Differential induction of apoptosis in human colonic carcinoma cells (Caco-2) by Atopobium, and commensal, probiotic and enteropathogenic bacteria: mediation by the mitochondrial pathway

The induction of apoptosis in mammalian cells by bacteria is well reported. This process may assist infection by pathogens whereas for non-pathogens apoptosis induction within carcinoma cells protects against colon cancer. Here, apoptosis induction by a major new gut bacterium, Atopobium minutum, was compared with induction by commensal (Escherichia coli K-12 strains), probiotic (Lactobacillus rhamnosus, Bifidobacterium latis) and pathogenic (E. coli: EPEC and VTEC) gut bacteria within the colon cancer cell line, Caco-2. The results show a major apoptotic effect for the pathogens, mild effects for the probiotic strains and A. minutum, but no effect for commensal E. coli. The mild apoptotic effects observed are consistent with the beneficial roles of probotics in protection against colon cancer and suggest, for the first time, that A. minutum possesses similar advantageous, anti-cancerous activity. Although bacterial infection increased Caco-2 membrane FAS levels, caspase-8 was not activated indicating that apoptosis is FAS independent. Instead, in all cases, apoptosis was induced through the mitochondrial pathway as indicated by BAX translocation, cytorchrome c release, and caspase-9 and -3 cleavage. This suggests that an intracellular stimulus initiates the observed apoptosis responses.

Soft topographic map for clustering and classification of bacteria

In this work a new method for clustering and building a topographic representation of a bacteria taxonomy is presented. The method is based on the analysis of stable parts of the genome, the so-called “housekeeping genes”. The proposed method generates topographic maps of the bacteria taxonomy, where relations among different type strains can be visually inspected and verified. Two well known DNA alignement algorithms are applied to the genomic sequences. Topographic maps are optimized to represent the similarity among the sequences according to their evolutionary distances. The experimental analysis is carried out on 147 type strains of the Gammaprotebacteria class by means of the 16S rRNA housekeeping gene. Complete sequences of the gene have been retrieved from the NCBI public database. In the experimental tests the maps show clusters of homologous type strains and present some singular cases potentially due to incorrect classification or erroneous annotations in the database.

Optimal protection of stabilised dry live bacteria from bile toxicity in oral dosage forms by bile acid adsorbent resins

We previously found that dried live bacteria of a vaccine strain can be temporarily sensitive to bile acids and suggested that Bile Adsorbing Resins (BAR) can be used in oral vaccine tablets to protect dried bacteria from intestinal bile. Here, we report a quantitative analysis of the ability of BAR to exclude the dye bromophenol blue from penetrating into matrix tablets and also sections of hard capsule shells. Based on this quantitative analysis, we made a fully optimised formulation, comprising 25% w/w of cholestyramine in Vcaps™ HPMC capsules. This gave effectively 100% protection of viability from 4% bile, with 4200-fold more live bacteria recovered from this formulation compared to unprotected dry bacteria. From the image analysis, we found that the filler material or compaction force used had no measurable effect on dye exclusion but did affect the rate of tablet hydration. Increasing the mass fraction of BAR gave more exclusion of dye up to 25% w/w, after which a plateau was reached and no further dye exclusion was seen. More effective dye exclusion was seen with smaller particle sizes (i.e. cholestyramine) and when the BAR was thoroughly dried and disaggregated. Similar results were found when imaging dye penetration into capsule sections or tablets. The predictions of the dye penetration study were tested using capsules filled with dried attenuated Salmonella vaccine plus different BAR types, and the expected protection from bile was found, validating the imaging study. Surprisingly, depending on the capsule shell material, some protection was given by the capsule alone without adding BAR, with Vcaps™ HPMC capsules providing up to 174-fold protection against 1% bile; faster releasing Vcaps Plus™ HPMC capsules and Coni Snap™ gelatin capsules gave less protection.

Formulation of a live bacterial vaccine for stable room temperature storage results in loss of acid, bile and bile salt resistance

Live bacterial vaccines have great promise both as vaccines against enteric pathogens and as heterologous antigen vectors against diverse diseases. Ideally, room temperature stable dry formulations of live bacterial vaccines will allow oral vaccination without cold-chain storage or injections. Attenuated Salmonella can cross the intestinal wall and deliver replicating antigen plus innate immune activation signals directly to the intestinal immune tissues, however the ingested bacteria must survive firstly gastric acid and secondly the antimicrobial defences of the small intestine. We found that the way in which cells are grown prior to formulation markedly affects sensitivity to acid and bile. Using a previously published stable storage formulation that maintained over 10% viability after 56 days storage at room temperature, we found dried samples of an attenuated S. typhimurium vaccine lost acid and bile resistance compared to the same bacteria taken from fresh culture. The stable formulation utilised osmotic preconditioning in defined medium plus elevated salt concentration to induce intracellular trehalose accumulation before drying. Dried bacteria grown in rich media without osmotic preconditioning showed more resistance to bile, but less stability during storage, suggesting a trade-off between bile resistance and stability. Further optimization is needed to produce the ultimate room-temperature stable oral live bacterial vaccine formulation.

Protection of live bacteria from bile acid toxicity using bile acid adsorbing resins

We previously demonstrated that a dry, room temperature stable formulation of a live bacterial vaccine was highly susceptible to bile, and suggested that this will lead to significant loss of viability of any live bacterial formulation released into the intestine using an enteric coating or capsule. We found that bile and acid tolerance is very rapidly recovered after rehydration with buffer or water, raising the possibility that rehydration in the absence of bile prior to release into the intestine might solve the problem of bile toxicity to dried cells. We describe here a novel formulation that combines extensively studied bile acid adsorbent resins with the dried bacteria, to temporarily adsorb bile acids and allow rehydration and recovery of bile resistance of bacteria in the intestine before release. Tablets containing the bile acid adsorbent cholestyramine release 250-fold more live bacteria when dissolved in a bile solution, compared to control tablets without cholestyramine or with a control resin that does not bind bile acids. We propose that a simple enteric coated oral dosage form containing bile acid adsorbent resins will allow improved live bacterial delivery to the intestine via the oral route, a major step towards room temperature stable, easily administered and distributed vaccine pills and other bacterial therapeutics

Microbial control of diamondback moth, Plutella xylostella L. (Lepidoptera:Yponomeutidae) using bacteria (Xenorhabdus nematophila) and its metabolites from the entomopathogenic nematode Steinernema carpocapsae

Cells and cell-free solutions of the culture filtrate of the bacterial symbiont, Xenorhabdus nematophila taken from the entomopathogenic nematode Steinernema carpocapsae in aqueous broth suspensions were lethal to larvae of the diamondback moth Plutella xylostella. Their application on leaves of Chinese cabbage indicated that the cells can penetrate into the insects in the absence of the nematode vector. Cell-free solutions containing metabolites were also proved as effective as bacterial cells suspension. The application of aqueous suspensions of cells of X. nematophila or solutions containing its toxic metabolites to the leaves represents a possible new strategy for controlling insect pests on foliage.

Biological control of black vine weevil (Otiorhynchus sulcatus, Coleoptera: Curculionidae) by entomopathogenic bacteria and their cell-free toxic metabolites

Biocontrol agents such as Xeiwrhabduf, nemalophilci and X. nematophila ssp. bovienii and their cell-free protein toxin complexes were lethal to larvae of O. sulcatus when applied to potting compost in the absence of plants. Similarly, strawberry plants infected with 0. sulcaitfi larvae were protected from damage by applications of both cell suspensions of the bacteria and solutions of their cell-free toxic metabolites, indicating that it is the protein toxins, which are responsible for the lethal effects observed. These toxic metabolites were found more effective against 0. sulccitus larvae when treated in soil microflora. Insect mortality is increased by increasing temperature and bacterial concentration. The toxins remained pathogenic for several months when stored in potting soil either at 15 or 20°C, however, bacterial cells were not as persistent as the toxins. It is therefore suggested that these bacteria and their toxic metabolites can he applied in soil for insect pest control.

Soilborne fungi and bacteria symbiotically associated with Steinernema spp. acting as biological agents against Fusarium wilt of tomato

An isolate of Gliocladium virens from disease affected soil in a commercial tomato greenhouse proved highly antagonistic to Fusarium oxysporum f.sp. lycopersici, used together with an isolate of the nematophagus fungus Verticillium chlamydosporium. Significant disease control was obtained when young mycelial preparation (on a food-base culture) of the G. virens together with V. chlamydosporium was applied in potting medium. Similar results were observed when a Trichoderma harzianum isolate was treated in combination with the V. chlamydosporium isolate. Most promising, in terms of minimizing the Fusarium wilt of tomato incidence, was also the effect of the bacteria associated with entomopathogenic nematodes (Steinernema spp.), Pseudomonas oryzihabitans and Xenorhabdus nematophilus.

Intracellular signalling pathways regulating the adaptation of skeletal muscle to exercise and nutritional changes

The focus of the present review is to assimilate current knowledge concerning the differing signalling transduction cascades that control muscle mass development and affect skeletal muscle phenotype following exercise or nutritional uptake. Effects of mechanical loading on protein synthesis are discussed. Muscle growth control is regulated by the interplay of growth promoting and growth suppressing factors, which act in concert. Much emphasis has been placed on understanding how increases in the rate of protein synthesis are induced in skeletal muscle during the adaptive process. One key point to emerge is that protein synthesis following resistance exercise or increased nutrient availability is mediated through changes in signal transduction involving the phosphorylation of mTOR and sequential activation of downstream targets. On the other hand, AMPK activation plays an important role in the inhibition of protein synthesis by suppressing the function of multiple translation regulators of the mTOR signalling pathway in response to cellular energy depletion and low metabolic conditions. The effects of exercise and/or nutritional uptake on the activation of signalling molecules that regulate protein synthesis are highlighted, providing a better understanding of the molecular changes in the cell.