month-old Bangladeshi children: one healthy and the other underweight. The undernourished donor's bacterial collection contained an enterotoxigenic Bacteroides fragilis strain (ETBF), whereas the healthy donor's bacterial collection contained two nontoxigenic strains of B. fragilis (NTBF). Analyses of mice harboring either the unmanipulated culture collections or systematically manipulated versions revealed that ETBF was causally related to weight loss in the context of its native community but not when introduced into the healthy donor's community. This phenotype was transmissible from the dams to their offspring and was associated with derangements in host energy metabolism manifested by impaired tricarboxylic acid cycle activity and decreased acyl-coenzyme A utilization. NTBF reduced ETBF's expression of its enterotoxin and mitigated the effects of ETBF on the transcriptomes of other healthy donor community members. These results illustrate how intraspecific (ETBF-NTBF) and interspecific interactions influence the effects of harboring B. fragilis.

Gut bacteria that prevent growth impairments transmitted by microbiota from malnourished children.

Undernourished children exhibit impaired development of their gut microbiota. Transplanting microbiota from 6- and 18-month-old healthy or undernourished Malawian donors into young germ-free mice that were fed a Malawian diet revealed that immature microbiota from undernourished infants and children transmit impaired growth phenotypes. The representation of several age-discriminatory taxa in recipient animals correlated with lean body mass gain; liver, muscle, and brain metabolism; and bone morphology. Mice were cohoused shortly after receiving microbiota from healthy or severely stunted and underweight infants; age- and growth-discriminatory taxa from the microbiota of the former were able to invade that of the latter, which prevented growth impairments in recipient animals. Adding two invasive species, Ruminococcus gnavus and Clostridium symbiosum, to the microbiota from undernourished donors also ameliorated growth and metabolic abnormalities in recipient animals. These results provide evidence that microbiota immaturity is causally related to undernutrition and reveal potential therapeutic targets and agents.