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      Intestinal transgene delivery with native E. coli chassis allows persistent physiological changes


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          Live bacterial therapeutics (LBTs) could reverse diseases by engrafting in the gut and providing persistent beneficial functions in the host. However, attempts to functionally manipulate the gut microbiome of conventionally raised (CR) hosts have been unsuccessful because engineered microbial organisms (i.e., chassis) have difficulty in colonizing the hostile luminal environment. In this proof-of-concept study, we use native bacteria as chassis for transgene delivery to impact CR host physiology. Native Escherichia coli bacteria isolated from the stool cultures of CR mice were modified to express functional genes. The reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect physiology of and reverse pathology in CR hosts months after administration. Thus, using native bacteria as chassis to “knock in” specific functions allows mechanistic studies of specific microbial activities in the microbiome of CR hosts and enables LBT with curative intent.

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          In brief

          Native E. coli strains isolated from mouse stool are genetically engineered for long-term engraftment in the conventional mouse gut and enable long-term systemic effects on the host, such as improvements in insulin sensitivity in mouse models of type 2 diabetes.

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          Most cited references73

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          Structure, Function and Diversity of the Healthy Human Microbiome

          Studies of the human microbiome have revealed that even healthy individuals differ remarkably in the microbes that occupy habitats such as the gut, skin, and vagina. Much of this diversity remains unexplained, although diet, environment, host genetics, and early microbial exposure have all been implicated. Accordingly, to characterize the ecology of human-associated microbial communities, the Human Microbiome Project has analyzed the largest cohort and set of distinct, clinically relevant body habitats to date. We found the diversity and abundance of each habitat’s signature microbes to vary widely even among healthy subjects, with strong niche specialization both within and among individuals. The project encountered an estimated 81–99% of the genera, enzyme families, and community configurations occupied by the healthy Western microbiome. Metagenomic carriage of metabolic pathways was stable among individuals despite variation in community structure, and ethnic/racial background proved to be one of the strongest associations of both pathways and microbes with clinical metadata. These results thus delineate the range of structural and functional configurations normal in the microbial communities of a healthy population, enabling future characterization of the epidemiology, ecology, and translational applications of the human microbiome.
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            Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies

            16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of ‘best available’ primer pairs for Bacteria and Archaea for three amplicon size classes (100–400, 400–1000, ≥1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.
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              A core gut microbiome in obese and lean twins

              The human distal gut harbors a vast ensemble of microbes (the microbiota) that provide us with important metabolic capabilities, including the ability to extract energy from otherwise indigestible dietary polysaccharides1–6. Studies of a small number of unrelated, healthy adults have revealed substantial diversity in their gut communities, as measured by sequencing 16S rRNA genes6–8, yet how this diversity relates to function and to the rest of the genes in the collective genomes of the microbiota (the gut microbiome) remains obscure. Studies of lean and obese mice suggest that the gut microbiota affects energy balance by influencing the efficiency of calorie harvest from the diet, and how this harvested energy is utilized and stored3–5. To address the question of how host genotype, environmental exposures, and host adiposity influence the gut microbiome, we have characterized the fecal microbial communities of adult female monozygotic and dizygotic twin pairs concordant for leanness or obesity, and their mothers. Analysis of 154 individuals yielded 9,920 near full-length and 1,937,461 partial bacterial 16S rRNA sequences, plus 2.14 gigabases from their microbiomes. The results reveal that the human gut microbiome is shared among family members, but that each person’s gut microbial community varies in the specific bacterial lineages present, with a comparable degree of co-variation between adult monozygotic and dizygotic twin pairs. However, there was a wide array of shared microbial genes among sampled individuals, comprising an extensive, identifiable ‘core microbiome’ at the gene, rather than at the organismal lineage level. Obesity is associated with phylum-level changes in the microbiota, reduced bacterial diversity, and altered representation of bacterial genes and metabolic pathways. These results demonstrate that a diversity of organismal assemblages can nonetheless yield a core microbiome at a functional level, and that deviations from this core are associated with different physiologic states (obese versus lean).

                Author and article information

                8 September 2022
                18 August 2022
                04 August 2022
                12 September 2022
                : 185
                : 17
                : 3263-3277.e15
                [1 ]Division of Gastroenterology, University of California, San Diego, La Jolla, CA 92093, USA
                [2 ]Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
                [3 ]Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA
                [4 ]Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA
                [5 ]VA Health Sciences San Diego, La Jolla, CA 92161, USA
                [6 ]BioCircuits Institute, University of California, San Diego, La Jolla, CA 92093, USA
                [7 ]Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA
                [8 ]Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA
                [9 ]Center for Microbiome Innovation, University of California, San Diego, La Jolla, CA 92093, USA
                [10 ]Department of Computer Science and Engineering, University of California, San Diego, La Jolla, CA 92093, USA
                [11 ]Lead contact
                Author notes


                Conceptualization, S.D.B., B.J.R., and A.Z.; methodology, investigation, and validation, S.D.B., B.J.R., N.S., A.R.S., I.M., A.L., E.S.M., C.S., L.-A.R., Y.M., A.F.M.P., and A.Z.; software, A.C.D.M. and R.A.R.; formal analysis, B.J.R., N.S., L.-A.R., C.S., A.C.D.M., R.A.R., and A.Z.; resources, S.B.H., L.E., D.J.G., A.S., R.K., and A.Z.; data curation, R.A.R.; writing – original draft preparation, B.J.R. and A.Z.; writing – reviewing and editing, B.J.R., S.D.B., R.A.R., S.B.H., L.E., J.H., D.J.G., A.S., R.K., and A.Z.; visualization, B.J.R. and A.Z.; supervision, A.Z.; project administration, A.Z.; funding acquisition, A.Z.

                [* ]Correspondence: azarrinpar@ 123456ucsd.edu

                This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/).


                Cell biology
                Cell biology


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