Maternal diet modulates the infant microbiome and intestinal Flt3L necessary for dendritic cell development and immunity to respiratory infection.

Poor maternal diet during pregnancy is a risk factor for severe lower respiratory infections (sLRIs) in the offspring, but the underlying mechanisms remain elusive. Here, we demonstrate that in mice a maternal low-fiber diet (LFD) led to enhanced LRI severity in infants because of delayed plasmacytoid dendritic cell (pDC) recruitment and perturbation of regulatory T cell expansion in the lungs. LFD altered the composition of the maternal milk microbiome and assembling infant gut microbiome. These microbial changes reduced the secretion of the DC growth factor Flt3L by neonatal intestinal epithelial cells and impaired downstream pDC hematopoiesis. Therapy with a propionate-producing bacteria isolated from the milk of high-fiber diet-fed mothers, or supplementation with propionate, conferred protection against sLRI by restoring gut Flt3L expression and pDC hematopoiesis. Our findings identify a microbiome-dependent Flt3L axis in the gut that promotes pDC hematopoiesis in early life and confers disease resistance against sLRIs.

Culture- and metagenomics-enabled analyses of the Methanosphaera genus reveals their monophyletic origin and differentiation according to genome size.

The genus Methanosphaera is a well-recognized but poorly characterized member of the mammalian gut microbiome, and distinctive from Methanobrevibacter smithii for its ability to induce a pro-inflammatory response in humans. Here we have used a combination of culture- and metagenomics-based approaches to expand the representation and information for the genus, which has supported the examination of their phylogeny and physiological capacity. Novel isolates of the genus Methanosphaera were recovered from bovine rumen digesta and human stool, with the bovine isolate remarkable for its large genome size relative to other Methanosphaera isolates from monogastric hosts. To substantiate this observation, we then recovered seven high-quality Methanosphaera-affiliated population genomes from ruminant and human gut metagenomic datasets. Our analyses confirm a monophyletic origin of Methanosphaera spp. and that the colonization of monogastric and ruminant hosts favors representatives of the genus with different genome sizes, reflecting differences in the genome content needed to persist in these different habitats.

Colonic thioguanine pro-drug: Investigation of microbiome and novel host metabolism.

Thiopurines are analogues of endogenous purines. They are pro-drugs which require the purine salvage pathway to convert them to the active drug nucleotides (TGN). These drugs are used to maintain clinical remission in patients with inflammatory bowel diseases. In our recent Gut paper, we showed that thioguanine worked quickly to improve colitis in the absence in the host animal of the key guanine salvage enzyme, hypoxanthine-guanine-phosphoribosyltransferase (HPRT). Current evidence favours the proposition that active drug delivery to the host lacking HPRT requires translocation of TGN-loaded bacteria across the inflamed mucosal barrier, and most likely delivery by phagocytosis. Alternatively, the efficacy of thioguanine in treating colitis could be mediated by modulation of the community of the microbiota in the intestine, or there are novel host pathways for conversion of the thioguanine pro-drug to TGN.

The Influence of the Microbiome on Early-Life Severe Viral Lower Respiratory Infections and Asthma-Food for Thought?

Severe viral lower respiratory infections are a major cause of infant morbidity. In developing countries, respiratory syncytial virus (RSV)-bronchiolitis induces significant mortality, whereas in developed nations the disease represents a major risk factor for subsequent asthma. Susceptibility to severe RSV-bronchiolitis is governed by gene-environmental interactions that affect the host response to RSV infection. Emerging evidence suggests that the excessive inflammatory response and ensuing immunopathology, typically as a consequence of insufficient immunoregulation, leads to long-term changes in immune cells and structural cells that render the host susceptible to subsequent environmental incursions. Thus, the initial host response to RSV may represent a tipping point in the balance between long-term respiratory health or chronic disease (e.g., asthma). The composition and diversity of the microbiota, which in humans stabilizes in the first year of life, critically affects the development and function of the immune system. Hence, perturbations to the maternal and/or infant microbiota are likely to have a profound impact on the host response to RSV and susceptibility to childhood asthma. Here, we review recent insights describing the effects of the microbiota on immune system homeostasis and respiratory disease and discuss the environmental factors that promote microbial dysbiosis in infancy. Ultimately, this knowledge will be harnessed for the prevention and treatment of severe viral bronchiolitis as a strategy to prevent the onset and development of asthma.

High Fat Diets Induce Colonic Epithelial Cell Stress and Inflammation that is Reversed by IL-22.

Prolonged high fat diets (HFD) induce low-grade chronic intestinal inflammation in mice, and diets high in saturated fat are a risk factor for the development of human inflammatory bowel diseases. We hypothesized that HFD-induced endoplasmic reticulum (ER)/oxidative stress occur in intestinal secretory goblet cells, triggering inflammatory signaling and reducing synthesis/secretion of proteins that form the protective mucus barrier. In cultured intestinal cells non-esterified long-chain saturated fatty acids directly increased oxidative/ER stress leading to protein misfolding. A prolonged HFD elevated the intestinal inflammatory cytokine signature, alongside compromised mucosal barrier integrity with a decrease in goblet cell differentiation and Muc2, a loss in the tight junction protein, claudin-1 and increased serum endotoxin levels. In Winnie mice, that develop spontaneous colitis, HFD-feeding increased ER stress, further compromised the mucosal barrier and increased the severity of colitis. In obese mice IL-22 reduced ER/oxidative stress and improved the integrity of the mucosal barrier, and reversed microbial changes associated with obesity with an increase in Akkermansia muciniphila. Consistent with epidemiological studies, our experiments suggest that HFDs are likely to impair intestinal barrier function, particularly in early life, which partially involves direct effects of free-fatty acids on intestinal cells, and this can be reversed by IL-22 therapy.

Differences down-under: alcohol-fueled methanogenesis by archaea present in Australian macropodids.

The Australian macropodids (kangaroos and wallabies) possess a distinctive foregut microbiota that contributes to their reduced methane emissions. However, methanogenic archaea are present within the macropodid foregut, although there is scant understanding of these microbes. Here, an isolate taxonomically assigned to the Methanosphaera genus (Methanosphaera sp. WGK6) was recovered from the anterior sacciform forestomach contents of a Western grey kangaroo (Macropus fuliginosus). Like the human gut isolate Methanosphaera stadtmanae DSMZ 3091(T), strain WGK6 is a methylotroph with no capacity for autotrophic growth. In contrast, though with the human isolate, strain WGK6 was found to utilize ethanol to support growth, but principally as a source of reducing power. Both the WGK6 and DSMZ 3091(T) genomes are very similar in terms of their size, synteny and G:C content. However, the WGK6 genome was found to encode contiguous genes encoding putative alcohol and aldehyde dehydrogenases, which are absent from the DSMZ 3091(T) genome. Interestingly, homologs of these genes are present in the genomes for several other members of the Methanobacteriales. In WGK6, these genes are cotranscribed under both growth conditions, and we propose the two genes provide a plausible explanation for the ability of WGK6 to utilize ethanol for methanol reduction to methane. Furthermore, our in vitro studies suggest that ethanol supports a greater cell yield per mol of methane formed compared to hydrogen-dependent growth. Taken together, this expansion in metabolic versatility can explain the persistence of these archaea in the kangaroo foregut, and their abundance in these 'low-methane-emitting' herbivores.

Towards an integrated understanding of the therapeutic utility of exclusive enteral nutrition in the treatment of Crohn's disease.

Crohn's disease (CD) is a chronic disease characterized by episodic and disabling inflammation of the gastrointestinal tract in genetically susceptible individuals. The incidence and prevalence of CD is rising rapidly across the world emphasising that disease risk is also influenced by environmental and lifestyle factors, as well as the microbial community resident in the gut. Childhood-onset CD is associated with an aggressive disease course that can adversely impact patient growth and development. There is no cure for CD however new onset and recurrent cases of paediatric CD are often responsive to exclusive enteral nutrition (EEN) treatment. EEN treatment involves the exclusive consumption of an elemental or polymeric formula for several weeks and it is well established as a primary intervention strategy. EEN treatments typically achieve remission rates of over 80% and importantly they are associated with a high rate of mucosal healing, far superior to steroids, which is prognostic of improved long-term health outcomes. Furthermore, they are safe, have few side effects, and improve nutritional status and linear growth. Surprisingly, despite the utility of EEN our understanding of the host-microbe-diet interactions that underpin clinical remission and mucosal healing are limited. Here, we review the current state of knowledge and propose that the induction of autophagy, in addition to modulation of the microbiota and coordinated effects on inflammation and epithelial cell biology, may be critical for the therapeutic effects associated with EEN. A better understanding of EEN treatment will provide new opportunities to restore gut homeostasis and prolong periods of remission, as well as provide new insights into the factors that trigger and perhaps prevent CD.

Isolation of Genetically Tractable Most-Wanted Bacteria by Metaparental Mating.

Metagenomics has rapidly advanced our inventory and appreciation of the genetic potential inherent to the gut microbiome. However it is widely accepted that two key constraints to further genetic dissection of the gut microbiota and host-microbe interactions have been our inability to recover new isolates from the human gut, and the paucity of genetically tractable gut microbes. To address this challenge we developed a modular RP4 mobilisable recombinant vector system and an approach termed metaparental mating to support the rapid and directed isolation of genetically tractable fastidious gut bacteria. Using this approach we isolated transconjugants affiliated with Clostridium cluster IV (Faecalibacterium and Oscillibacter spp.), Clostridium cluster XI (Anaerococcus) and Clostridium XIVa (Blautia spp.) and group 2 ruminococci amongst others, and demonstrated that the recombinant vectors were stably maintained in their recipient hosts. By a similar approach we constructed fluorescently labelled bacterial transconjugants affiliated with Clostridium cluster IV (including Flavonifractor and Pseudoflavonifractor spp.), Clostridium XIVa (Blautia spp.) and Clostridium cluster XVIII (Clostridium ramosum) that expressed a flavin mononucleotide-based reporter gene (evoglow-C-Bs2). Our approach will advance the integration of bacterial genetics with metagenomics and realize new directions to support a more mechanistic dissection of host-microbe associations relevant to human health and disease.

Draft genome sequence of Bacteroides vulgatus PC510, a strain isolated from human feces.

Although Bacteroides vulgatus is one of the most prevalent microorganisms in the human gastrointestinal tract, little is known about the genetic potential of this species. Here, we describe the annotated draft genome sequence of B. vulgatus PC510 isolated from human feces.

Draft genome sequence of Turicibacter sanguinis PC909, isolated from human feces.

While the microbiota resident in the human gut is now known to provide a range of functions relevant to host health, many of the microbial members of the community have not yet been cultured or are represented by a limited number of isolates. We describe here the draft genome sequence of Turicibacter sanguinis PC909, isolated from a pooled healthy human fecal sample as part of the Australian Human Gut Microbiome Project.

Numerical ecology validates a biogeographical distribution and gender-based effect on mucosa-associated bacteria along the human colon.

We applied constrained ordination numerical ecology methods to data produced with a human intestinal tract-specific phylogenetic microarray (the Aus-HIT Chip) to examine the microbial diversity associated with matched biopsy tissue samples taken from the caecum, transverse colon, sigmoid colon and rectum of 10 healthy patients. Consistent with previous studies, the profiles revealed a marked intersubject variability; however, the numerical ecology methods of analysis allowed the subtraction of the subject effect from the data and revealed, for the first time, evidence of a longitudinal gradient for specific microbes along the colorectum. In particular, probes targeting Streptococcus and Enterococcus spp. produced strongest signals with caecal and transverse colon samples, with a gradual decline through to the rectum. Conversely, the analyses suggest that several members of the Enterobacteriaceae increase in relative abundance towards the rectum. These collective differences were substantiated by the multivariate analysis of quantitative PCR data. We were also able to identify differences in the microarray profiles, especially for the streptococci and Faecalibacterium prausnitzii, on the basis of gender. The results derived by these multivariate analyses are biologically intuitive and suggest that the biogeography of the colonic mucosa can be monitored for changes through cross-sectional and/or inception cohort studies.

The hmuUV genes of Sinorhizobium meliloti 2011 encode the permease and ATPase components of an ABC transport system for the utilization of both haem and the hydroxamate siderophores, ferrichrome and ferrioxamine B.

Sinorhizobium meliloti, the endosymbiont of Medicago sativa, can use haem compounds, including haemoglobin and leghaemoglobin, when growing in the free-living state. The components of the system involved in haem acquisition were confirmed to be ShmR, an outer membrane receptor, and HmuTUV, predicted to be an ABC transport system comprising a periplasmic protein, a permease and an ATPase respectively. The roles of HmuTUV in haem transport were confirmed in a heterologous expression system in Escherichia coli in conjunction with HasR, the outer membrane haem receptor of Serratia marcescens. hmuTUV mutants of S. meliloti showed a reduced capacity to acquire haem, suggesting the presence of a second haem acquisition system in the organism. S. meliloti can also acquire iron from xenosiderophores and the genes encoding the outer membrane receptors for ferrichrome and ferrioxamine B, fhuA1 and fhuA2, respectively, were identified. In light of this it is proposed that fhuA2 should be renamed foxA in the S. meliloti 1021 genome sequence. A siderophore reductase, FhuF, with the capacity to complement an E. coli ferrioxamine B reductase mutant, was identified encoded by a gene next to fhuA2. In the same transcriptional unit as fhuF the gene fhuP was identified and shown to encode a protein necessary for transport of ferrichrome and ferrioxamine B and predicted to be periplasmic. Interestingly, the remaining components of the transport system for the siderophores are HmuU and HmuV. Ferrichrome, ferrioxamine B and haem compounds therefore share components of the same transport system in S. meliloti.

FoxB of Pseudomonas aeruginosa functions in the utilization of the xenosiderophores ferrichrome, ferrioxamine B, and schizokinen: evidence for transport redundancy at the inner membrane.

Expression of the inner membrane protein FoxB (PA2465) of Pseudomonas aeruginosa in mutants of Sinorhizobium meliloti that are defective in the utilization of ferrichrome, ferrioxamine B, and schizokinen resulted in the restoration of siderophore utilization. Mutagenesis of foxB in P. aeruginosa did not abolish siderophore utilization, suggesting that the function is redundant.

Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli: purification, biochemical and kinetic characterisation.

We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl peptidase type-1 (PAP1). The cloned nucleotide sequence has an ORF coding for a primary sequence of 209 amino acid residues, which displays 98% identity with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary between the two sequences. The recombinant bovine PAP1 with a C-terminal His(6) tag (rBtaPAP1(6H)) was expressed in Escherichia coli XL10-Gold cells and purified by immobilised nickel ion affinity chromatography resulting in a yield of 2.6 mg of PAP1 per litre of culture. Purified rBtaPAP1(6H) had a specific activity of 3633 units mg(-1). SDS-PAGE revealed a band for bovine PAP1 with a molecular weight of approximately 24 kDa, which is in good agreement with previously reported data on PAP1. The K (m) and k (cat) values obtained for rBtaPAP1(6H) were 59 muM and 3.5 s(-1), respectively. The optimum pH for activity was 9.0-9.5 and the optimum temperature was 37 degrees C. rBtaPAP1(6H) was found to have an absolute requirement for the thiol-reducing agent DTT, consistent with the expected property of a cysteine protease. Kinetic studies using the peptides pGlu-His-Pro-NH(2) (TRH), pGlu-Ala and pGlu-Val revealed K (i) values of 44.1, 141 and 652.17 microM, respectively. The lowest K (i), observed for Thyrotropin-releasing Hormone (TRH), indicates that rBtaPAP1(6H) has a higher affinity for tripeptides over dipeptides.

Identification and characterization of an iron-regulated gene, chtA, required for the utilization of the xenosiderophores aerobactin, rhizobactin 1021 and schizokinen by Pseudomonas aeruginosa.

Pseudomonas aeruginosa utilizes several xenosiderophores under conditions of iron limitation, including the citrate hydroxamate siderophore aerobactin. Analysis of the P. aeruginosa genome sequence revealed the presence of two genes, chtA (PA4675) and PA1365, encoding proteins displaying significant similarity to the aerobactin outer-membrane receptor, IutA, of Escherichia coli. The chtA and PA1365 genes were mutated by insertional inactivation and it was demonstrated that ChtA is the outer-membrane receptor for aerobactin. ChtA also mediated the utilization of rhizobactin 1021 and schizokinen, which are structurally similar to aerobactin. In contrast to the utilization of other xenosiderophores by P. aeruginosa, there was no apparent redundancy in the utilization of aerobactin, rhizobactin 1021 and schizokinen. The utilization of citrate hydroxamate siderophores by P. aeruginosa was demonstrated to be TonB1 dependent. A Fur box was identified in the region directly upstream of chtA and it was demonstrated by the in vivo Fur titration assay that this region is capable of binding Fur and accordingly that expression of chtA is iron regulated. The PA1365 mutant was unaffected in the utilization of citrate hydroxamate siderophores.

Novel mobilizable prokaryotic two-hybrid system vectors for high-throughput protein interaction mapping in Escherichia coli by bacterial conjugation.

Since its initial description, the yeast two-hybrid (Y2H) system has been widely used for the detection and analysis of protein-protein interactions. Mating-based strategies have been developed permitting its application for automated proteomic interaction mapping projects using both exhaustive and high-throughput strategies. More recently, a number of prokaryotic two-hybrid (P2H) systems have been developed but, despite the many advantages such Escherichia coli-based systems have over the Y2H system, they have not yet been widely implemented for proteomic interaction mapping. This may be largely due to the fact that high-throughput strategies employing bacterial transformation are not as amenable to automation as Y2H mating-based strategies. Here, we describe the construction of novel conjugative P2H system vectors. These vectors carry a mobilization element of the IncPalpha group plasmid RP4 and can therefore be mobilized with high efficiency from an E.coli donor strain encoding all of the required transport functions in trans. We demonstrate how these vectors permit the exploitation of bacterial conjugation for technically simplified and automated proteomic interaction mapping strategies in E.coli, analogous to the mating-based strategies developed for the Y2H system.

Identification of rhtX and fptX, novel genes encoding proteins that show homology and function in the utilization of the siderophores rhizobactin 1021 by Sinorhizobium meliloti and pyochelin by Pseudomonas aeruginosa, respectively.

Rhizobactin 1021 is a hydroxymate siderophore produced by the soil bacterium Sinorhizobium meliloti 2011. A regulon comprising rhtA, encoding the outer membrane receptor protein for the ferrisiderophore; the biosynthesis operon rhbABCDEF; and rhrA, the Ara-C-like regulator of the receptor and biosynthesis genes has been previously described. We report the discovery of a gene, located upstream of rhbA and named rhtX (for "rhizobactin transport"), which is required, in addition to rhtA, to confer the ability to utilize rhizobactin 1021 on a strain of S. meliloti that does not naturally utilize the siderophore. Rhizobactin 1021 is structurally similar to aerobactin, which is transported in Escherichia coli via the IutA outer membrane receptor and the FhuCDB inner membrane transport system. E. coli expressing iutA and fhuCDB was found to also transport rhizobactin 1021. We demonstrated that RhtX alone could substitute for FhuCDB to transport rhizobactin 1021 in E. coli. RhtX shows similarity to a number of uncharacterized proteins which are encoded proximal to genes that are either known to be or predicted to be involved in iron acquisition. Among these is PA4218 of Pseudomonas aeruginosa, which is located close to the gene cluster that functions in pyochelin biosynthesis and outer membrane transport. PA4218 was mutated by allelic replacement, and the mutant was found to have a pyochelin utilization-defective phenotype. It is proposed that PA4218 be named fptX (for "ferripyochelin transport"). RhtX and FptX appear to be members of a novel family of permeases that function as single-subunit transporters of siderophores.