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Recent metagenomic studies have demonstrated that the overall functional potential of the intestinal microbiome is rather conserved between healthy individuals. Here we assessed the biological processes undertaken in-vivo by microbes and the host in the intestinal tract by conducting a metaproteome analysis from a total of 48 faecal samples of 16 healthy adults participating in a placebo-controlled probiotic intervention trial. Half of the subjects received placebo and the other half consumed Lactobacillus rhamnosus GG for three weeks (1010 cfu per day). Faecal samples were collected just before and at the end of the consumption phase as well as after a three-week follow-up period, and were processed for microbial composition and metaproteome analysis. A common core of shared microbial protein functions could be identified in all subjects. Furthermore, we observed marked differences in expressed proteins between subjects that resulted in the definition of a stable and personalized microbiome both at the mass-spectrometry-based proteome level and the functional level based on the KEGG pathway analysis. No significant changes in the metaproteome were attributable to the probiotic intervention. A detailed taxonomic assignment of peptides and comparison to phylogenetic microarray data made it possible to evaluate the activity of the main phyla as well as key species, including Faecalibacterium prausnitzii. Several correlations were identified between human and bacterial proteins. Proteins of the human host accounted for approximately 14% of the identified metaproteome and displayed variations both between and within individuals. The individually different human intestinal proteomes point to personalized host-microbiota interactions. Our findings indicate that analysis of the intestinal metaproteome can complement gene-based analysis and contributes to a thorough understanding of the activities of the microbiome and the relevant pathways in health and disease.

IgA secretion at mucosal sites is important for host defence against pathogens as well as maintaining the symbiosis with microorganisms present in the small intestine that affect IgA production. In the present study, we tested the ability of 5 strains of lactic acid bacteria stimulating IgA production, being Pediococcus acidilactici K15 selected as the most effective on inducing this protective immunoglobulin. We found that this response was mainly induced via IL-10, as efficiently as IL-6, secreted by K15-stimulated dendritic cells. Furthermore, bacterial RNA was largely responsible for the induction of these cytokines; double-stranded RNA was a major causative molecule for IL-6 production whereas single-stranded RNA was critical factor for IL-10 production. In a randomized, double-blind, placebo-controlled clinical trial, ingestion of K15 significantly increased the secretory IgA (sIgA) concentration in saliva compared with the basal level observed before this intervention. These results indicate that functional lactic acid bacteria induce IL-6 and IL-10 production by dendritic cells, which contribute to upregulating the sIgA concentration at mucosal sites in humans.

The majority of research on the physiological effects of dietary resistant starch type 2 (RS2) has focused on sources derived from high-amylose maize. In this study, we conduct a double-blind, randomized, placebo-controlled, crossover trial investigating the effects of RS2 from wheat on glycemic response, an important indicator of metabolic health, and the gut microbiota. Overall, consumption of RS2-enriched wheat rolls for one week resulted in reduced postprandial glucose and insulin responses relative to conventional wheat when participants were provided with a standard breakfast meal containing the respective treatment rolls (RS2-enriched or conventional wheat). This was accompanied by an increase in the proportions of bacterial taxa Ruminococcus and Gemmiger in the fecal contents, reflecting the composition in the distal intestine. Additionally, fasting breath hydrogen and methane were increased during RS2-enriched wheat consumption. However, although changes in fecal short-chain fatty acid (SCFA) concentrations were not significant between control and RS-enriched wheat roll consumption, butyrate and total SCFAs were positively correlated with relative abundance of Faecalibacterium, Ruminoccocus, Roseburia, and Barnesiellaceae. These effects show that RS2-enriched wheat consumption results in a reduction in postprandial glycemia, altered gut microbial composition, and increased fermentation activity relative to wild-type wheat.

Background Small RNAs (sRNAs) are non-coding RNAs known to regulate various biological functions such as stress adaptation, metabolism, virulence as well as pathogenicity across a wide range of bacteria, mainly by controlling mRNA stabilization or regulating translation. Identification and functional characterization of sRNAs has been carried out in various plant growth-promoting bacteria and they have been shown to help the cells cope up with environmental stress. No study has been carried out to uncover these regulatory molecules in the diazotrophic alpha-proteobacterium Azospirillum brasilense Sp245 to date. Results Expression-based sRNA identification (RNA-seq) revealed the first list of ~ 468 sRNA candidate genes in A. brasilense Sp245 that were differentially expressed in nitrogen starvation versus non-starved conditions. In parallel, in silico tools also identified 2 of the above as candidate sRNAs. Altogether, putative candidates were stringently curated from RNA-seq data based on known sRNA parameters (size, location, secondary structure, and abundance). In total, ~ 59 significantly expressed sRNAs were identified in this study of which 53 are potentially novel sRNAs as they have no Rfam and BSRD homologs. Sixteen sRNAs were randomly selected and validated for differential expression, which largely was found to be in congruence with the RNA-seq data. Conclusions Differential expression of 468 A. brasilense sRNAs was indicated by RNA-seq data, a subset of which was confirmed by expression analysis. Four of the significantly expressed sRNAs were not observed in nitrogen starvation while 16 sRNAs were found to be exclusively expressed in nitrogen depletion. Putative candidate sRNAs identified have potential mRNA targets primarily involved in stress (abiotic and biotic) adaptability; regulation of bacterial cellular, biological and molecular pathways such as nitrogen fixation, polyhydroxybutyrate synthesis, chemotaxis, biofilm formation and transcriptional regulation. In addition to directly influencing bacteria, some of these sRNAs also have targets influencing plant-microbe interactions through adhesion of bacteria to plant roots directly, suppressing host response, inducing plant defence and signalling.

There is little data on human milk oligosaccharide (HMO) composition in Sub-Saharan Africa. Iron fortificants adversely affect the infant gut microbiota, while co-provision of prebiotic galacto-oligosaccharides (GOS) mitigates most of the adverse effects. Whether variations in maternal HMO profile can influence the infant response to iron and/or GOS fortificants is unknown. The aim of this study was to determine HMO profiles and the secretor/non-secretor phenotype of lactating Kenyan mothers and investigate their effects on the maternal and infant gut microbiota, and on the infant response to a fortification intervention with 5 mg iron (2.5 mg as sodium iron ethylenediaminetetraacetate and 2.5 mg as ferrous fumarate) and 7.5 g GOS. We studied mother–infant pairs (n = 80) participating in a 4-month intervention trial in which the infants (aged 6.5–9.5 months) received daily a micronutrient powder without iron, with iron or with iron and GOS. We assessed: (1) maternal secretor status and HMO composition; (2) effects of secretor status on the maternal and infant gut microbiota in a cross-sectional analysis at baseline of the intervention trial; and (3) interactions between secretor status and intervention groups during the intervention trial on the infant gut microbiota, gut inflammation, iron status, growth and infectious morbidity. Secretor prevalence was 72% and HMOs differed between secretors and non-secretors and over time of lactation. Secretor status did not predict the baseline composition of the maternal and infant gut microbiota. There was a secretor-status-by-intervention-group interaction on Bifidobacterium (p = 0.021), Z-scores for length-for-age (p = 0.022) and weight-for-age (p = 0.018), and soluble transferrin receptor (p = 0.041). In the no iron group, longitudinal prevalence of diarrhea was higher among infants of non-secretors (23.8%) than of secretors (10.4%) (p = 0.001). In conclusion, HMO profile may modulate the infant gut microbiota response to fortificant iron; compared to infants of secretor mothers, infants of non-secretor mothers may be more vulnerable to the adverse effect of iron but also benefit more from the co-provision of GOS.

Recent advances in culture-independent approaches have enabled insights into the diversity, complexity, and individual variability of gut microbial communities. To examine the effect of oral administration of Saccharomyces (S.) boulardii and mode of delivery on the intestinal microbial community in preterm infants. Stool samples were collected from preterm newborns randomly divided into two groups: a probiotic-receiving group (n = 18) or a placebo group (n = 21). Samples were collected before probiotic intake (day 0), and after 2 and 6 weeks of supplementation. The composition of colonizing bacteria was assessed by 16S ribosomal RNA (rRNA) gene sequencing of fecal samples using the Ion 16S Metagenomics Kit and the Ion Torrent Personal Genome Machine platform. A total of 11932257 reads were generated, and were clustered into 459, 187, and 176 operational taxonomic units at 0 days, 2 weeks, and 6 weeks, respectively. Of the 17 identified phyla, Firmicutes Actinobacteria, Proteobacteria, and Bacteroidetes were universal. The microbial community differed at day 0 compared with at 2 weeks and 6 weeks. There was a tendency for increased bacterial diversity at 2 weeks and 6 weeks compared with day 0, and infants with a gestational age of 31 weeks or higher presented increased bacterial diversity prior to S. boulardii administration. Firmicutes and Proteobacteria remained stable during the observation period, whereas Actinobacteria and Bacteroidetes increased in abundance, the latter particularly more sharply in vaginally delivered infants. While the mode of delivery may influence the development of a microbial community, this study had not enough power to detect statistical differences between cohorts supplemented with probiotics, and in a consequence, to speculate on S. boulardii effect on gut microbiome composition in preterm newborns.

Rhizobial infection and root nodule formation in legumes require recognition of signal molecules produced by the bacteria and their hosts. Here, we show that rhizobial transfer RNA (tRNA)-derived small RNA fragments (tRFs) are signal molecules that modulate host nodulation. Three families of rhizobial tRFs were confirmed to regulate host genes associated with nodule initiation and development through hijacking the host RNA-interference machinery that involves ARGONAUTE 1. Silencing individual tRFs with the use of short tandem target mimics or by overexpressing their targets represses root hair curling and nodule formation, whereas repressing these targets with artificial microRNAs identical to the respective tRFs or mutating these targets with CRISPR-Cas9 promotes nodulation. Our findings thus uncover a bacterial small RNA–mediated mechanism for prokaryote-eukaryote interaction and may pave the way for enhancing nodulation efficiency in legumes.

Medical male circumcision (MMC) reduces the risk of genital ulcer disease (GUD) in men by 50%. In Ugandan and Kenyan trials, a sexually transmissible agent was not identified in 50-60% of GUD specimens by polymerase chain reaction (PCR) assay. We sought to better define the etiology of GUD in men participating in the Kenyan trial and examine how MMC affects GUD etiology. We defined GUD of unknown etiology as negative for HSV (type 1 and type 2), T. pallidum, and H. ducreyi by PCR, and negative for HSV-2 and T. pallidum by serology. We identified bacterial microbiota in a subset of 59 GUD specimens using multitag pyrosequencing of the 16S rRNA gene, and compared results by unknown vs. STI-associated etiology. Statistical analysis employed Bray-Curtis similarity measure of bacterial community by etiology, hierarchical clustering and logistic regression. In 59 GUD specimens from 59 men, 23 (39%) had unknown etiology. Bacterial diversity was greater in GUD of unknown than STI etiology (p = 0.01). Fusobacteria (Fusobacterium spp. and Sneathia spp.) were more commonly detected in men with GUD of unknown etiology [adjusted OR = 5.67; 95% CI: 1.63-19.8] as were Oxobacter spp. and Anaerovorax spp. [adjusted OR = 3.12; 95% CI: 0.83-11.7]. Sequences from these four anaerobic bacterial taxa were more often detected in uncircumcised men than circumcised men (p<0.05). Anaerobic bacteria are more common in genital ulcers of uncircumcised men. The specific anaerobic bacteria associated with GUD of unknown etiology have cytotoxic properties that can exacerbate epithelial disruptions leading to ulcer-like appearance. MMC may reduce GUD through a reduction in these anaerobic bacteria.

Conventional CRISPR–Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a notable inversion of this paradigm, in which bacterial Tn 7 -like transposons have co-opted nuclease-deficient CRISPR–Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn 6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair. A programmable transposase integrates donor DNA at user-defined genomic target sites with high fidelity, revealing a new approach for genetic engineering that obviates the need for DNA double-strand breaks and homologous recombination. 

The CRISPR-associated protein Cpf1 from Francisella novicida is a novel enzyme with specific, dual-endoribonuclease–endonuclease activities in precursor crRNA processing and crRNA-programmable cleavage of target DNA. Cpf1 enzyme in CRISPR immunity The bacterial immune system, CRISPR, utilizes a small RNA guide, or crRNA, to target a nucleolytic CRISPR complex to DNA with a complementary sequence. This process has been widely exploited for various types of genome engineering. Previously described CRISPR systems utilize one nuclease, such as Cas6, to generate the mature crRNA, and a second, such as Cas9, to cleave the target DNA. Two studies illustrate a different approach that involves the Cpf1 protein. Emmanuelle Charpentier and colleagues report that type V-A Cpf1 protein from Francisella novicida functions as a minimalistic CRISPR system. It is a dual-nuclease enzyme that can perform both the pre-crRNA processing and DNA cleavage activities, having distinct active domains for the two substrates. Zhiwei Huang and colleagues solve the crystal structure of monomeric Lachnospiraceae bacterium Cpf1 protein bound to crRNA, showing how binding induces conformational changes in the nuclease. CRISPR–Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA 1 . The well-studied types I, II and III utilize a set of distinct CRISPR-associated (Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA) 2 , 3 , 4 , 5 and the mature crRNAs then guide a complex of Cas proteins (Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA 6 , 7 , 8 , 9 , 10 , 11 , 12 . In type II systems, RNase III cleaves pre-crRNA base-paired with trans -activating crRNA (tracrRNA) in the presence of Cas9 (refs 13 , 14 ). The mature tracrRNA–crRNA duplex then guides Cas9 to cleave target DNA 15 . Here, we demonstrate a novel mechanism in CRISPR–Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5′-YTN-3′ protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5′ overhang 16 . The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V-A constitutes the most minimalistic of the CRISPR–Cas systems so far described.