Chronic Stress Dampens Lactobacillus Johnsonii-Mediated Tumor Suppression to Enhance Colorectal Cancer Progression.

Colorectal cancer development and outcome are impacted by modifiable risk factors, including psychologic stress. The gut microbiota has also been shown to be linked to psychologic factors. Here, we found a marked deteriorative effect of chronic stress in multiple colorectal cancer models, including chemically induced (AOM/DSS), genetically engineered (APCmin/+), and xenograft tumor mouse models. RNA sequencing data from colon tissues revealed that expression of stemness-related genes was upregulated in the stressed colorectal cancer group by activated ß-catenin signaling, which was further confirmed by results from ex vivo organoid analyses as well as in vitro and in vivo cell tumorigenicity assays. 16S rRNA sequencing of the gut microbiota showed that chronic stress disrupted gut microbes, and antibiotic treatment and fecal microbiota transplantation abolished the stimulatory effects of chronic stress on colorectal cancer progression. Stressed colorectal cancer mice displayed a significant decrease in Lactobacillus johnsonii (L. johnsonii) abundance, which was inversely correlated with tumor load. Moreover, protocatechuic acid (PCA) was identified as a beneficial metabolite produced by L. johnsonii based on metabolome sequencing and LC/MS-MS analysis. Replenishment of L. johnsonii or PCA blocked chronic stress-induced colorectal cancer progression by decreasing ß-catenin expression. Furthermore, PCA activated the cGMP pathway, and the cGMP agonist sildenafil abolished the effects of chronic stress on colorectal cancer. Altogether, these data identify that stress impacts the gut microbiome to support colorectal cancer progression. SIGNIFICANCE: Chronic stress stimulates cancer stemness by reducing the intestinal abundance of L. johnsonii and its metabolite PCA to enhance ß-catenin signaling, forming a basis for potential strategies to circumvent stress-induced cancer aggressiveness. See related commentary by McCollum and Shah, p. 645.

Nanovesicles From Lactobacillus johnsonii N6.2 Reduce Apoptosis in Human Beta Cells by Promoting AHR Translocation and IL10 Secretion.

L. johnsonii N6.2 releases nano-sized vesicles (NVs) with distinct protein and lipid contents. We hypothesized that these NVs play a central role in the delivery of bioactive molecules that may act as mechanistic effectors in immune modulation. In this report, we observed that addition of NVs to the human pancreatic cell line ßlox5 reduced cytokine-induced apoptosis. Through RNAseq analyses, increased expression of CYP1A1, CYP1B1, AHRR, and TIPARP genes in the aryl hydrocarbon receptor (AHR) pathways were found to be significantly induced in presence of NVs. AHR nuclear translocation was confirmed by confocal microscopy. The role of NVs on beta cell function was further evaluated using primary human pancreatic islets. It was found that NVs significantly increased insulin secretion in presence of high glucose concentrations. These increases positively correlated with increased GLUT6 and SREBF1 mRNA and coincided with reduced oxidative stress markers. Furthermore, incubation of NVs with THP-1 macrophages promoted the M2 tolerogenic phenotype through STAT3 activation, expression of AHR-dependent genes and secretion of IL10. Altogether, our findings indicate that bacterial NVs have the potential to modulate glucose homeostasis in the host by directly affecting insulin secretion by islets and through the induction of a tolerogenic immune phenotype.

Identification of Gut Bacteria such as Lactobacillus johnsonii that Disseminate to Systemic Tissues of Wild Type and MyD88-/- Mice.

In healthy hosts the gut microbiota is restricted to gut tissues by several barriers some of which require MyD88-dependent innate immune sensor pathways. Nevertheless, some gut taxa have been reported to disseminate to systemic tissues. However, the extent to which this normally occurs during homeostasis in healthy organisms is still unknown. In this study, we recovered viable gut bacteria from systemic tissues of healthy wild type (WT) and MyD88-/- mice. Shotgun metagenomic-sequencing revealed a marked increase in the relative abundance of L. johnsonii in intestinal tissues of MyD88-/- mice compared to WT mice. Lactobacillus johnsonii was detected most frequently from multiple systemic tissues and at higher levels in MyD88-/- mice compared to WT mice. Viable L. johnsonii strains were recovered from different cell types sorted from intestinal and systemic tissues of WT and MyD88-/- mice. L. johnsonii could persist in dendritic cells and may represent murine immunomodulatory endosymbionts.

Cholesterol-lowering effect of bile salt hydrolase from a Lactobacillus johnsonii strain mediated by FXR pathway regulation.

Hypercholesterolemia is a major risk factor for cardiovascular diseases worldwide. Healthy intestinal microbiota can contribute to reducing the high cholesterol symptoms by producing bile salt hydrolase (BSH). In this study, recombinant BSH from the strain L. johnsonii 334 with high cholesterol reduction ability was selected to study the cholesterol-lowering mechanism mediated by farnesoid X receptor (FXR) regulation in mice. In the presence of recombinant BSH, mice had a larger bile acid pool. Analysis of individual bile acids revealed that bile acid composition was affected not only by recombinant BSH but also by the modulated gut microbiota. We confirmed a marked reduction in the transcription of FXR and its molecular targets in the ileum and a significant increase in the transcription of cholesterol 7a-hydroxylase (CYP7A1), which resulted in the increased bile acid synthesis and cholesterol-lowering effects. Notably, our work reveals the importance of BSH substrate specificity.

Immune Responses in Pregnant Sows Induced by Recombinant Lactobacillus johnsonii Expressing the COE Protein of Porcine Epidemic Diarrhea Virus Provide Protection for Piglets against PEDV Infection.

Porcine epidemic diarrhea (PED) induced by porcine epidemic diarrhea virus (PEDV) is an intestinal infectious disease in pigs that causes serious economic losses to the pig industry. To develop an effective oral vaccine against PEDV infection, we used a swine-origin Lactobacillus johnsonii (L. johnsonii) as an antigen delivery carrier. A recombinant strain pPG-T7g10-COE/L. johnsonii (L. johnsonii-COE) expressing COE protein (a neutralizing epitope of the viral spike protein) was generated. The immunomodulatory effect on dendritic cell in vitro and immunogenicity in pregnant sows was evaluated following oral administration. L. johnsonii-COE could activate monocyte-derived dendritic cell (MoDC) maturation and triggered cell immune responses. After oral vaccination with L. johnsonii-COE, levels of anti-PEDV-specific serum IgG, IgA, and IgM antibodies as well as mucosal secretory immunoglobulin A (SIgA) antibody were induced in pregnant sows. High levels of PEDV-specific SIgA and IgG antibodies were detected in the maternal milk, which provide effective protection for the piglets against PEDV infection. In summary, oral L. johnsonii-COE was able to efficiently activate anti-PEDV humoral and cellular immune responses, demonstrating potential as a vaccine for use in sows to provide protection of their piglets against PEDV.

Detection of a Novel, and Likely Ancestral, Tn916-Like Element from a Human Saliva Metagenomic Library.

Tn916 is a conjugative transposon (CTn) and the first reported and most well characterised of the Tn916/Tn1545 family of CTns. Tn916-like elements have a characteristic modular structure and different members of this family have been identified based on similarities and variations in these modules. In addition to carrying genes encoding proteins required for their conjugation, Tn916-like elements also carry accessory, antimicrobial resistance genes; most commonly the tetracycline resistance gene, tet(M). Our study aimed to identify and characterise tetracycline resistance genes from the human saliva metagenome using a functional metagenomic approach. We identified a tetracycline-resistant clone, TT31, the sequencing of which revealed it to encode both tet(M) and tet(L). Comparison of the TT31 sequence with the accessory, regulation, and recombination modules of other Tn916-like elements indicated that a partial Tn916-like element encoding a truncated orf9 was cloned in TT31. Analysis indicated that a previous insertion within the truncated orf9 created the full length orf9 found in most Tn916-like transposons; demonstrating that orf9 is, in fact, the result of a gene fusion event. Thus, we hypothesise that the Tn916-like element cloned in TT31 likely represents an ancestral Tn916.

Identification of Genes Required for Glucan Exopolysaccharide Production in Lactobacillus johnsonii Suggests a Novel Biosynthesis Mechanism.

Lactobacillus johnsonii FI9785 makes two capsular exopolysaccharides-a heteropolysaccharide (EPS2) encoded by the eps operon and a branched glucan homopolysaccharide (EPS1). The homopolysaccharide is synthesized in the absence of sucrose, and there are no typical glucansucrase genes in the genome. Quantitative proteomics was used to compare the wild type to a mutant where EPS production was reduced to attempt to identify proteins associated with EPS1 biosynthesis. A putative bactoprenol glycosyltransferase, FI9785_242 (242), was less abundant in the Δeps_cluster mutant strain than in the wild type. Nuclear magnetic resonance (NMR) analysis of isolated EPS showed that deletion of the FI9785_242 gene (242) prevented the accumulation of EPS1, without affecting EPS2 synthesis, while plasmid complementation restored EPS1 production. The deletion of 242 also produced a slow-growth phenotype, which could be rescued by complementation. 242 shows amino acid homology to bactoprenol glycosyltransferase GtrB, involved in O-antigen glycosylation, while in silico analysis of the neighboring gene 241 suggested that it encodes a putative flippase with homology to the GtrA superfamily. Deletion of 241 also prevented production of EPS1 and again caused a slow-growth phenotype, while plasmid complementation reinstated EPS1 synthesis. Both genes are highly conserved in L. johnsonii strains isolated from different environments. These results suggest that there may be a novel mechanism for homopolysaccharide synthesis in the Gram-positive L. johnsoniiIMPORTANCE Exopolysaccharides are key components of the surfaces of their bacterial producers, contributing to protection, microbial and host interactions, and even virulence. They also have significant applications in industry, and understanding their biosynthetic mechanisms may allow improved production of novel and valuable polymers. Four categories of bacterial exopolysaccharide biosynthesis have been described in detail, but novel enzymes and glycosylation mechanisms are still being described. Our findings that a putative bactoprenol glycosyltransferase and flippase are essential to homopolysaccharide biosynthesis in Lactobacillus johnsonii FI9785 indicate that there may be an alternative mechanism of glucan biosynthesis to the glucansucrase pathway. Disturbance of this synthesis leads to a slow-growth phenotype. Further elucidation of this biosynthesis may give insight into exopolysaccharide production and its impact on the bacterial cell.

Vaginal microbiome in early pregnancy and subsequent risk of spontaneous preterm birth: a case-control study.

OBJECTIVES: To explore differences in the vaginal microbiome between preterm and term deliveries. DESIGN: Nested case-control study in 3D cohort (design, develop, discover). SETTING: Quebec, Canada. SAMPLE: Ninety-four women with spontaneous preterm birth as cases [17 early (<34 weeks) and 77 late (34-36 weeks) preterm birth] and 356 women as controls with term delivery (≥37 weeks). METHODS: To assess the vaginal microbiome by sequencing the V4 region of the 16S ribosomal RNA (rRNA) gene in swabs self-collected during early pregnancy. MAIN OUTCOME MEASURES: Comparison of relative abundance of bacterial operational taxonomic units and oligotypes and identifying vaginal community state types (CSTs) in early or late spontaneous preterm and term deliveries. RESULTS: Lactobacillus gasseri/ Lactobacillus johnsonii (coefficient -5.36, 95% CI -8.07 to -2.65), Lactobacillus crispatus (99%)/ Lactobacillus acidophilus (99%) (-4.58, 95% CI -6.20 to -2.96), Lactobacillus iners (99%)/ Ralstonia solanacearum (99%) (-3.98, 95% CI -6.48 to -1.47) and Bifidobacterium longum/ Bifidobacterium breve (-8.84, 95% CI -12.96 to -4.73) were associated with decreased risk of early but not late preterm birth. Six vaginal CSTs were identified: four dominated by Lactobacillus; one with presence of bacterial vaginosis-associated bacteria (Gardnerella vaginalis, Atopobium vaginae and Veillonellaceae bacterium) (CST IV); and one with nondominance of Lactobacillus (CST VI). CST IV was associated with increased risk of early (4.22, 95% CI 1.24-24.85) but not late (1.63, 95% CI 0.68-5.04) preterm birth, compared with CST VI. CONCLUSIONS: Lactobacillus gasseri/L. johnsonii, L. crispatus/L. acidophilus, L. iners/R. solanacearum and B. longum/B. breve may be associated with decreased risk of early preterm birth. A bacterial vaginosis-related vaginal CST versus a CST nondominated by Lactobacillus may be associated with increased risk of early preterm birth. TWEETABLE ABSTRACT: Largest study of its kind finds certain species of vaginal Lactobacillus + Bifidobacterium may relate to lower risk of preterm birth.

Identification of a mouse Lactobacillus johnsonii strain with deconjugase activity against the FXR antagonist T-ß-MCA.

Bile salt hydrolase (BSH) activity against the bile acid tauro-beta-muricholic acid (T-ß-MCA) was recently reported to mediate host bile acid, glucose, and lipid homeostasis via the farnesoid X receptor (FXR) signaling pathway. An earlier study correlated decreased Lactobacillus abundance in the cecum with increased concentrations of intestinal T-ß-MCA, an FXR antagonist. While several studies have characterized BSHs in lactobacilli, deconjugation of T-ß-MCA remains poorly characterized among members of this genus, and therefore it was unclear what strain(s) were responsible for this activity. Here, a strain of L. johnsonii with robust BSH activity against T-ß-MCA in vitro was isolated from the cecum of a C57BL/6J mouse. A screening assay performed on a collection of 14 Lactobacillus strains from nine different species identified BSH substrate specificity for T-ß-MCA only in two of three L. johnsonii strains. Genomic analysis of the two strains with this BSH activity revealed the presence of three bsh genes that are homologous to bsh genes in the previously sequenced human-associated strain L. johnsonii NCC533. Heterologous expression of several bsh genes in E. coli followed by enzymatic assays revealed broad differences in substrate specificity even among closely related bsh homologs, and suggests that the phylogeny of these enzymes does not closely correlate with substrate specificity. Predictive modeling allowed us to propose a potential mechanism driving differences in BSH activity for T-ß-MCA in these homologs. Our data suggests that L. johnsonii regulates T-ß-MCA levels in the mouse intestinal environment, and that this species may play a central role in FXR signaling in the mouse.

EpsA is an essential gene in exopolysaccharide production in Lactobacillus johnsonii FI9785.

Lactobacillus johnsonii FI9785 has an eps gene cluster which is required for the biosynthesis of homopolymeric exopolysaccharides (EPS)-1 and heteropolymeric EPS-2 as a capsular layer. The first gene of the cluster, epsA, is the putative transcriptional regulator. In this study we showed the crucial role of epsA in EPS biosynthesis by demonstrating that deletion of epsA resulted in complete loss of both EPS-1 and EPS-2 on the cell surface. Plasmid complementation of the epsA gene fully restored EPS production, as confirmed by transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, this complementation resulted in a twofold increase in the expression levels of this gene, which almost doubled amounts of EPS production in comparison with the wild-type strain. Analysis of EPS by NMR showed an increased ratio of the heteropolysaccharide to homopolysaccharide in the complemented strain and allowed identification of the acetylated residue in EPS-2 as the (1,4)-linked ßGlcp unit, with the acetyl group located at O-6. These findings indicate that epsA is a positive regulator of EPS production and that EPS production can be manipulated by altering its expression.