Bacillus of nitrate and nitrite. B. subtilis

Bacillus subtilis (B. subtilis), also known as the hay bacillus or grass bacillus first discovered in 1835 by Christian Gottfried Ehrenberg (originally named Vibrio subtilis) and was renamed by Ferdinand Cohn in 1877 (McLoon, Guttenplan et al. 2011). B. subtilis is a gram positive, spore former and anaerobic bacteria found in soil and human intestinal tract. It is a rod shape and catalase positive bacteria. It is considered as an obligate aerobic bacterium though it can also grow and function anaerobically in the presence of nitrate and nitrite. B. subtilis is not toxic or pathogenic and it is considered safe for human consumption. The size of B. subtilis cells are between 4-10 ?m long and between 0.25–1.0 ?m in diameter (Allen, Loo et al. 2014). It can divide both symmetric resulting in two daughter cells or asymmetric, results in formation an endospore. Spore or endo spore formation is triggered by changes in environmental conditions that can be lethal to vegetative form i.e. nutrient limitation, changes in temperature, desiccation, and radiation. Spores have been shown able to survive indefinitely in the environment. Once the environment is unfavourable to survive, B. subtilis cell will enter a pathway where the cell divides asymmetrically in near extreme pole, to form two different cells, a smaller forespore and larger mother cell. The mother cells nurture the developing forespore that is destined to become a spore. At the early stage of sporulation, the mother cell and forespore lie side by side, however later in the development, the mother cell fully swallows the forespore by migration its membrane around the spore and create a cell within a cell. The inner forespore will then mature and become a spore and eventually, the mother cell will liberate the spore by lysing. Spore can stay inert for very long time and once the good environmental condition returns, it can germinate and produce vegetative cells. The shape of mature B. subtilis spores are ellipsoidal and their sizes are approximately 1.2 ?m in length (Ricca and Cutting 2003).  B. subtilis spores made of three different layers and these layers can be seen by using transmission electron microscopy. The most inner and central part of the spore is the core that contains the chromosome. The internal core is surrounded by a thin layer of peptidoglycan called cortex which is involved in dehydration state of spores. The next layer surrounding the cortex is called coat layer that sub-divides into an inner coat and an outer coat part. The inner coat is a thin layer about 70 nm wide, though the outer coat is thicker and it ranges from 70 to 200 nm wide (Driks 1999).  Coat layer, consist of the inner and outer layer, is a proteinaceous shell around the spore that is important for protection and survival of the spore. Both layers have a critical role in protecting the spore. The coat layer showen to be important for protecting cortex from lysozyme. Spores from B. subtilis strain that has a mutated cotE, which lacks the electron-dense outer coat, compared to wild type spores that have both inner and outer coat, showed higher sensitivity to enzyme lysozyme (Driks 1999). Coat layer also protected the spore from being digest once it has been ingested, in another word it makes the spore “eat resistance”. For example, when wild-type B. subtilis spores (strain PS533) and cotE mutant were incubated with Tetrahymena thermophila that consume bacteria by ingesting them, there was no decrease in spore titer of wild type after 48h incubation, whereas cotE mutant spores showed 100-fold reduction in spore titer (Klobutcher, Ragkousi et al. 2006). The coat layer is also involved in changing the state of the spore form dormancy back to vegetative form in a process called germination. Mutant cotE spores with a defective outer coat and mutant gerE spores, that don’t have an inner coat with a severe defect in the outer coat, are highly deficient in germination (JAMES and MANDELSTAM 1985, Driks 1999). In B. subtilis spores, 25% of total protein are coat protein which is 10% of the total weight of a single dry spore. As many as 25 different proteins on both layers of spore coat of B. subtilis has been identified (Munoz, Sadaie et al. 1978). CotA (65 kDa), CotB (59 kDa), CotG (24 kDa), CotC (11 kDa) and CotF (8 kDa) are the principle polypeptides that belong to outer coat layer. CotB, CotC, and CotG are possibly the most abundant proteins on outer coat layer and there assembly depends on CotE protein (Potot, Serra et al. 2010). These proteins were shown to be quite useful in industry, a strategy where these are used to display a heterologous protein on the spore surface (Ricca and Cutting 2003). The ability of engineering B. subtilis spores that display heterologous antigen has been documented (Isticato, Cangiano et al. 2001, Permpoonpattana, Hong et al. 2011, Nguyen, Huynh et al. 2013). To engineer spores that display protein on their spore surface, a heterologous protein (passenger protein) is fused to a protein on spore outer coat layer which serves as a carrier protein, and thus the expressed chimeric protein can be displayed on spore surface (Figure 2). Two coat proteins, CotB and CotC were initially used as the carrier protein since these proteins were not necessary for the formation of normal spores. To be able to display chimeric protein fused to CotC and CotB there are two points needs to be considered; first and foremost is that for the contraction of translation fusion the promoter and gene of CotB or CotC must be used. Second, the fusion gene should be integrated into the coding sequence of a non-essential gene, though it is also possible to integrate the fusion into the coding sequence of an essential gene, such as thymidylate A (thyA) gene that is responsible for producing thymine, if the growing media is supplemented with missing substance. Fusing 459 amino acid of C-terminal fragment of the tetanus toxin (TTFC) and the 103-amino acid B subunit of the toxin of enterotoxigenic strains of Escherichia coli (LTB), both are 51.8 and 12 kDa respectively, to the C-terminal of CotB resulted in misfolded chimeric proteins, whereas both proteins when fused to N-terminus and the middle of CotB, both showed correct assembly. However, fusing both of this proteins to C-terminus of CotC resulted in the correct assembly of these proteins on the spore surface. Ease of production, low coast and the robustness of bacterial spore are some advantages of spore display system. Another import advantage is the Stability of Spore-Displayed Proteins. It has been shown that no differences seen in TTFC displayed on spore surface when stored at -80°C, -20°C, +4°C and at room temperature for 12 weeks, compared to freshly prepared spores. Probiotics are living, ingestible microorganisms that provide health benefits to host by improving colonic balance, producing substance with systemic effect and improving the immune function (Guo, Wu et al. 2017). Two genera, Lactobacillus and Bifidobacterium have been used as probiotics against pathogens in the gut, however their sensitivity to temperature gastric acid and their slow growth were their limitation, hence the search for probiotics with better efficiency was necessary such as resistance to Hydrochloric acid (HCl) in the stomach that protects the body from pathogens. 


Bacillus species especially B. subtilis and some of its close relatives have been widely used as probiotic. The features of B. subtilis spores of being robust and their resistance to the stomach HCl, making them an attractive model as probiotic. As these spores arrive in the small intestine, they germinate and proliferate once they sense that the environment is favourable, and it is here that they convey benefits to the host. Prevention of intestinal inflammation, antidiarrheal effect, production antimicrobial substance against pathogens, exclusion of pathogens, and also normalisation of colonic flora are some of the advantages introduced by B. subtilis as probiotics. B. subtilis is also recognised as safe for human consumption as food and drug administration by European Food Safety Authority (EFSA) (Suva, Sureja et al. 2016). These advantages make B. subtilis the most intriguing probiotic species for treatment of different clinical diseases.  B. subtilis spores as probiotic have been used to treat many conditions in the human cause by pathogen or conditions such as food allergy. Experimentally infected mice by Clostridium difficile (C. difficile) that causes diarrhoea, when dosed with probiotic B. subtilis spores PXN21 both after and post infection showed attenuated symptoms of infection, although the administration of PXN21 spores post infection produced better suppression of the C. difficile infection (CDI). The mechanism of protection was suggested to be through the innate immunity by upregulation the toll like receptor 2 (TLR2) once PXN21 spore was germinated and the peptidoglycan, carried by cortex, that induces the TLR2 is released (Colenutt and Cutting 2014). Clostridium perfringens is a pathogenic bacterium that causes a common poultry disease called Necrotic enteritis (NE). This disease has a huge effect on the profitability of commercial broiler production. Traditionally, to overcome the pathogenic effect of NE, antibiotic feed supplements were administered. However, the growth promoting antibiotic got restricted in the European Union and therefore the search for an alternative treatment became necessary. In one study B subtilis spores of strain, QST 713 were tested to determine their effect in broilers that had NE. they showed that the NE-induced broiler chickens that were not dosed with B. subtilis spores had high mortality whereas the NE-induced birds that were administered the spores have greatly reduced the mortality rate (Tactacan, Schmidt et al. 2013).  As well as normal wild type spore, there is a huge interest in recombinant spore that can be used as probiotics. An example of this is a recombinant B. subtilis spores which display the mucosal adjuvant cholera toxin B subunit (CTB) fused with the peanut major allergen Ara h2 tp that could help human that have an allergy to peanut (Zhou, Song et al. 2015). The intestinal Mucosa is constantly exposed to foreign antigens and is an important line of defence against the enteric pathogen. Vaccination is a way to trigger an immune response in an individual to develop adapted immunity to microbes. Since most pathogens first infect the mucosal surface, there is an increasing interest in the development of vaccines that induce protective mucosal immunity via mucosal routs. An advantage of the mucosal vaccine is that it does not require injection and it easily administered i.e. orally or via the nasal cavity. An efficient delivery system is important for the development of mucosal vaccines. B. subtilis spores are used in the generation of the orally administered mucosal vaccine, by acting as a platform for the presentation of heterologous proteins (antigen) on their spore surface, and this species has attracted noticeable attention as it is safe for human consumption, its shelf life, and its probiotics effect. Many studies, both on human and murine, have been conducted that used recombine B. subtilis spore as a mucosal vaccine. Tuberculosis (TB) that is caused by a pathogen called Mycobacterium tuberculosis (Mtb) is an infectious disease that mainly affects the lungs and has high morbidity and mortality in different parts of the world. A vaccine called BCG was designed about eight decades ago for protection against TB and still being administered, however, it is incapable of creating full protection against the disease. Thus, a more effect vaccine is necessary. Using B. subtilis spore as a delivery vehicle a strategy to produce recombinant spores in which a major immunodominant antigens, Ag85B from Mtb was displayed on the spore surface. Mice that were dosed with recombinant spores showed increasing level in Ag85B specific IFN-g producing cell and a higher level of Ag85B specific IgG antibodies in the serum compare to mice that were dosed with naked wildtype spores (Das, Thomas et al. 2016). IFN-g has shown to inhibit the growth and replication of Mtb (Szabo, Sullivan et al. 2002). Therefore, the delivery of Mtb antigen on the surface of B. subtilis spore proves that it can create an immune response, and this can be a potential vaccine strategy against TB. Despite the potentiality of using recombinant spores that express drugs, for certain diseases, as treatment, it has not got much attention to it. B. subtilis was showen that it can be used to deliver anti-tumour compounds. Nguyen, et al. constructed killed Bacillus spores that can be engineered to display cetuximab which is a monoclonal antibody that recognises epidermal growth factor receptor (EFGR) expressed on cancer cells. These spores thereby could be loaded with an anti-cancer drug called Paclitaxel on their surface and were able to specifically target cancer cells in vitro, resulting in inhibition of cancer cell growth (Nguyen, Huynh et al. 2013). Organisms with a change or changes in their genepool in a way which it can’t occur naturally are regarded as genetically modified organism (GMO). GMO includes genetically modified animals (GM animals), microorganisms (GMMs) and plants (GM plants). The deliberate modification of and the resulting entities, beside from the benefits, has always been considered a threat both to human and the environment. For instance, GMMs can be used in agriculture as a biopesticide, nitrogen fixation or plant growth promoter, yet when introduced into the environment they could have environmental consequences and have more pronounced ecological roles in comparison to wildtype (Heuer and Smalla 2007). Also, it is possible that the organism acquires another characteristic as a result of DNA modification and it might not be only limited to a characteristic of the replaced gene. It is crucial to ensure that when GMOs are released into nature, they are not harmful to both human and environment. Thus it is necessary to assess the environmental risks that may be caused by recombinant organism once they are introduced into natural environment (Johnson, Raybould et al. 2007). Insertion of a single gene into a microorganism genome could impact the entire genome of the host resulting in different unintended characteristics, and not all these characteristics can be recognised at the same time. Thus, prediction of all types of risks is after a gene insertion is difficult. Some of the types of risks using GMO has been identified are: i) the possibility of GMO to inbreed with the wildtype resulting in disappearing of the novel trait  in wildtype; ii)  GMOs have competitive advantage over other organisms as a result of faster growth, which possibly allows them to spread (become invasive) into new habitat and cause damage to economy and ecology; iii) horizontal transfer, via transformation or conjugation, of recombinant genes to other microorganisms and this can be particularly problematic when an antibiotic resistance gene is transferred or conjugated to a pathogen that can cause disease in human and animal (Bennett, Livesey et al. 2004); iv) have adverse effect on human or animal health by increasing the pathogenicity or emergence of new diseases. published a risk assessment guideline in 2006 which is used to identify and evaluate the potential adverse effects by GMMs on human, animals and environment, whether these adverse effects are direct or indirect, or they are immediate or delayed (Committee 2007).  The risk assessment is based on identification and characterization of hazard(s) caused by GMMs and their likelihood and severity of having an adverse effect toward human and environment following exposure, and characterisation of the risk(s) which is the probability of occurrence and severity of adverse effect (EPoGM 2011).


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