Identification of Biomarkers for Systemic Distribution of Nanovesicles From Lactobacillus johnsonii N6.2.

The ability of bacterial extracellular vesicles (EV) to transport biological molecules has increased the research to determine their potential as therapeutic agents. In this study, Lactobacillus johnsonii N6.2-derived nanovesicles (NV) were characterized to identify components that may serve as biomarkers in host-microbe interactions. Comparative proteomic and lipidomic analyses of L. johnsonii N6.2 NV and cell membrane (CM) were performed. The lipidomic profiles indicated that both fractions contained similar lipids, however, significant differences were observed in several classes. LC-MS/MS proteomic analysis indicated that NV contained several unique and differentially expressed proteins when compared to the CM. Analysis of Gene Ontology (GO) terms, based on cellular component, showed significant enrichment of proteins in the cytoplasm/intracellular space category for the NV fraction. Based on these results, the proteins T285_RS00825 (named Sdp), Eno3 and LexA were selected for studies of localization and as potential biomarkers for host-microbe interactions. Immunogold staining, followed by scanning and transmission electron microscopy (SEM and TEM, respectively), revealed that Sdp was preferentially localized along the cell wall/membrane, and on NV-like structures surrounding the bacteria. These results were confirmed using immunofluorescence staining in Caco-2 cells incubated with NV. Consequently, we evaluated the potential for NV surface-exposed proteins to generate an immune response in the host. Plasma from individuals administered L. johnsonii N6.2 showed that IgA and IgG antibodies were generated against NV and Sdp domains in vivo. Altogether, these results show that L. johnsonii N6.2 NV have the potential to mediate host interactions through immune modulation.

Anti-inflammatory effects of Lactobacillus johnsonii L531 in a pig model of Salmonella Infantis infection involves modulation of CCR6+ T cell responses and ER stress.

Probiotic pretreatment is an effective non-antibiotic strategy for preventing or controlling Salmonella infections. We found that Lactobacillus johnsonii L531, isolated from the colon of a clinically healthy weaned piglet, effectively prevented infection with Salmonella enterica serovar Infantis in a pig model. Newly weaned piglets were intragastrically administered Lactobacillus johnsonii L531 at 1.0 × 1010 CFU/day for 1 week before S. Infantis challenge. Pretreatment with L. johnsonii L531 lessened the severity of diarrhea and ileal inflammation in S. Infantis-infected piglets. Lactobacilli were more abundant in the ileum than jejunum after L. johnsonii L531 pretreatment. Treatment with L. johnsonii L531 reduced the abundance of total bacteria in the ileal mucosa and the production of lipocalin 2 in the jejunum of piglets challenged with Salmonella. Both intestinal morphology and transmission electron microscopy results indicated that L. johnsonii L531 alleviated intestinal tissue damage following S. Infantis challenge, especially in the villus and endoplasmic reticulum (ER). ER stress induced by S. Infantis was attenuated by L. johnsonii L531 treatment. The number of CD4- CCR6+ T cells decreased following S. Infantis challenge, but the percentage of CCR6- IFNγ+ T cells in peripheral blood increased. In intestinal mesenteric lymph nodes, S. Infantis increased the proportion of CCR6+ IFNγ+ T cells, whereas L. johnsonii L531 induced an increase in the proportion of CD4+ CCR6+ T cells in response to S. Infantis infection. Our data thus suggest that L. johnsonii L531 contributes to the maintenance of intestinal homeostasis by modulating T-cell responses and ER stress.

Lactobacillus johnsonii glycolipids, their structure and immunoreactivity with sera from inflammatory bowel disease patients.

Structural studies of the major glycolipids produced by two Lactobacillus johnsonii (LJ) strains, LJ 151 isolated from intestinal tract of healthy mice and LJ 142 isolated from mice with experimentally induced inflammatory bowel disease (IBD), were performed. Two major glycolipids, GL1 and GL2, were present in lipid extracts from L. johnsonii 142 and 151 strains. Glycolipid GL1 has been identified as ß-D-Glcp-(1→6)-α-D-Galp-(1→2)-α-D-Glcp-diglyceride and GL2 as α-D-Galp-(1→2)-α-D-Glcp-diglyceride. The main fatty acid residues identified by gas-liquid chromatography-mass spectrometry were palmitic, stearic and lactobacillic acids. Besides structural elucidation of the major glycolipids, the aim of this study was to determine the immunochemical properties of these glycolipids and to compare their immunoreactivity to that of polysaccharides obtained from the same strains. Sera from rabbits immunized with bacterial cells possessed much higher serological reactivity with polysaccharides than with glycolipids. Inversely, reactivity of the glycolipids with human sera from patients with IBD was much higher than that determined for the polysaccharides, while reactivity of glycolipids with human sera from healthy individuals was much lower than one measured for the polysaccharides. Results indicate that glycoconjugates from Lactobacillus cell wall act as antigens and may represent new IBD diagnostic biomarkers.