Bacillus Subtilis Delays Neurodegeneration and Behavioral Impairment in the Alzheimer's Disease Model Caenorhabditis Elegans.

Multiple causes, apart from genetic inheritance, predispose to the production and aggregation of amyloid-ß (Aß) peptide and Alzheimer's disease (AD) development in the older population. There is currently no therapy or medicine to prevent or delay AD progression. One novel strategy against AD might involve the use of psychobiotics, probiotic gut bacteria with specific mental health benefits. Here, we report the neuronal and behavioral protective effects of the probiotic bacterium Bacillus subtilis in a Caenorhabditis elegans AD model. Aging and neuronal deterioration constitute important risk factors for AD development, and we showed that B. subtilis significantly delayed both detrimental processes in the wild-type C. elegans strain N2 compared with N2 worms colonized by the non-probiotic Escherichia coli OP50 strain. Importantly, B. subtilis alleviated the AD-related paralysis phenotype of the transgenic C. elegans strains CL2120 and GMC101 that express, in body wall muscle cells, the toxic peptides Aß3-42 and Aß1-42, respectively. B. subtilis-colonized CL2355 worms were protected from the behavioral deficits (e.g., poor chemotactic response and decreased body bends) produced by pan-neuronal Aß1-42 expression. Notably, B. subtilis restored the lifespan level of C. elegans strains that express Aß to values similar to the life expectancy of the wild-type strain N2 fed on E. coli OP50 cells. The B. subtilis proficiencies in quorum-sensing peptide (i.e., the Competence Sporulation Factor, CSF) synthesis and gut-associated biofilm formation (related to the anti-aging effect of the probiotic) play a crucial role in the anti-AD effects of B. subtilis. These novel results are discussed in the context of how B. subtilis might exert its beneficial effects from the gut to the brain of people with or at risk of developing AD.

Microbial flora, probiotics, Bacillus subtilis and the search for a long and healthy human longevity.

Probiotics are live microorganisms that have beneficial effects on host health, including extended lifespan, when they are administered or present in adequate quantities. However, the mechanisms by which probiotics stimulate host longevity remain unclear and very poorly understood. In a recent study (Nat. Commun. 8, 14332 (2017) doi: 10.1038/ncomms14332), we used the spore-forming probiotic bacterium Bacillus subtilis and the model organism Caenorhabditis elegans to study the mechanism by which a probiotic bacterium affects host longevity. We found that biofilm-proficient B. subtilis colonized the C. elegans gut and extended the worm lifespan significantly longer than did biofilm-deficient isogenic strains. In addition to biofilm proficiency, the quorum-sensing pentapeptide CSF and nitric oxide (NO) represent the entire B. subtilis repertoire responsible for the extended longevity of C. elegans. B. subtilis grown under biofilm-supporting conditions synthesized higher levels of NO and CSF than under planktonic growth conditions, emphasizing the key role of the biofilm in slowing host aging. Significantly, the prolongevity effect of B. subtilis was primarily due to a downregulation of the insulin-like signaling system that precisely is a key partaker in the healthy longevity of human centenarians. These findings open the possibility to test if the regular consumption of B. subtilis incorporated in foods and beverages could significantly extend human life expectancy and contribute to stop the development of age-related diseases.

Transcriptional regulation of adhesive properties of Bacillus subtilis to extracellular matrix proteins through the fibronectin-binding protein YloA.

Bacterial adherence to extracellular matrix proteins (ECMp) plays important roles during host-pathogen interaction, however its genetic regulation remains poorly understood. yloA of the model bacterium Bacillus subtilis shows high homology to genes encoding fibronectin-binding proteins of Gram-positive pathogens. Here, we characterized the regulatory network of YloA-dependent adhesive properties of the probiotic B. subtilis natto (Bsn). YloA-proficient, but not YloA-deficient, Bsn specifically bound to ECMp in a concentration-dependent manner and were proficient in biofilm formation. yloA expression showed a continuous increase in activity during the growth phase and decreased during the stationary phase. The transcription factors AbrB and DegU downregulated yloA expression during the logarithmic and stationary growth phases respectively. Analysis of the yloA promoter region revealed the presence of AT-rich direct and inverted repeats previously reported to function as DegU-recognized binding sites. In spo0A cells, yloA expression was completely turned off because of upregulation of AbrB throughout growth. Accordingly, DNase I footprinting analysis confirmed that AbrB bound to the promoter region of yloA. Interestingly, Bsn bound fibronectin with higher affinity, lower Kd, than several bacterial pathogens and competitively excluded them from binding to immobilized-fibronectin, a finding that might be important for the anti-infective properties of B. subtilis and its relatives.

Bacillus subtilis biofilm extends Caenorhabditis elegans longevity through downregulation of the insulin-like signalling pathway.

Beneficial bacteria have been shown to affect host longevity, but the molecular mechanisms mediating such effects remain largely unclear. Here we show that formation of Bacillus subtilis biofilms increases Caenorhabditis elegans lifespan. Biofilm-proficient B. subtilis colonizes the C. elegans gut and extends worm lifespan more than biofilm-deficient isogenic strains. Two molecules produced by B. subtilis - the quorum-sensing pentapeptide CSF and nitric oxide (NO) - are sufficient to extend C. elegans longevity. When B. subtilis is cultured under biofilm-supporting conditions, the synthesis of NO and CSF is increased in comparison with their production under planktonic growth conditions. We further show that the prolongevity effect of B. subtilis biofilms depends on the DAF-2/DAF-16/HSF-1 signalling axis and the downregulation of the insulin-like signalling (ILS) pathway.

Culturing Bacteria from Caenorhabditis elegans Gut to Assess Colonization Proficiency.

Determining an accurate count of intestinal bacteria from Caenorhabditis elegans is one critical way to assess colonization proficiency by a given bacteria. This can be accomplished by culturing appropriate dilutions of worm gut bacteria on selective or differential agarized media. Because of the high concentration of bacteria in gut worm, dilution is necessary before plating onto growth media. Serial dilutions can reduce the concentration of the original intestinal sample to levels low enough for single colonies to be grown on media plates, allowing for the calculation of the initial counts of bacteria in the intestinal sample.

Determination of NO and CSF Levels Produced by Bacillus subtilis.

The cell-to-cell communication and division of labour that occurs inside a beneficial biofilm produce significant differences in gene expression compared with the gene expression pattern of cells grew under planktonic conditions. In this sense, the levels of NO (nitric oxide) and CSF (Competence Sporulation Stimulating Factor) produced in Bacillus subtilis cultures have been measured only under planktonic growth conditions. We sought to determine whether NO and/or CSF production is affected in B. subtilis cells that develop as a biofilm. To measure the production levels of the two prolongevity molecules, we grew B. subtilis cells under planktonic and biofilm supporting condition.