Complete Genome Sequencing And Probiotic Properties Of Lactobacillus Taiwanensis CLG01

Background and Aims:Lactic acid bacteria(LAB)are a group of Gram-positive bacteria that produce lactic acid as a major fermentation metabolite.The genus Lactobacillus is the important member of LAB and common commensal bacteria in the mammalian intestine,which were generally regarded as the probiotics.It has been reported that Lactobacillus species can exert a wide range of benefits to the host,such as enhancing immune responses against intestinal infection,preventing colon carcinoma and stabilizing the gut mucosal barrier.It is noteworthy that the beneficial characteristics of probiotics can be species-specific.Probiotics may play distinct roles in diverse physiological and pathophysiological states of the host.Therefore,the development and investigation of novel probiotics are still necessary.Lactobacillus taiwanensis was originally isolated and identified by Taiwanese from silage cattle feed and was abundant in mammalian small intestine.L.taiwanensis has long been proposed to be a potential probiotic by researchers.Previous studies have shown that L.taiwanensis may provide protective effects in the colorectal tumorgenesis and infectious diseases.However,probiotic properties of L.taiwanensis have not been systematically studied.No study has yet provided evidence to the role it plays in modulating intestinal immune responses.Meanwhile,the current genome sequence of L.taiwanensis is fairly limited,which inhibit the functional mining of this species.In this study,we isolated and identified a new strain CLG01 of L.taiwanensis from Peyer’s patches of small intestinal of healthy C57BL/6 mice and evaluated its probiotic characteristics both in vivo and in vitro.Complete genome sequencing of L.taiwanensis CLG01 was also performed to unravel its genomic features,phylogenetic position and the functional genes related to its probiotic traits.This study aims at evaluating the probiotic properties and immunomodulatory activities of L.taiwanensis so as to provide theoretical support for the development of new probiotic and its therapeutic application in gastrointestinal diseases.MethodsPart Ⅰ1.Isolation and identification of dominant Lactobacillus strains in Peyer’s patchesThe dominant Lactobacillus strains in Peyer’s patches of healthy C57BL/6 mice were isolated and purified by traditional microbiological methods.The morphological identification,physiological and biochemical characteristics and 16S rRNA sequence alignment analysis of the isolates were conducted.2.The probiotic properties of L.taiwanensis CLG01The resistance of acid and bile salt and the adhesive ability to intestinal epithelial cells were determined by agar plate dilution method.The anti-pathogenic activity of L.taiwanensis CLG01 were tested by well agar diffsion method.The antibiotic susceptibility was assessed by paper diffusion method,RT-qPCR and ELISA were used to determine the ability of heat-killed L.taiwanensis CLG01 and its metabolites to induce the cytokines production in the mouse macrophage cells.Part Ⅱ1.Complete genome sequencing and analysis of L.taiwanensis CLG01The complete genome sequencing of L.taiwanensis CLG01 was performed with the second-generation high-throughput sequencing platforms.The sequencing data were compared with databases including NR,COG,CAZy databases to obtain the functional gene annotations and secondary metabolites prediction.2.Comparative genomic analysis of L.taiwanensis CLG01Ten representative strains of Lactobacillus species,one strain of Enterococcus faecalis and one strain of Escherichia coli with complete genome were utilized as the reference strains.A phylogenetic tree was constructed based on single copy,orthologous genes.Part Ⅲ1.Role of L.taiwanensis CLG01 in intestinal immunity under physiological conditionsThe healthy C57BL/6 mice were given live L.taiwanensis CLG01 for 2 weeks and the levels of ILCs,T cells and related cytokines in lamina propria the were detected by flow cytometry.2.Role of L.taiwanensis CLG01 in intestinal immunity under inflammatory conditions.The mouse model of acute colitis was constructed by C.rodentium infection and treated with L.taiwanensis CLG01 for 2 weeks after the infection.the levels of ILCs,T cells and related cytokines in lamina propria the were detected by flow cytometry.ResultsPart Ⅰ1.A total of 9 strains of Gram-positive,rod-shaped bacteria were isolated from Peyer’s patches and named as CLG01-CLG09.The results of initial species identifications by 16S rRNA sequencing showed that the strain CLGO1 belonged to L.taiwanensis.2.L.taiwanensis CLG01 could maintain a high survival rate at pH 2.0 and 3.0 and tolerate 0-0.2%sodium cholate.L.taiwanensis CLG01 adhered much better to LS 174T cells than Caco-2 cells.The cell-free supernatants of L.taiwanensis CLG01 were capable of inhibiting the growth of multiple pathogenic microorganisms.Both heat-killed bacteria and the metabolites of L.taiwanensis CLG01 could activate macrophage cells and upregulate the expression of TNF-α and IL-6 both in mRNA and protein level.Statistically significant increase in the IL-10 production was observed when LPS co-treatment was administered.Part Ⅱ1.The complete genome of L.taiwanensis CLG01 was composed of a single,circular chromosome and two circular plasmids.The genome of L.taiwanensis CLG01 encoded multiple functional genes related to its stress responses,adhesive ability and immune activation capacity and contained the gene clusters related to bacteriocin,LAP and lanthipeptide biosynthesis.2.The genome-wide phylogenetic analysis of CLG01 showed the closest relatedness with L.taiwanensis DSM 21401.Part Ⅲ1.L.taiwanensis CLG01 treatment could up-regulate the level of IL-22 in colon under physiological conditions.2.L.taiwanensis CLG01 could promote the secretion of IgA and downregulate the expression of inflammatory factor IFN-y caused by C.rodentium infection in colon of mouse model.We collected the supernatant of fecal samples and the levels of IgA in the fecal samples were evaluated by ELISA.Conclusions1.L.taiwanensis CLG01 showed good tolerance to the simulated gastrointestinal niches and a promising antibacterial activity against a broad spectrum of pathogenic bacteria.L.taiwanensis CLG01 could also activate macrophages.L.taiwanensis may be regarded as a potential probiotic candidate.2.The genome of L.taiwanensis CLG01 encoded a series of proteins and secondary metabolites associated with its beneficial effects,which revealed the molecular mechanisms of its probiotic properties.3.L.taiwanensis CLG01 can be considered as a potential probiotic which can downregulate the inflammatory level induced by C.rodentium infection.