Differential peripheral immune signatures elicited by vegan versus ketogenic diets in humans.

Nutrition has broad impacts on all physiological processes. However, how nutrition affects human immunity remains largely unknown. Here we explored the impact of a dietary intervention on both immunity and the microbiota by performing a post hoc analysis of a clinical trial in which each of the 20 participants sequentially consumed vegan or ketogenic diets for 2 weeks ( NCT03878108 ). Using a multiomics approach including multidimensional flow cytometry, transcriptomic, proteomic, metabolomic and metagenomic datasets, we assessed the impact of each diet, and dietary switch, on host immunity and the microbiota. Our data revealed that overall, a ketogenic diet was associated with a significant upregulation of pathways and enrichment in cells associated with the adaptive immune system. In contrast, a vegan diet had a significant impact on the innate immune system, including upregulation of pathways associated with antiviral immunity. Both diets significantly and differentially impacted the microbiome and host-associated amino acid metabolism, with a strong downregulation of most microbial pathways following ketogenic diet compared with baseline and vegan diet. Despite the diversity of participants, we also observed a tightly connected network between datasets driven by compounds associated with amino acids, lipids and the immune system. Collectively, this work demonstrates that in diverse participants 2 weeks of controlled dietary intervention is sufficient to significantly and divergently impact host immunity, which could have implications for precision nutritional interventions. ClinicalTrials.gov registration: NCT03878108 .

Colchicine effects on the gut microbiome in adults with metabolic syndrome.

Obesity-induced inflammation plays a substantial role in the development of insulin resistance and type 2 diabetes. The altered gut flora in obesity can also contribute to metabolic dysregulation and systemic inflammation. However, it remains unclear how dysregulation of systemic inflammation in obesity affects the gut microbiome. We hypothesized that colchicine's systemic anti-inflammatory effects in obesity would be associated with improvements in gut microbial diversity. We conducted a secondary analysis of a double-blind randomized placebo-controlled trial, in which 40 adults with obesity, high C-reactive protein (CRP) (≥2.0 mg/L), insulin resistance (homeostatic model of insulin resistance: HOMA-IR ≥2.6 mg/L), and metabolic syndrome (MetS) were randomized to three months of colchicine 0.6 mg or placebo tablets twice daily. Serum and stool samples were collected at baseline and final visit. Gut microbiota composition was characterized from stool DNA by dual-index amplification and sequencing of 16S ribosomal RNA. Pre- and post-intervention stool samples were available for 15 colchicine- and 12 placebo-treated subjects. Circulating high sensitivity CRP (hsCRP), interleukin-6, resistin, white blood count, and neutrophils were significantly decreased in the colchicine arm as compared to placebo. However, changes in stool microbiome alpha diversity, as assessed by the Chao1, Shannon, and Pielou indices, were not significant between groups. Amplicon sequence variant counts were unchanged among all examined phyla or families. Oscillibacter was the only genus to demonstrate even a nominally significant change. Among adults with obesity and MetS, colchicine significantly improved systemic inflammation. However, this anti-inflammatory effect was not associated with significant changes in the gut microbiome. Further studies are warranted to investigate this relationship.

The systemic anti-microbiota IgG repertoire can identify gut bacteria that translocate across gut barrier surfaces.

Unique gut microbiota compositions have been associated with inflammatory diseases, but identifying gut bacterial functions linked to immune activation in humans remains challenging. Translocation of pathogens from mucosal surfaces into peripheral tissues can elicit immune activation, although whether and which gut commensal bacteria translocate in inflammatory diseases is difficult to assess. We report that a subset of commensal gut microbiota constituents that translocate across the gut barrier in mice and humans are associated with heightened systemic immunoglobulin G (IgG) responses. We present a modified high-throughput, culture-independent approach to quantify systemic IgG against gut commensal bacteria in human serum samples without the need for paired stool samples. Using this approach, we highlight several commensal bacterial species that elicit elevated IgG responses in patients with inflammatory bowel disease (IBD) including taxa within the clades Collinsella, Bifidobacterium, Lachnospiraceae, and Ruminococcaceae. These and other taxa identified as translocating bacteria or targets of systemic immunity in IBD concomitantly exhibited heightened transcriptional activity and growth rates in IBD patient gut microbiomes. Our approach represents a complementary tool to illuminate interactions between the host and its gut microbiota and may provide an additional method to identify microbes linked to inflammatory disease.

The vaginal microbiome and preterm birth.

The incidence of preterm birth exceeds 10% worldwide. There are significant disparities in the frequency of preterm birth among populations within countries, and women of African ancestry disproportionately bear the burden of risk in the United States. In the present study, we report a community resource that includes 'omics' data from approximately 12,000 samples as part of the integrative Human Microbiome Project. Longitudinal analyses of 16S ribosomal RNA, metagenomic, metatranscriptomic and cytokine profiles from 45 preterm and 90 term birth controls identified harbingers of preterm birth in this cohort of women predominantly of African ancestry. Women who delivered preterm exhibited significantly lower vaginal levels of Lactobacillus crispatus and higher levels of BVAB1, Sneathia amnii, TM7-H1, a group of Prevotella species and nine additional taxa. The first representative genomes of BVAB1 and TM7-H1 are described. Preterm-birth-associated taxa were correlated with proinflammatory cytokines in vaginal fluid. These findings highlight new opportunities for assessment of the risk of preterm birth.

Racioethnic diversity in the dynamics of the vaginal microbiome during pregnancy.

The microbiome of the female reproductive tract has implications for women's reproductive health. We examined the vaginal microbiome in two cohorts of women who experienced normal term births: a cross-sectionally sampled cohort of 613 pregnant and 1,969 non-pregnant women, focusing on 300 pregnant and 300 non-pregnant women of African, Hispanic or European ancestry case-matched for race, gestational age and household income; and a longitudinally sampled cohort of 90 pregnant women of African or non-African ancestry. In these women, the vaginal microbiome shifted during pregnancy toward Lactobacillus-dominated profiles at the expense of taxa often associated with vaginal dysbiosis. The shifts occurred early in pregnancy, followed predictable patterns, were associated with simplification of the metabolic capacity of the microbiome and were significant only in women of African or Hispanic ancestry. Both genomic and environmental factors are likely contributors to these trends, with socioeconomic status as a likely environmental influence.

Conserved regions of gonococcal TbpB are critical for surface exposure and transferrin iron utilization.

The transferrin-binding proteins TbpA and TbpB enable Neisseria gonorrhoeae to obtain iron from human transferrin. The lipoprotein TbpB facilitates, but is not strictly required for, TbpA-mediated iron acquisition. The goal of the current study was to determine the contribution of two conserved regions within TbpB to the function of this protein. Using site-directed mutagenesis, the first mutation we constructed replaced the lipobox (LSAC) of TbpB with a signal I peptidase cleavage site (LAAA), while the second mutation deleted a conserved stretch of glycine residues immediately downstream of the lipobox. We then evaluated the resulting mutants for effects on TbpB expression, surface exposure, and transferrin iron utilization. Western blot analysis and palmitate labeling indicated that the lipobox, but not the glycine-rich motif, is required for lipidation of TbpB and tethering to the outer membrane. TbpB was released into the supernatant by the mutant that produces TbpB LSAC. Neither mutation disrupted the transport of TbpB across the bacterial cell envelope. When these mutant TbpB proteins were produced in a strain expressing a form of TbpA that requires TbpB for iron acquisition, growth on transferrin was either abrogated or dramatically diminished. We conclude that surface tethering of TbpB is required for optimal performance of the transferrin iron acquisition system, while the presence of the polyglycine stretch near the amino terminus of TbpB contributes significantly to transferrin iron transport function. Overall, these results provide important insights into the functional roles of two conserved motifs of TbpB, enhancing our understanding of this critical iron uptake system.

Evidence of Fe3+ interaction with the plug domain of the outer membrane transferrin receptor protein of Neisseria gonorrhoeae: implications for Fe transport.

Neisseria gonorrhoeae is an obligate pathogen that hijacks iron from the human iron transport protein, holo-transferrin (Fe(2)-Tf), by expressing TonB-dependent outer membrane receptor proteins, TbpA and TbpB. Homologous to other TonB-dependent outer membrane transporters, TbpA is thought to consist of a ß-barrel with an N-terminal plug domain. Previous reports by our laboratories show that the sequence EIEYE in the plug domain is highly conserved among various bacterial species that express TbpA and plays a crucial role in iron utilization for gonococci. We hypothesize that this highly conserved EIEYE sequence in the TbpA plug, rich in hard oxygen donor groups, binds with Fe(3+) through the transport process across the outer membrane through the ß-barrel. Sequestration of Fe(3+) by the TbpA-plug supports the paradigm that the ferric iron must always remain chelated and controlled throughout the transport process. In order to test this hypothesis here we describe the ability of both the recombinant wild-type plug, and three small peptides that encompass the sequence EIEYE of the plug, to bind Fe(3+). This is the first report of the expression/isolation of the recombinant wild-type TbpA plug. Although CD and SUPREX spectroscopies suggest that a non-native structure is observed for the recombinant plug, fluorescence quenching titrations indicate that the wild-type recombinant TbpA plug binds Fe (3+) with a conditional log K(d) = 7 at pH 7.5, with no evidence of binding at pH 6.3. A recombinant TbpA plug with mutated sequence (NEIEYEN → NEIAAAN) shows no evidence of Fe(3+) binding under our experimental set up. Interestingly, in silico modeling with the wild-type plug also predicts a flexible loop structure for the EIEYE sequence under native conditions which once again supports the Fe(3+) binding hypothesis. These in vitro observations are consistent with the hypothesis that the EIEYE sequence in the wild-type TbpA plug binds Fe(3+) during the outer membrane transport process in vivo.

Functional expression of Escherichia coli fhuA gene in Rhizobium spp. of Cajanus cajan provides growth advantage in presence of Fe3+: ferrichrome as iron source.

Cajanus cajan rhizobial isolates were found to be unable to utilize iron bound to ferrichrome, desferrioxamine B or rhodotorulic acid, all being hydroxamate type siderophores. A broad host range expression vector containing the Escherichia coli fhuA gene, encoding the outer membrane receptor for Fe-ferrichrome, was constructed. The plasmid construct (pGR1), designed to express fhuA under the lac promoter of E. coli, complemented E. coli MB97 DeltafhuA mutant for ferri-ferrichrome utilization and also allowed Rhizobium spp. ST1 and Rhizobium spp. IC3123 to grow using iron bound to ferrichrome. Sensitivity to the antibiotic albomycin, transported via the FhuA receptor, was found in case of MB97 as well as rhizobial transformants harboring pGR1. The rhizobial transformants expressing fhuA showed growth stimulation when co-inoculated with Ustilago maydis, a fungal species known to produce ferrichrome under iron starved conditions. Growth stimulation was also observed in the presence of externally supplied ferrichrome. The significance of these findings in terms of the potential for improving the survivability of rhizobial bioinoculant strains in natural soils is discussed.