Isolation And Identification Of The Predominant Lactobacillus Spp.from Calorie-restricted Mice Gut And Evaluation Of Their Effects On Aging-associated Inflammation

The homeostasis of gut microbiota that resides in the gastrointestinal tract is tightly linked to the health of their host.Gut microbiota not only plays a critical role in the development of some diseases but also affects the process of aging.With invertebrate model systems,such as the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans,scientists have demonstrated that some bacterial variants serendipitously determined the host lifespan,and small molecules secreted by the bacteria have been linked to host longevity in the context of specific bacterial backgrounds.But,in mammals,identifying the specific members involved in driving or slowing the ageing process from a complex gut microbial community and determining their mechanisms of actions is still very challenging.Calorie restriction(CR)without malnutrition exerts a potent anti-inflammatory effects and extends lifespan in various experimental models.Our previous life-long mice study showed that CR induced dramatic changes in gut microbiota,especially enriching the beneficial bacteria Lactobacillus,and the CR-modulated gut microbiota showed significant correlation with the lifespan and the metabolic phenotypes in midlife.However,whether the modulated gut microbiota contributes to the attenuation of inflammation and elongation of lifespan during CR is unknown,as are the members of the microbial community that may be key mediators of this process.First,SPF C57BL/6J male mice were employed to reveal the dynamic responses of the gut microbiota to CR.Mice were randomly assigned into two groups: the first group received a normal chow diet ad libitum(NC group),while the second was fed 70% of the ad libitum chow(CR group)for 84 days to explore the effects of short-term CR on metabolic health and monitor the dynamic changes of gut microbiota by high-throughput sequencing.Here we showed that 84-day CR significantly improved the metabolic health in mice,including enhanced glucose-insulin homeostasis,decreased serum levels of leptin and increased serum levels of adiponectin.A unique Lactobacillus-dominated gut microbiota was rapidly attained in mice within 2 weeks of CR,which significantly decreased the levels of circulating microbial antigens,as indicated by serum levels of lipopolysaccharide-binding protein(LBP),and systemic inflammatory markers such as TNF-α.Co-abundance group(CAG)network analysis of the 50 key OTUs responding to CR showed that the most abundant Lactobacillus OTU in CR mice showed negative correlation with most of the key OTUs decreased by CR.CR rapidly created a Lactobacillus-dominated gut microbiota and decreased the systemic inflammation in mice,but it remains to be elucidated whether these predominant Lactobacillus spp.contributes to the host health benefits induced by CR.Then,with a ‘sequence-guided isolation’ scheme,we obtained two isolates(CR141 and CR147)that represented the most abundant Lactobacillus OTU from the feces of mice on CR for about two weeks.The two strains of Lactobacillus spp.were subjected to whole-genome sequencing for further identification and comparative analysis.The two Lactobacillus strains were identified as members of Lactobacillus murinus based on that they shared average nucleotide identity(ANI)values of greater than 96% with the available genomes of L.murinus.Pairwise whole-genome comparison showed that CR141 and CR147 shared high sequence similarity and a conserved genetic background,but there existed a 1.6-Mb chromosomal inversion.Moreover,40 and 46 strain-specific coding sequences(CDSs)were identified in the CR141 and CR147 genomes,respectively.The functions of the two strains of L.murinus,which predominated in the CR mice gut and showed highly similar in genome sequence,need to be carefully studied.Various experimental systems were utilized to evaluate the anti-inflammatory and anti-ageing effects of these two CR-enriched L.murinus CR141 or CR147.TNF-α-stimulated Caco-2 cells were used as an in vitro model of inflammation,and adding 10% bacterial culture supernatant(BCS)from L.murinus CR147 significantly downregulated the interleukin-8(IL-8)production.In the nematode C.elegans model,L.murinus was mixed with the standard food E.coli OP50 to feed worms,and we showed that 1:9 mixture of E.coli OP50 and L.murinus CR147 significantly increased the lifespan and the brood sizes of the worms.By contrast,L.murinus CR141 showed no similar health-promoting effects both in vitro and in vivo.Then we colonized 10-monthold germ-free mice with the microbiota from 18-month-old SPF mice and found that additional L.murinus CR147 supplementation significantly reduced the intestinal permeability and serum endotoxin load,consequently attenuating the inflammation induced by the old microbiota.These results indicated that the strain L.murinus CR147 may function as a key mediator in CR-modulated gut microbiota to contribute to host health.Taken together,our study demonstrated that the predominant L.murinus strain isolated from the feces of CR mice was one of the key members of the microbiota that contributed to the beneficial effects of CR in mammalian hosts.Such key beneficial bacteria may become an attractive target for improving gut barrier function,mitigating age-associated inflammation and elongating lifespan in humans.