Tunable control of B. infantis abundance and gut metabolites by co-administration of human milk oligosaccharides.

Precision engineering of the gut microbiome holds promise as an effective therapeutic approach for diseases associated with a disruption in this microbial community. Engrafting a live biotherapeutic product (LBP) in a predictable, controllable manner is key to the consistent success of this approach and has remained a challenge for most LBPs under development. We recently demonstrated high-level engraftment of Bifidobacterium longum subsp. infantis (B. infantis) in adults when co-dosed with a specific prebiotic, human milk oligosaccharides (HMO). Here, we present a cellular kinetic-pharmacodynamic approach, analogous to pharmacokinetic-pharmacodynamic-based analyses of small molecule- and biologic-based drugs, to establish how HMO controls expansion, abundance, and metabolic output of B. infantis in a human microbiota-based model in gnotobiotic mice. Our data demonstrate that the HMO dose controls steady-state abundance of B. infantis in the microbiome, and that B. infantis together with HMO impacts gut metabolite levels in a targeted, HMO-dependent manner. We also found that HMO creates a privileged niche for B. infantis expansion across a 5-log range of bacterial inocula. These results demonstrate remarkable control of both B. infantis levels and the microbiome community metabolic outputs using this synbiotic approach, and pave the way for precision engineering of desirable microbes and metabolites to treat a range of diseases.

Precision modulation of dysbiotic adult microbiomes with a human-milk-derived synbiotic reshapes gut microbial composition and metabolites.

Manipulation of the gut microbiome using live biotherapeutic products shows promise for clinical applications but remains challenging to achieve. Here, we induced dysbiosis in 56 healthy volunteers using antibiotics to test a synbiotic comprising the infant gut microbe, Bifidobacterium longum subspecies infantis (B. infantis), and human milk oligosaccharides (HMOs). B. infantis engrafted in 76% of subjects in an HMO-dependent manner, reaching a relative abundance of up to 81%. Changes in microbiome composition and gut metabolites reflect altered recovery of engrafted subjects compared with controls. Engraftment associates with increases in lactate-consuming Veillonella, faster acetate recovery, and changes in indolelactate and p-cresol sulfate, metabolites that impact host inflammatory status. Furthermore, Veillonella co-cultured in vitro and in vivo with B. infantis and HMO converts lactate produced by B. infantis to propionate, an important mediator of host physiology. These results suggest that the synbiotic reproducibly and predictably modulates recovery of a dysbiotic microbiome.

Manufacturing Processes of a Purified Microbiome Therapeutic Reduce Risk of Transmission of Potential Bacterial Pathogens in Donor Stool.

BACKGROUND: Although fecal microbiota transplant has been used to prevent recurrent Clostridioides difficile infection (rCDI), documented pathogen transmissions highlight inherent safety risks of minimally processed stool. We describe manufacturing processes for fecal microbiota spores, live (VOWST; VOS, formerly SER-109), a microbiota-based oral therapeutic of Firmicutes spores. METHODS: Bacterial inactivation kill curves were obtained after ethanol exposure for 4 model organisms spiked into process intermediates. RESULTS: Bacterial log reduction factors ranged from 6.5 log10 to 7.4 log10 and lysis of spiked organisms occurred rapidly within 30 seconds. CONCLUSIONS: These experiments demonstrate substantial and rapid inactivation of representative organisms, supporting the potential benefit of VOS manufacturing processes to mitigate risk.

Dosing a synbiotic of human milk oligosaccharides and B. infantis leads to reversible engraftment in healthy adult microbiomes without antibiotics.

Predictable and sustainable engraftment of live biotherapeutic products into the human gut microbiome is being explored as a promising way to modulate the human gut microbiome. We utilize a synbiotic approach pairing the infant gut microbe Bifidobacterium longum subspecies infantis (B. infantis) and human milk oligosaccharides (HMO). B. infantis, which is typically absent in adults, engrafts into healthy adult microbiomes in an HMO-dependent manner at a relative abundance of up to 25% of the bacterial population without antibiotic pretreatment or adverse effects. Corresponding changes in metabolites are detected. Germ-free mice transplanted with dysbiotic human microbiomes also successfully engraft with B. infantis in an HMO-dependent manner, and the synbiotic augments butyrate levels both in this in vivo model and in in vitro cocultures of the synbiotic with specific Firmicutes species. Finally, the synbiotic inhibits the growth of enteropathogens in vitro. Our findings point to a potential safe mechanism for ameliorating dysbioses characteristic of numerous human diseases.

Characterization of the TnsD-attTn7 complex that promotes site-specific insertion of Tn7.

The bacterial transposon Tn7 is distinguished by its ability to recognize a specific site called attTn7, and insert just downstream of the highly conserved chromosomal glmS gene. TnsD is one of four transposon-encoded polypeptides (TnsABC+D) required for site-specific insertion of Tn7 into attTn7, and is the target site-selector that binds to a highly conserved sequence in the end of the glmS protein coding region. In this study, we identified important nucleotides within this region that are crucial for TnsD-attTn7 interaction. We also probed the regions of TnsD that interact with attTn7 and found that there are important DNA-binding determinants throughout the entire length of the protein, including an amino-terminal CCCH zinc-finger motif. A key role of TnsD is to recruit the non-sequence specific DNA-binding protein TnsC to attTn7; TnsC also interacts with and controls both the TnsA and TnsB subunits of the Tn7 transposase. TnsC stimulates the binding of TnsD to attTn7 in vivo, and TnsCD and TnsD can also interact in the absence of DNA and localize their interaction domains to the N-terminal region of each protein.

An SOS-regulated type 2 toxin-antitoxin system.

The Escherichia coli chromosome encodes seven demonstrated type 2 toxin-antitoxin (TA) systems: cassettes of two or three cotranscribed genes, one encoding a stable toxin protein that can cause cell stasis or death, another encoding a labile antitoxin protein, and sometimes a third regulatory protein. We demonstrate that the yafNO genes constitute an additional chromosomal type 2 TA system that is upregulated during the SOS DNA damage response. The yafNOP genes are part of the dinB operon, of which dinB underlies stress-induced mutagenesis mechanisms. yafN was identified as a putative antitoxin by homology to known antitoxins, implicating yafO (and/or yafP) as a putative toxin. Using phage-mediated cotransduction assays for linkage disruption, we show first that yafN is an essential gene and second that it is essential only when yafO is present. Third, yafP is not a necessary part of either the toxin or the antitoxin. Fourth, although DinB is required, the yafNOP genes are not required for stress-induced mutagenesis in the Escherichia coli Lac assay. These results imply that yafN encodes an antitoxin that protects cells against a yafO-encoded toxin and show a protein-based TA system upregulated by the SOS response.

Fast, easy and efficient: site-specific insertion of transgenes into enterobacterial chromosomes using Tn7 without need for selection of the insertion event.

BACKGROUND: Inserting transgenes into bacterial chromosomes is generally quite involved, requiring a selection for cells carrying the insertion, usually for drug-resistance, or multiple cumbersome manipulations, or both. Several approaches use phage lambda red recombination, which allows for the possibility of mutagenesis of the transgene during a PCR step. RESULTS: We present a simple, rapid and highly efficient method for transgene insertion into the chromosome of Escherichia coli, Salmonella or Shigella at a benign chromosomal site using the site-specific recombination machinery of the transposon Tn7. This method requires very few manipulations. The transgene is cloned into a temperature-sensitive delivery plasmid and transformed into bacterial cells. Growth at the permissive temperature with induction of the recombination machinery leads to transgene insertion, and subsequent growth at the nonpermissive temperature cures the delivery plasmid. Transgene insertion is highly site-specific, generating insertions solely at the Tn7 attachment site and so efficient that it is not necessary to select for the insertion. CONCLUSION: This method is more efficient and straightforward than other techniques for transgene insertion available for E. coli and related bacteria, making moving transgenes from plasmids to a chromosomal location a simple matter. The non-requirement for selection is particularly well suited for use in development of unmarked strains for environmental release, such as live-vector vaccine strains, and also for promoter-fusion studies, and experiments in which every bacterial cell must express a transgene construct.

The dinB operon and spontaneous mutation in Escherichia coli.

Apparently conflicting data regarding the role of SOS-inducible, error-prone DNA polymerase IV (DinB) in spontaneous mutation are resolved by the finding that mutation is reduced by a polar allele with which dinB and neighboring yafN are deleted but not by two nonpolar dinB alleles. We demonstrate the existence of a dinB operon that contains four genes, dinB-yafN-yafO-yafP. The results imply a role for yafN, yafO, and/or yafP in spontaneous mutation.

Long CTG tracts from the myotonic dystrophy gene induce deletions and rearrangements during recombination at the APRT locus in CHO cells.

Expansion of CTG triplet repeats in the 3' untranslated region of the DMPK gene causes the autosomal dominant disorder myotonic dystrophy. Instability of CTG repeats is thought to arise from their capacity to form hairpin DNA structures. How these structures interact with various aspects of DNA metabolism has been studied intensely for Escherichia coli and Saccharomyces cerevisiae but is relatively uncharacterized in mammalian cells. To examine the stability of (CTG)(17), (CTG)(98), and (CTG)(183) repeats during homologous recombination, we placed them in the second intron of one copy of a tandemly duplicated pair of APRT genes. Cells selected for homologous recombination between the two copies of the APRT gene displayed distinctive patterns of change. Among recombinants from cells with (CTG)(98) and (CTG)(183), 5% had lost large numbers of repeats and 10% had suffered rearrangements, a frequency more than 50-fold above normal levels. Analysis of individual rearrangements confirmed the involvement of the CTG repeats. Similar changes were not observed in proliferating (CTG)(98) and (CTG)(183) cells that were not recombinant at APRT. Instead, they displayed high frequencies of small changes in repeat number. The (CTG)(17) repeats were stable in all assays. These studies indicate that homologous recombination strongly destabilizes long tracts of CTG repeats.