Development of a live biotherapeutic throat spray with lactobacilli targeting respiratory viral infections

Respiratory viruses such as influenza viruses, respiratory syncytial virus (RSV), and coronaviruses initiate infection at the mucosal surfaces of the upper respiratory tract (URT), where the resident respiratory microbiome has an important gatekeeper function. In contrast to gut-targeting administration of beneficial bacteria against respiratory viral disease, topical URT administration of probiotics is currently underexplored, especially for the prevention and/or treatment of viral infections. Here, we report the formulation of a throat spray with live lactobacilli exhibiting several in vitro mechanisms of action against respiratory viral infections, including induction of interferon regulatory pathways and direct inhibition of respiratory viruses. Rational selection of Lactobacillaceae strains was based on previously documented beneficial properties, up-scaling and industrial production characteristics, clinical safety parameters, and potential antiviral and immunostimulatory efficacy in the URT demonstrated in this study. Using a three-step selection strategy, three strains were selected and further tested in vitro antiviral assays and in formulations: Lacticaseibacillus casei AMBR2 as a promising endogenous candidate URT probiotic with previously reported barrier-enhancing and anti-pathogenic properties and the two well-studied model strains Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum WCFS1 that display immunomodulatory capacities. The three strains and their combination significantly reduced the cytopathogenic effects of RSV, influenza A/H1N1 and B viruses, and HCoV-229E coronavirus in co-culture models with bacteria, virus, and host cells. Subsequently, these strains were formulated in a throat spray and human monocytes were employed to confirm the formulation process did not reduce the interferon regulatory pathway-inducing capacity. Administration of the throat spray in healthy volunteers revealed that the lactobacilli were capable of temporary colonization of the throat in a metabolically active form. Thus, the developed spray with live lactobacilli will be further explored in the clinic as a potential broad-acting live biotherapeutic strategy against respiratory viral diseases.     

The phosphatidylcholine-transfer protein catalyzed import of phosphatidylcholine into isolated rat liver mitochondria

In order to study the individual steps involved in the import of phosphatidylcholine (PC) into rat liver mitochondria, a number of PC analogues were introduced into the outer membrane of isolated mitochondria. Two fluorescent PC species, i.e. 1-palmitoyl-2-(16-bimanylthio)hexadecanoyl-PC (bimane-PC) and 1-palmitoyl-2-(10-pyrene)decanoyl-PC (pyrene-PC), and one radiolabeled PC species, i.e. 1-palmitoyl-2-[1-14C]oleoyl-PC (14C-POPC), were studied. The PC analogues were introduced from small unilamellar vesicles with the use of PC-specific transfer protein. The amount of PC imported was quantified by reisolation of the mitochondria. Import of the fluorescent PC species was monitored by on-line fluorescence spectroscopy. The distribution of the newly inserted PC between the outer and the inner membrane was assessed by separation of the two membranes using digitonin treatment. All analogues tested remained exclusively localized in the outer membrane thereby suggesting that additional (extramitochondrial) factors are required to initiate transfer of PC to the inner membrane.     

Stability function in the Bacillus subtilis plasmid pTA 1060

Plasmid pBB2 (11.3 kb) was constructed by genetically labeling the cryptic Bacillus subtilis plasmid pTA 1060 with the pC194-derived CmR and the pUB110-derived KmR markers. In nonselective media pBB2 was segregationally almost completely stable (loss rates less than or equal to 0.02% per cell generation). In contrast, pBB3, obtained by deleting from pBB2 a region consisting of two ClaI fragments (1.45 and 0.20 kb, respectively), was unstable (loss rates greater than or equal to 0.5% per cell generation). This indicates that a genetic element required for stability is located on one or both of these fragments. In pBB3 cop, a mutant with a two- to threefold increased copy number, the rate of plasmid loss was reduced compared to that of pBB3. The insertion of a 4.2-kb Escherichia coli DNA fragment reduced the stability of pBB2 only slightly, suggesting that this vector may be useful for the cloning of relatively large fragments.     

Identification of enteroendocrine cells that express TRPA1 channels in the mouse intestine

TRPA1 is an ion channel that detects specific chemicals in food and also transduces mechanical, cold and chemical stimulation. Its presence in sensory nerve endings is well known and recent evidence indicates that it is expressed by some gastrointestinal enteroendocrine cells (EEC). The purpose of the present work is to identify and quantify EEC that express TRPA1 in the mouse gastrointestinal tract. Combined in situ hybridisation histochemistry for TRPA1 and immunofluorescence for EEC hormones was used. TRPA1 expressing EEC were common in the duodenum and jejunum, were rare in the distal small intestine and were absent from the stomach and large intestine. In the duodenum and jejunum, TRPA1 occurred in EEC that contained both cholecystokinin (CCK) and 5-hydroxytryptamine (5HT) and in a small number of cells expressing 5HT but not CCK. TRPA1 was absent from CCK cells that did not express 5HT and from EEC containing glucagon-like insulinotropic peptide. Thus TRPA1 is contained in very specific EEC populations. It is suggested that foods such as garlic and cinnamon that contain TRPA1 stimulants may aid digestion by facilitating the release of CCK.     

Signal peptide peptidase- and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins.

Signal peptides direct the export of secretory proteins from the cytoplasm. After processing by signal peptidase, they are degraded in the membrane and cytoplasm. The resulting fragments can have signaling functions. These observations suggest important roles for signal peptide peptidases. The present studies show that the Gram-positive eubacterium Bacillus subtilis contains two genes for proteins, denoted SppA and TepA, with similarity to the signal peptide peptidase A of Escherichia coli. Notably, TepA also shows similarity to ClpP proteases. SppA of B. subtilis was only required for efficient processing of pre-proteins under conditions of hyper-secretion. In contrast, TepA depletion had a strong effect on pre-protein translocation across the membrane and subsequent processing, not only under conditions of hyper-secretion. Unlike SppA, which is a typical membrane protein, TepA appears to have a cytosolic localization, which is consistent with the observation that TepA is involved in early stages of the secretion process. Our observations demonstrate that SppA and TepA have a role in protein secretion in B. subtilis. Based on their similarity to known proteases, it seems likely that SppA and TepA are specifically required for the degradation of proteins or (signal) peptides that are inhibitory to protein translocation.