Metabolic reconstitution of germ-free mice by a gnotobiotic microbiota varies over the circadian cycle.

The capacity of the intestinal microbiota to degrade otherwise indigestible diet components is known to greatly improve the recovery of energy from food. This has led to the hypothesis that increased digestive efficiency may underlie the contribution of the microbiota to obesity. OligoMM12-colonized gnotobiotic mice have a consistently higher fat mass than germ-free (GF) or fully colonized counterparts. We therefore investigated their food intake, digestion efficiency, energy expenditure, and respiratory quotient using a novel isolator-housed metabolic cage system, which allows long-term measurements without contamination risk. This demonstrated that microbiota-released calories are perfectly balanced by decreased food intake in fully colonized versus gnotobiotic OligoMM12 and GF mice fed a standard chow diet, i.e., microbiota-released calories can in fact be well integrated into appetite control. We also observed no significant difference in energy expenditure after normalization by lean mass between the different microbiota groups, suggesting that cumulative small differences in energy balance, or altered energy storage, must underlie fat accumulation in OligoMM12 mice. Consistent with altered energy storage, major differences were observed in the type of respiratory substrates used in metabolism over the circadian cycle: In GF mice, the respiratory exchange ratio (RER) was consistently lower than that of fully colonized mice at all times of day, indicative of more reliance on fat and less on glucose metabolism. Intriguingly, the RER of OligoMM12-colonized gnotobiotic mice phenocopied fully colonized mice during the dark (active/eating) phase but phenocopied GF mice during the light (fasting/resting) phase. Further, OligoMM12-colonized mice showed a GF-like drop in liver glycogen storage during the light phase and both liver and plasma metabolomes of OligoMM12 mice clustered closely with GF mice. This implies the existence of microbiota functions that are required to maintain normal host metabolism during the resting/fasting phase of circadian cycle and which are absent in the OligoMM12 consortium

“EvoVax” – A rationally designed inactivated Salmonella Typhimurium vaccine induces strong and long-lasting immune responses in pigs

Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm) poses a considerable threat to public health due to its zoonotic potential. Human infections are mostly foodborne, and pork and pork products are ranked among the top culprits for transmission. In addition, the high percentage of antibiotic resistance, especially in monophasic S.Tm, limits treatment options when needed. Better S.Tm control would therefore be of benefit both for farm animals and for safety of the human food chain. A promising pre-harvest intervention is vaccination. In this study we tested safety and immunogenicity of an oral inactivated S.Tm vaccine, which has been recently shown to generate an "evolutionary trap" and to massively reduce S.Tm colonization and transmission in mice. We show that this vaccine is highly immunogenic and safe in post-weaning pigs and that administration of a single oral dose results in a strong and long-lasting serum IgG response. This has several advantages over existing - mainly live - vaccines against S.Tm, both in improved seroconversion and reduced risk of vaccine-strain persistence and reversion to virulence

Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells

Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has established itself as a key technology to study protein-protein interactions in living cells. A widespread approach, BioID, uses an abortive variant of the E. coli BirA biotin protein ligase, a quite bulky enzyme with slow labeling kinetics. To improve PDB versatility and speed, various enzymes have been developed by different approaches. Here we present a small-size engineered enzyme: ultraID. We show its practical use to probe the interactome of Argonaute-2 after a 10 min labeling pulse and expression at physiological levels. Moreover, using ultraID, we provide a membrane-associated interactome of coatomer, the coat protein complex of COPI vesicles. To date, ultraID is the smallest and most efficient biotin ligase available for PDB and offers the possibility of investigating interactomes at a high temporal resolution.ISSN:2399-364

Metabolic reconstitution of germ-free mice by a gnotobiotic microbiota varies over the circadian cycle

The capacity of the intestinal microbiota to degrade otherwise indigestible diet components is known to greatly improve the recovery of energy from food. This has led to the hypothesis that increased digestive efficiency may underlie the contribution of the microbiota to obesity. OligoMM12-colonized gnotobiotic mice have a consistently higher fat mass than germ-free (GF) or fully colonized counterparts. We therefore investigated their food intake, digestion efficiency, energy expenditure, and respiratory quotient using a novel isolator-housed metabolic cage system, which allows long-term measurements without contamination risk. This demonstrated that microbiota-released calories are perfectly balanced by decreased food intake in fully colonized versus gnotobiotic OligoMM12 and GF mice fed a standard chow diet, i.e., microbiota-released calories can in fact be well integrated into appetite control. We also observed no significant difference in energy expenditure after normalization by lean mass between the different microbiota groups, suggesting that cumulative small differences in energy balance, or altered energy storage, must underlie fat accumulation in OligoMM12 mice. Consistent with altered energy storage, major differences were observed in the type of respiratory substrates used in metabolism over the circadian cycle: In GF mice, the respiratory exchange ratio (RER) was consistently lower than that of fully colonized mice at all times of day, indicative of more reliance on fat and less on glucose metabolism. Intriguingly, the RER of OligoMM12-colonized gnotobiotic mice phenocopied fully colonized mice during the dark (active/eating) phase but phenocopied GF mice during the light (fasting/resting) phase. Further, OligoMM12-colonized mice showed a GF-like drop in liver glycogen storage during the light phase and both liver and plasma metabolomes of OligoMM12 mice clustered closely with GF mice. This implies the existence of microbiota functions that are required to maintain normal host metabolism during the resting/fasting phase of circadian cycle and which are absent in the OligoMM12 consortium.ISSN:1544-9173ISSN:1545-788

OligoMM12 mice have increased fat mass compared to GF mice and SPF C57B6/J mice.

(A) Schematic representation of isolator-based indirect calorimetry system, with a TSE PhenoMaster calorimeter connected to 2 flexible surgical isolators with 4 metabolic cages each. (B) Pictures of isolator-based indirect calorimetry system inside the facility. (C) Cecal mass (tissue including luminal content). (D) Total body mass at the end of the experiment and before cecum removal. (E) Total body mass after cecum removal. (F) Lean body mass acquired by EchoMRI before cecum removal (N of mice per group with EchoMRI and indirect calorimetry measurements: GF = 12, OligoMM12 = 8, SPF = 11). (G) Fat mass from iBAT, iWAT, and vWAT. Number of mice per group in all figures unless otherwise specified: GF = 16, OligoMM12 = 12, SPF = 11. p-values obtained by Tukey’s honest significance test. Data underlying this figure are supplied in S1 Data. GF, germ-free; iBAT, interscapular brown adipose tissue; iWAT, inguinal white adipose tissue; SPF, specific-opportunistic-pathogen-free; vWAT, visceral white adipose tissue.</p

Metabolic profile comparison of GF, OligoMM12, and SPF C57B6/J mice by UPLC/MS in liver.

Manually curated list of compounds obtained by targeted peak extraction from differentially expressed pathways in liver samples during the light phase (ZT 5) and dark phase (ZT 16). p-values obtained by Tukey’s honest significance test after log2 transformation of area value. Number of mice per group: ZT5: GF = 4, OligoMM12 = 6, SPF = 7; ZT16: GF = 4, OligoMM12 = 6, SPF = 7. Data underlying this figure are supplied in S1 Data. GF, germ-free; SPF, specific-opportunistic-pathogen-free; UPLC/MS, ultraperformance liquid chromatography coupled with mass spectrometry; ZT, Zeitgeber time. (TIF)</p

Sterility test in isolator-based indirect calorimetry system.

(A) OD measurement of BHIS liquid cultures incubated overnight in aerobic and anaerobic conditions. (B-C) Representative (B) BHI-blood and (C) YPD plates streaked with GF and SPF cecum content and incubated for 3 d. (D) Representative histograms bacteria flow cytometry plots of PBS, GF, and SPF cecum content stained with SYBR Gold. Data underlying this figure are supplied in S1 Data. GF, germ-free; OD, optical density; SPF, specific-opportunistic-pathogen-free. (TIF)</p

Cecal mass interferes with normalization of energy expenditure.

(A-B) Comparison of circadian changes in energy expenditure (without normalization) among GF, OligoMM12, and SPF C57B6/J mice. (A) Circadian variation in average energy expenditure per time point and (B) overlayed curves obtained by smoothing function of data obtained every 24 min per mouse over 10 d. (C-E) Energy expenditure values obtained by “classical” ratio-based normalization methods (dividing energy expenditure values per phase by mass). (C) Area-under-curve after normalization by total mass after cecal dissection. (D) Area-under-curve after normalization by lean body mass (EchoMRI). (E) Area-under-curve after normalization by total body mass before cecal dissection. Number of mice per group in all figures unless otherwise specified: GF = 9, OligoMM12 = 8, SPF = 10. p-values obtained by Tukey’s honest significance test. Data underlying this figure are supplied in S1 Data. GF, germ-free; SPF, specific-opportunistic-pathogen-free. (TIF)</p

List of metabolites identified by targeted peak extraction in the UPLC/MS data.

Table indicates compound name, KEGG entry number, type of column was used for UPLC and if the peak ID matched the retention time and MS2 spectra identified with the chemical standard in liver and plasma samples. Data of all compounds in liver and plasma samples during the light phase (ZT 5) and dark phase (ZT 16) available in S1 Data. UPLC/MS, ultraperformance liquid chromatography coupled with mass spectrometry; ZT, Zeitgeber time. (DOCX)</p