Anti-inflammatory and wound healing potential of medicinal maggot excretions/secretions at the ocular surface.

PURPOSE: In the skin, Lucilia sericata maggot excretions/secretions (ES) accelerate wound healing and limit inflammation. This study aimed to determine whether ES have similar beneficial effects at the ocular surface. METHODS: Human corneal epithelial cells (HCEC) were cultured with ES and cell viability was determined by the MTT assay. Additionally, mRNA expression of growth factors, antimicrobial peptides (AMPs) and cytokines was assessed by qPCR. ES ability to modulate TLR-induced IL-6 and IL-8 expression was determined by qPCR and ELISA. ES potential to promote corneal healing was evaluated in vitro by a migration assay in HCEC, and in vivo using a mouse model. RESULTS: ES did not impair HCEC viability up to 25 µg/ml. Among the factors evaluated, only hBD-2 was upregulated (2.5-fold) by 1.5 µg/ml ES after 6 hrs (P = 0.04). In HCEC, ES reduced Poly I:C-induced IL-6 and IL-8 mRNA (P ≤ 0.001) and protein (P ≤ 0.0001) expression. A similar effect was observed with Flagellin (TLR5 agonist) but it was less robust for FSL-1 (TLR2/6 agonist) and Pam3CSK4 (TLR1/2 agonist). The greatest in vitro migration effect was observed with 6.2 µg/ml ES after 44 hrs where gap area compared to vehicle was 53.3 ± 3.7% vs. 72.6 ± 5.4% (P = 0.001). In the mouse model, the maximum healing effect was present with 1.5 µg/ml ES after 12 hrs with a wound area of 19.0 ± 2.7% vs. 60.1 ± 21.6% (P = 0.003) or 77% reduction of the wound area compared to the negative control. CONCLUSIONS: ES significantly reduce in vitro TLR-induced production of inflammatory cytokines and promote corneal wound healing.

Secondary metabolite production and the safety of industrially important members of the Bacillus subtilis group.

Members of the 'Bacillus subtilis group' include some of the most commercially important bacteria, used for the production of a wide range of industrial enzymes and fine biochemicals. Increasingly, group members have been developed for use as animal feed enhancers and antifungal biocontrol agents. The group has long been recognised to produce a range of secondary metabolites and, despite their long history of safe usage, this has resulted in an increased focus on their safety. Traditional methods used to detect the production of secondary metabolites and other potentially harmful compounds have relied on phenotypic tests. Such approaches are time consuming and, in some cases, lack specificity. Nowadays, accessibility to genome data and associated bioinformatical tools provides a powerful means for identifying gene clusters associated with the synthesis of secondary metabolites. This review focuses primarily on well-characterised strains of B. subtilis and B. licheniformis and their synthesis of non-ribosomally synthesised peptides and polyketides. Where known, the activities and toxicities of their secondary metabolites are discussed, together with the limitations of assays currently used to assess their toxicity. Finally, the regulatory framework under which such strains are authorised for use in the production of food and feed enzymes is also reviewed.

The Identification of Intrinsic Chloramphenicol and Tetracycline Resistance Genes in Members of the Bacillus cereus Group (sensu lato).

Bacillus toyonensis strain BCT-7112T (NCIMB 14858T) has been widely used as an additive in animal nutrition for more than 30 years without reports of adverse toxigenic effects. However, this strain is resistant to chloramphenicol and tetracycline and it is generally considered inadvisable to introduce into the food chain resistance determinants capable of being transferred to other bacterial strains, thereby adding to the pool of such determinants in the gastro-enteric systems of livestock species. We therefore characterized the resistance phenotypes of this strain and its close relatives to determine whether they were of recent origin, and therefore likely to be transmissible. To this end we identified the genes responsible for chloramphenicol (catQ) and tetracycline (tetM) resistance and confirmed the presence of homologs in other members of the B. toyonensis taxonomic unit. Unexpectedly, closely related strains encoding these genes did not exhibit chloramphenicol and tetracycline resistance phenotypes. To understand the differences in the behaviors, we cloned and expressed the genes, together with their upstream regulatory regions, into Bacillus subtilis. The data showed that the genes encoded functional proteins, but were expressed inefficiently from their native promoters. B. toyonensis is a taxonomic unit member of the Bacillus cereus group (sensu lato). We therefore extended the analysis to determine the extent to which homologous chloramphenicol and tetracycline resistance genes were present in other species within this group. This analysis revealed that homologous genes were present in nearly all representative species within the B. cereus group (sensu lato). The absence of known transposition elements and the observations that they are found at the same genomic locations, indicates that these chloramphenicol and tetracycline resistance genes are of ancient origin and intrinsic to this taxonomic group, rather than recent acquisitions. In this context we discuss definitions of what are and are not intrinsic genes, an issue that is of fundamental importance to both Regulatory Authorities, and the animal feed and related industries.

Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data.

INTRODUCTION: Standardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP). METHODS: The adaptation focused on different stages of production, from cell isolation steps to cell culturing and cryopreservation. The origin and quality of materials and reagents were considered and steps for avoiding microbiological and endotoxin contamination of the final cell product were implemented. Cell isolation efficiency, MSCs surface markers and genetic profiles, originating from the use of different medium supplements, were compared. The ATMP-compliant UCX® product was also cryopreserved avoiding the use of dimethyl sulfoxide, an added benefit for the use of these cells as an ATMP. Cells were analyzed for expansion capacity and longevity. The final cell product was further characterized by flow cytometry, differentiation potential, and tested for contaminants at various passages. Finally, genetic stability and immune properties were also analyzed. RESULTS: The isolation efficiency of UCX® was not affected by the introduction of clinical grade enzymes. Furthermore, isolation efficiencies and phenotype analyses revealed advantages in the use of human serum in cell culture as opposed to human platelet lysate. Initial decontamination of the tissue followed by the use of mycoplasma- and endotoxin-free materials and reagents in cell isolation and subsequent culture, enabled the removal of antibiotics during cell expansion. UCX®-ATMP maintained a significant expansion potential of 2.5 population doublings per week up to passage 15 (P15). They were also efficiently cryopreserved in a DMSO-free cryoprotectant medium with approximately 100% recovery and 98% viability post-thaw. Additionally, UCX®-ATMP were genetically stable upon expansion (up to P15) and maintained their immunomodulatory properties. CONCLUSIONS: We have successfully adapted a method to consistently isolate, expand and cryopreserve a well-characterized population of human umbilical cord tissue-derived MSCs (UCX®), in order to obtain a cell product that is compliant with cell therapy. Here, we present quality and safety data that support the use of the UCX® as an ATMP, according to existing international guidelines.

Proteomic analysis of Bacillus subtilis strains engineered for improved production of heterologous proteins.

The use of bacterial systems for recombinant protein production has advantages of simplicity, time and cost over competing systems. However, widely used bacterial expression systems (e.g. Escherichia coli, Pseudomonas fluorescens) are not able to secrete soluble proteins directly into the culture medium. This limits yields and increases downstream processing time and costs. In contrast, Bacillus spp. secrete native enzymes directly into the culture medium at grams-per-litre quantities, although the yields of some recombinant proteins are severely limited. We have engineered the Bacillus subtilis genome to generate novel strains with precise deletions in the genes encoding ten extracytoplasmic proteases that affect recombinant protein secretion, which lack chromosomal antibiotic resistance genes. The deletion sites and presence of single nucleotide polymorphisms were confirmed by sequencing. The strains are stable and were used in industrial-scale fermenters for the production of the Bacillus anthracis vaccine protein, protective antigen, the productivity of which is extremely low in the unmodified strain. We also show that the deletion of so-called quality control proteases appears to influence cell-wall synthesis, resulting in the induction of the cell-wall stress regulon that encodes another quality control protease.

Global network reorganization during dynamic adaptations of Bacillus subtilis metabolism.

Adaptation of cells to environmental changes requires dynamic interactions between metabolic and regulatory networks, but studies typically address only one or a few layers of regulation. For nutritional shifts between two preferred carbon sources of Bacillus subtilis, we combined statistical and model-based data analyses of dynamic transcript, protein, and metabolite abundances and promoter activities. Adaptation to malate was rapid and primarily controlled posttranscriptionally compared with the slow, mainly transcriptionally controlled adaptation to glucose that entailed nearly half of the known transcription regulation network. Interactions across multiple levels of regulation were involved in adaptive changes that could also be achieved by controlling single genes. Our analysis suggests that global trade-offs and evolutionary constraints provide incentives to favor complex control programs.

Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis.

Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.

Comparative analysis of the responses of related pathogenic and environmental bacteria to oxidative stress.

Bacillus anthracis, the causative agent of anthrax, is exposed to host-mediated antibacterial activities, such as reactive oxygen species (ROS), during the early stages of its disease process. The ability to resist these host-mediated stresses is an essential characteristic of a successful pathogen while it is generally assumed that non-pathogenic environmental bacteria succumb to these antimicrobial activities. In order to gain insights into the underlying mechanisms that pathogens use to resist host-mediated oxidative stress, we have compared the oxidative stress responses of B. anthracis and Bacillus subtilis, a well-studied environmental bacterium. Among the four putative catalases encoded by B. anthracis we identified KatB as the main vegetative catalase. Comparative analysis of catalase production in B. anthracis and B. subtilis in response to superoxide and peroxide stress reveals different expression profiles, even though both are regulated by the PerR repressor, which senses and responds to peroxide stress. A B. anthracis perR deletion mutant exhibits enhanced KatB activity and is hyper-resistant to peroxide stress. Superoxide dismutase A1 (SodA1) is the main contributor to the intracellular superoxide dismutase activity in vegetative cells and the gene encoding this enzyme is constitutively expressed. Although aspects of the ROS detoxifying systems of B. anthracis and B. subtilis are similar, their responses to superoxide stress are different. The observed differences are likely to reflect adaptations to specific environmental niches.

Cellular iron distribution in Bacillus anthracis.

Although successful iron acquisition by pathogens within a host is a prerequisite for the establishment of infection, surprisingly little is known about the intracellular distribution of iron within bacterial pathogens. We have used a combination of anaerobic native liquid chromatography, inductively coupled plasma mass spectrometry, principal-component analysis, and peptide mass fingerprinting to investigate the cytosolic iron distribution in the pathogen Bacillus anthracis. Our studies identified three of the major iron pools as being associated with the electron transfer protein ferredoxin, the miniferritin Dps2, and the superoxide dismutase (SOD) enzymes SodA1 and SodA2. Although both SOD isozymes were predicted to utilize manganese cofactors, quantification of the metal ions associated with SodA1 and SodA2 in cell extracts established that SodA1 is associated with both manganese and iron, whereas SodA2 is bound exclusively to iron in vivo. These data were confirmed by in vitro assays using recombinant protein preparations, showing that SodA2 is active with an iron cofactor, while SodA1 is cambialistic, i.e., active with manganese or iron. Furthermore, we observe that B. anthracis cells exposed to superoxide stress increase their total iron content more than 2-fold over 60 min, while the manganese and zinc contents are unaffected. Notably, the acquired iron is not localized to the three identified cytosolic iron pools.

The iron-binding protein Dps2 confers peroxide stress resistance on Bacillus anthracis.

Iron is an essential nutrient that is implicated in most cellular oxidation reactions. However, iron is a highly reactive element that, if not appropriately chaperoned, can react with endogenously and exogenously generated oxidants such as hydrogen peroxide to generate highly toxic hydroxyl radicals. Dps proteins (DNA-binding proteins from starved cells) form a distinct class (the miniferritins) of iron-binding proteins within the ferritin superfamily. Bacillus anthracis encodes two Dps-like proteins, Dps1 and Dps2, the latter being one of the main iron-containing proteins in the cytoplasm. In this study, the function of Dps2 was characterized in vivo. A B. anthracis Δdps2 mutant was constructed by double-crossover mutagenesis. The growth of the Δdps2 mutant was unaffected by excess iron or iron-limiting conditions, indicating that the primary role of Dps2 is not that of iron sequestration and storage. However, the Δdps2 mutant was highly sensitive to H(2)O(2), and pretreatment of the cells with the iron chelator deferoxamine mesylate (DFM) significantly reduced its sensitivity to H(2)O(2) stress. In addition, the transcription of dps2 was upregulated by H(2)O(2) treatment and derepressed in a perR mutant, indicating that dps2 is a member of the regulon controlled by the PerR regulator. This indicates that the main role of Dps2 is to protect cells from peroxide stress by inhibiting the iron-catalyzed production of OH.

Combined proteomic and transcriptomic analysis of the response of Bacillus anthracis to oxidative stress.

The endospore-forming Gram-positive pathogen Bacillus anthracis is responsible for the usually fatal disease, inhalational anthrax. The success of this pathogen is dependent on its ability to subvert elements of the innate immune system of its animal hosts. B. anthracis spores, which are the main infective agent, are engulfed and germinate in patrolling alveolar macrophages. In order for the infection to progress, the resulting vegetative cells must resist the antimicrobial oxidative burst mounted by the host NADPH oxidase complex. The response of B. anthracis to this and other macrophage-related stresses is therefore of major importance to the success of this pathogen, and consequently we have analysed the superoxide and peroxide stress stimulons of B. anthracis strain UM23C1-2 by means of a combined transcriptomics and proteomics approach. The results show distinct patterns of expression in response to paraquat (endogenous superoxide) and hydrogen peroxide stress. While the main response to paraquat is the induction of iron uptake pathways, the response to peroxide predominantly involves the induction of protection and repair mechanisms. Comparisons between the responses of B. anthracis and related soil bacterium, B. subtilis, reveal differences that are likely to be relevant to their respective habitats.

Heterologous protein secretion by bacillus species from the cradle to the grave.

The Gram-positive bacterium Bacillus subtilis and some of its close relatives are widely used for the industrial production of enzymes for the detergents, food, and beverage industries. The choice of these organisms is based almost exclusively on the high capacity of their secretion systems that are, under the right conditions, able to secrete proteins at grams per liter concentrations. In contrast, there are relatively few examples of Bacillus species being used for the cytoplasmic production of proteins. The range of proteins that are capable of high-level production and secretion is limited by a combination of characteristics of both the target protein and the host bacterium. The secretion pathway includes checkpoints that are designed to validate the authenticity of pathway substrates. Although many of these checkpoints are known, only some can be overcome by reengineering the host. As a result, the yield of heterologous protein production is extremely variable. In this review, we consider the Bacillus protein secretion pathway from the synthesis of the target protein (cradle) to its emergence at the outer surface of the complex cell wall (grave), and discuss the roles of the various checkpoints both with respect to the target protein and their role on cell homeostasis.

Optimization of the binding properties of a synthetic anion receptor using rational and combinatorial strategies.

The solution conformations of tetrameric and hexameric cyclopeptides containing alternating L-proline and 6-aminopicolinic acid subunits strongly depend on solvent polarity. Whereas in polar solvents, such as d6-DMSO, both peptides prefer on average symmetric conformations with converging NH groups, in less polar chloroform intramolecular hydrogen bonds to the peptide NH groups stabilize other, and in the case of the hexapeptide, non-symmetrical conformations. Independent of the solvent, both peptides interact with anions via their NH groups but whereas anion binding requires a cleavage of the intramolecular hydrogen bonds accompanied by a conformational reorganization in chloroform, in polar solvents the peptides are already well preorganized for anion complexation. Complex formation between anions and the cyclic hexapeptide was even detected in highly competitive D2O/CD3OD or H2O/CH3CN mixtures, which was attributed to the special sandwich-type structure of the complexes formed. Stabilizing these 2:1 aggregates by covalently linking two cyclopeptide rings together affords ditopic receptors with a high anion affinity in protic solvents. Complex stability depends on the structure of the linker with which the two receptor moieties are connected and even more potent anion receptors were obtained by a dynamic combinatorial optimization of this linking unit.