Abstract

Intestinal microbiota is a key component of both the metabolism and immunity. These can be helpful in healthcare, especially for the management of digestive diseases and food-borne illnesses. Through genetic engineering it is possible to fully express biologically active copies of such powerful molecules from food and commensal bacteria. Genetically engineered probiotics can be used to treat inflammatory bowel disorders such as Crohn's disease, ulcerative colitis, as well as other disorders that result from an overactive immune system. Genetically modified (GM) probiotics are also used as food additives to control the release of human growth factors by the modified bacteria to fight against injury and inflammation in the gut. For example the use of a plant sugar called xylan to stimulate the genetically modified human gut probiotic bacterium Bacteroides ovatus to produce specific proteins that can repair damaged cells and dampen down the immune system in the intestine that causes inflammation and disease. Studies have confirmed that administration of xylan with the genetically engineered probiotic bacteria resulted in a significant improvement of colitis, reduced weight loss, improved stool consistency, reduced rectal bleeding and accelerated healing of damaged colonic cells. GM probiotics posses potential in clinical applications like delivery of antigens for vaccines and thus are more readily accepted. This would provide a safer method of vaccination than the use of attenuated pathogens e.g. GM, Lactococcus lactis, produces IL-10 in the mouse intestine. The safety of such organisms that produce very powerful bioactive substances is of major concern as excess production of these substances in a healthy individual may be detrimental. INTRODUCTION Probiotics have many potential therapeutic uses,
African Journal of Basic & Applied Sciences 6 (3): 57-64, 2014
ISSN 2079-2034
© IDOSI Publications, 2014
DOI: 10.5829/idosi.ajbas.2014.6.3.8595
Corresponding Author: Charu Gupta, Amity Institute for Herbal Research & Studies,
Amity University UP, Sector-125, Noida-201303, India.
E-mail: charumicro@gmail.com.
57
Genetically Engineered Probiotics
Charu Gupta, Dhan Prakash and Sneh Gupta
12
Amity Institute for Herbal Research and Studies,
1
Amity University UP, Sector-125, Noida-201303, India
Department of Zoology, R.G.P.G. College,
2
Chippi Tank, Meerut-250001, India
Abstract: Intestinal microbiota is a key component of both the metabolism and immunity. These can be helpful
in healthcare, especially for the management of digestive diseases and food-borne illnesses. Through genetic
engineering it is possible to fully express biologically active copies of such powerful molecules from food and
commensal bacteria. Genetically engineered probiotics can be used to treat inflammatory bowel disorders such
as Crohn's disease, ulcerative colitis, as well as other disorders that result from an overactive immune system.
Genetically modified (GM) probiotics are also used as food additives to control the release of human growth
factors by the modified bacteria to fight against injury and inflammation in the gut. For example the use of a
plant sugar called xylan to stimulate the genetically modified human gut probiotic bacterium Bacteroides ovatus
to produce specific proteins that can repair damaged cells and dampen down the immune system in the intestine
that causes inflammation and disease. Studies have confirmed that administration of xylan with the genetically
engineered probiotic bacteria resulted in a significant improvement of colitis, reduced weight loss,
improved stool consistency, reduced rectal bleeding and accelerated healing of damaged colonic cells.
GM probiotics posses potential in clinical applications like delivery of antigens for vaccines and thus are more
readily accepted. This would provide a safer method of vaccination than the use of attenuated pathogens
e.g. GM, Lactococcus lactis, produces IL-10 in the mouse intestine. The safety of such organisms that produce
very powerful bioactive substances is of major concern as excess production of these substances in a healthy
individual may be detrimental.
Key words: Bowel Disease Colitis Genetically Engineered Inflammation Probiotics
INTRODUCTION Probiotics have many potential therapeutic uses,
Probiotic micro-organisms have been used since
several years. Originating as food supplements, they are
now most often administered orally and offer an attractive
alternative for treatment of intestinal disorders. Lactic acid
bacteria (LAB) have proved to be effective mucosal
delivery vectors. Moreover, some specific strains of
LAB exert beneficial properties (known as probiotic
effect) on both human and animal health. Although
probiotic effects are strain-specific traits, it is
theoretically possible, using genetic engineering
techniques, to design strains that can exert a variety of
beneficial properties [1].
but have not been universally accepted due to the lack of
understanding of their action. LAB has been modified by
traditional and genetic engineering methods to produce
new varieties. Modern techniques of molecular biology
have facilitated the identification of probiotic lactic
acid bacterial strains, but only a few LAB have been
modified by recombinant-DNA technology because
of consumer resistance to their introduction to markets
[2].
Lactococcus lactis is a food-grade, non-colonizing,
non-invasive, Gram-positive bacterium, widely used in the
preparation of food products and recognized as being
African J. Basic & Appl. Sci., 6 (3): 57-64, 2014
58
completely safe for human consumption. These LAB can In addition, the treated mice had 90% less inflammation in
be genetically modified to efficiently express any desired their colon tissue than did their untreated counterparts
protein or peptide without disturbing their biological [6].
activity. Inflammatory bowel disease is one of the prime areas
When delivered in the gastrointestinal tract, of interest for probiotic treatment, but there is little
genetically modified L. lactis bacteria reside there for evidence to show that probiotics are effective in treatment
8–48 h, during which time they continuously release or prevention of Crohn’s disease. Some studies suggest
therapeutic amounts of biologically active proteins. that two different probiotic formulations can help prevent
New prototypes of genetically modified L. lactis strains recurrence of ulcerative colitis. The mechanism of action
expressing single as well as multiple human â cell-specific of how probiotic bacteria influence the gastrointestinal
Ag (s) are currently under investigation. Different safety system is still unknown but previous research suggests
agencies have already given their approval to the use of several possible mechanisms. Beneficial bacteria might
transgenic bacteria, so a phase I trial has been performed temporarily alter the ratio of good to bad bacteria that
in Crohn’s disease and the other is in progress for oral inhabit the intestine, or they might specifically block
mucositis [3]. activity of bad bacteria. Probiotics seem to influence the
A better understanding of the mechanisms by which immune system by stimulating protective immune cells or
these micro-organisms act has now opened up blocking detrimental activities of immune cells [7].
possibilities for designing new probiotic strains. Researchers looked in detail at the molecular effects
Through genetic engineering, it is possible not only to of the engineered bacteria and found that the production
strengthen the effects of existing strains, but also to of regulatory immune cells, rather than inflammatory
create completely new probiotics. These need not immune cells was enhanced. The regulatory T cells,
necessarily be composed only of bacterial products a type of immune cell, counteract the effects of harmful
but can also include elements of regulatory systems immune cells that attack the cells lining the gut [8].
or enzymes derived from a foreign-human-source. According to another study, probiotic treatments do
If designed carefully and with absolute attention to not have the same effects in humans as in animals and
biological safety, the development of genetically there is a big difference in showing protection in animal
modified probiotics like Lactococcus lactis has the models (by treating the animals before symptoms occur)
potential to revolutionize alimentary health by secreting versus treating ongoing human disease [9].
the actives of interest [4]. It is an elegant strategy for gut Before beginning clinical tests, it is important to
mucosal delivery. study the roles of more kinds of surface proteins in the
Type-1 diabetes is an autoimmune disease against engineered bacteria, to determine which are helpful and
pancreatic beta cells and the scientists have demonstrated which are harmful. If the molecules, produced by the
that oral treatment of diabetic mice with genetically bacteria, can be identified that regulate the immune
modified L. lactis strain to secrete human pro-insulin and system, they may be used to develop drugs that have a
the immuno-modulatory cytokine IL-10 that is able to similar effect. It is easier to modify and give
restore the tolerance of pancreatic beta cells in a safe, controlled doses of chemical compounds than live
specific and long-lasting way [5]. bacteria, which can behave unpredictably once ingested
In another study, scientists deleted a gene from the [4].
bacterium Lactobacillus acidophilus which is commonly Engineered probiotics as a treatment for colon
found in yoghurt. This gene is responsible for producing cancer can also be explored. In preliminary studies in mice
an enzyme that increases inflammation, a defining designed to mimic colon cancer, treatment with the
characteristic of Crohn’s disease and ulcerative colitis. modified bacteria reduced the number of polyps the
But the unaltered form of the bacterium also triggered animals developed by 90 percent. The scientists observed
production of a beneficial immune molecule, an average of only three small polyps in treated mice,
Interleukin-10 that helps to regulate the immune system. compared to about 35 to 50 in untreated [10].
The goal of engineering the microbes was to deliver the The bacteria’s ability to reduce inflammation is not
beneficial effects without the harmful impact [6]. limited to the gut but the regulatory cells migrate
When the engineered microbe was fed to mice throughout the body. It is hypothesized that these
suffering with colitis and inflammation of the colon, microbes may also be able to help treat other diseases
the engineered bacteria prevented the weight loss and linked to inflammation, such as rheumatoid arthritis and
bloody diarrhea that typically accompanies this condition. psoriasis [11].
African J. Basic & Appl. Sci., 6 (3): 57-64, 2014
59
Bioengineered probiotics expressing foreign gene modified probiotic while those that did not received
products to achieve inhibitory effects against food-borne remained infected. This probiotic is a food-grade bacterial
pathogens is highly desirable. Enteric infections account product that can be easily produced in large quantities in
for high morbidity and mortality and are considered to be a simple fermenter and it can be manufactured in a form
the fifth leading cause of death at all ages worldwide. that has a long shelf-life and is also cost effective [2].
Significant efforts have been directed toward the Bt is attractive because it is a well-understood,
detection, control and prevention of food-borne diseases. natural substance for controlling plant pests that is
Many antimicrobials including antibiotics have been used believed to be safe for animals and humans. It is
for their control and prevention. However, probiotics offer frequently sprayed on organic crops and is mainly
a potential alternative intervention strategy owing to their lethal to insects in their larva stage. Bt is also a
general health beneficial properties [12]. bacteria used in genetically engineered corn and
Genetically engineered probiotics are also useful in soybean to endow the crops with resistance to plant
the treatment of parasitic infections. In a study, pests. It can be used as an effective intervention for
laboratory animals that were fed a modified version of a intestinal worms as a treatment and not as a dietary
common genetically modified probiotic were completely supplement [12].
cured of intestinal worms that belong to a family of However, these results need to be replicated in other
parasites. It is believed that around 1.5 billion people are animals and also in humans but this is an important
currently infected with the intestinal worms (Annual development to find a safe, affordable and effective way
meeting of the American Society of Tropical Medicine and to confront a major global health problem.
Hygiene (ASTMH). Soil transmitted helminthes (STHs)
and other intestinal worms, though rarely fatal, are leading Inter-relationship Between Human and Bacteria:
contributors to disease in school-age children in The human gastrointestinal tract microbiota consists of a
low-income countries and are amongst the most large number of bacteria around 10-fold more than the
burdensome of the world's "neglected tropical diseases" total number of human cells that plays an important role
or NTDs. STHs are found in soil that has been in several physiological and metabolic processes and in
contaminated with human feces. Hookworms can linger in the development of immune system. The collective adult
the intestines for years, where they feed on blood and human gastrointestinal tract microbiota is composed of
tissue, robbing their hosts of iron and protein upto 1000-1200 bacterial species [7, 13] with Gram-positive
and interfering with absorption of critical nutrients. bacteria as the predominant species. Clostridium group is
They frequently cause stunting and cognitive delays in the most abundant in this complex ecosystem [13].
infected children. They also can have long-term effects on This micro-ecosystem is fundamental for the
educational achievement and productivity [4]. maintenance of homeostasis of a healthy individual [10].
The only drugs available to treat hookworms in This is a perfect example of mutualism between the host
humans were originally developed to combat parasites and bacteria where commensal bacteria provides essential
that infect farm animals. They were only partially effective nutrients to the host, metabolize indigestible compounds,
against the range of intestinal parasites that infect defend against colonization of opportunistic pathogens
humans. There is also evidence of re-infections occurring and contribute to the development of intestinal
rapidly after treatment and low levels of efficacy in some architecture and stimulates the immune system [14];
cases. whereas host provides nutrients and a stable environment
In an experiment, hamsters were deliberately infected to the bacteria [10].
with hookworms and then divided into two groups.
One group received a bacteria Bacillus subtilis, which is Advantages of Probiotics for Human: The concept of
often marketed as a "probiotic". The other group received probiotics is based on the observations made by Elie
the same probiotic, except the researchers modified it to Metchnikoff (1907) in which the regular consumption of
express a protein derived from a closely related bacterium, lactic acid bacteria in fermented dairy products such as
Bacillus thuringiensis or Bt, which is known to be safe in yoghurt was associated with enhanced health and
humans but potentially lethal to intestinal worms. After longevity in many people [15]. Most probiotics belong to
five days of administration of bacteria, the intestine of group of LAB but some species and genera belong to
the animals was examined for the presence of worms. other group also. Notable examples of probiotics are
There were no worms in the animals that received the Bfidobacterium sp., Escherichia coli Nissle 1917 and
African J. Basic & Appl. Sci., 6 (3): 57-64, 2014
60
east Saccharomyces boulardii [16]. The important criteria [25-27]. Moreover, LGG is also found effective in reducing
for a microorganism to be considered as probiotic are: i) it atopic dermatitis as it enhances the production of the
should have a clear beneficial effect on the host; ii) it anti-inflammatory cytokine IL-10 [28]. Oral administration
should be non-pathogenic; iii) it should be able to survive of L. cassei Shirota strain inhibits specific IgE production
transit through the GIT; iv) large number of viable bacteria [29].
must be able to survive prolonged periods upon storage There are various reports that have confirmed that
[17]. selected strains of probiotics that have been
Microbial Imbalances: Dysbiosis: Dysbiosis is microbial IBD, e.g. L. casei BL23 strain has shown anti-inflammatory
imbalance within the body. It usually occurs when the effects in an animal model, other probiotic strain like
homeostasis (balanced microbial ecosystem) is broken VSL#3, LGG, BIFICO, E. coli Nissle) have shown positive
due to overgrowth of opportunistic bacteria leading to a effects in human trials suffering with pouchitis, UC and
situation of illness. The apt correlation between dysbiosis CD [11]. Likewise Lactobacilllus acidophilus has a
and disease has been found with inflammatory bowel modulatory effect in intestinal pain [30].
disease (IBD) where the proportion of Firmicutes, From the above studies, it is pertinent to say that
particularly Faecalibacterium prausnitzii was found to equilibrium of the microbiota in the gastro-intestinal tract
be low in patients that exhibited endoscopic recurrence is very important as its disturbance can contribute to
6 months after surgery [6, 18]. The composition of human several intestinal diseases such as IBD. Gut microbiota
microbiota is different in each individual, changes in performs various functions like metabolize nutrients,
phylogenic distribution have been found in obese produce vitamins and degrade toxic products such as
and diabetic individuals versus normal ones [19, 20]. carcinogens, food additives, bile salts and cholesterol etc
The importance of human microbiota has been explained [12]. Probiotic bacteria are also helpful in intestinal
in hygiene hypothesis [21] which described that low disorders like IBS (irritable bowel syndrome), that is
exposure to infectious agents in early life explains the characterized by chronic abdominal pain, discomfort,
increased number of people suffering from allergies and bloating and alteration of bowel habits.
asthma in developed countries. This hypothesis
suggests that a well-balanced human microbiota is a Genetically Modified Microorganisms: New Prospects
factor that protects from such pathologies [22, 23]. Delivery of Therapeutic Molecules: Administration of
There is some inter-relationship between microbial probiotics is a natural way to treat dysbiosis within the
activities and health and disease. The production of GIT. Commensal bacteria can be genetically modified to
short chain fatty acids such as butyrate has been function as potential live delivery vectors for target
proposed to protect against different illnesses [9, 24, compounds such as food grade LAB at the mucosal level
25]. [31, 8]. Recombinant bacteria are used for producing the
Probiotics for Dysbiosis: The administration of beneficial as vaccines. The main advantage of using the genetically
microorganisms to restore the normal ecosystem is a engineered bacteria or viruses is that they can be used as
strategy to improve the health status of the patient and/or carriers to produce heterologous antigens that increase
to prevent a normal healthy individual from acquiring the immunogenicity of weakly immunogenic antigens.
dysbiosis in the future. There is scientific evidence on The live bacterial vectors have an additional advantage
the use of probiotics as therapeutics against over viruses that they have a genome that is able to
traveler’s diarrhea, irritable bowel syndrome (IBS), IBD, harbor many heterologous genes, in contrast to
lactose intolerance, peptic ulcers, allergy and autoimmune viruses where the capacity to encapsulate foreign DNA
disorders among others [24, 9]. The colonization of GIT is limited. This is an effective strategy to deliver
with Bifidobacteria properly shapes gut microbiota, therapeutic molecules to the mucosal tissues in order
induces oral tolerance and decreases the frequency of to avoid degradation and promote uptake of the
allergic disorders [25]. Perinatal administration of a antigen in situ and stimulate adaptive immune
probiotic strain of Lactobacillus rhamnosus GG (LGG) responses rather than the tolerogenic immune responses
reduces the development of atopic eczema in children due that are observed in feeding studies with soluble
to the anti-inflammatory properties of this probiotic antigens [32].
genetically modified has various therapeutic effects in
heterologous molecule of interest in vivo that can be used
African J. Basic & Appl. Sci., 6 (3): 57-64, 2014
61
There are two types of bacterial vectors used to Recently LAB secreting elafin have been tested in
deliver compounds at the mucosal level: attenuated chronic and acute colitis models and it was found that
pathogens and non-pathogenic bacteria. Gram-positive inflamed epithelium was protected from increased
commensal or food-grade bacteria constitute good intestinal permeability and from the release of cytokines
alternatives to pathogenic bacteria [33]. The reason and chemokines by LAB secreting elafin [39]. In addition
behind using food-grade LAB is that they are used in the to these recombinant probiotic strains, mutants in specific
fermentation and preservation of food and is categorized genes encoding potential probiotic functions (mucosal
under GRAS (Generally Recognized as Safe). The other adhesion factors, resistance to acid, specific cell wall
advantage of LAB as delivery vectors for vaccine components etc.) have been engineered to compare their
purposes is due to their potential to elicit both biological effects with that of their wild-type counterparts.
antigen-specific immune responses at mucosal surface Grangette et al. [40] has used a mutant of
and effective systemic immune responses. Studies Lactobacillus plantarum with impaired capacity to
have shown that LAB vaccines elicit antigen-specific incorporate D-alanine in teichoic acids [40]. This mutant
IgA response in faeces, saliva or broncho-alveolar was found to be more protective in a mouse model
and intestinal lavage fluids and antigen-specific IgA- of colitis than the wild type strain. Moreover,
secreting cells in the lungs and mesenteric lymph nodes the lipoteichoic acid deficient L. acidophilus enhances
[34]. IL-10 production by dendritic cells and macrophages and
Lactococcus lactis is a model LAB species that is down-regulates IL-12 and TNF-á under in vitro conditions
used for the heterologous expression of therapeutic [41].
proteins such as antigens, cytokines and enzymes. As discussed in the above paragraphs that
Recombinant LAB and Commensals in IBD: production of heterologous proteins and is considered as
The conventional therapies for IBD are anti-inflammatory the live delivery vector model [42]. But the main drawback
drugs combined with immunosuppressive agents [35]. is that this bacterium has a short survival time (~24 hrs) in
Recombinant L. lacis strain is used for producing and the human GIT thus leading to a reduced time of action.
delivering IL-10, an anti-inflammatory cytokine, in situ in As an alternative, Bacteroides ovatus was selected
different mouse models [36]. It has been shown that daily over L. lactis by Hamady et al. [43] to deliver the proteins.
mucosal administration of recombinant L. lactis secreting This bacterium has the ability to colonize the colon
IL-10 led to a 50% decrease of the colitis that was induced and can utilize xylan. They were able to develop a
by the administration of dextran sodium sulphate (DSS) in xylan-regulated delivery of human keratynocyte growth
mice. This beneficial effect was dependent on the in situ factor-2 to the inflamed colon [44] and the human TGF-b1
secretion of IL-10 by the live lactococci. The delivery of to treat colitis in mice [45]. These promising results
immunosuppressive cytokines by other bacteria has also confirm the potential of the use of recombinant
been studied e.g. probiotic potential of IL-10 expressing commensal for in vivo delivery.
E. coli Nissle 1917. The use of LAB to produce Trefoil
Factors (TFF) at the mucosal level to treat IBD has been Future Prospects: Public interest in probiotics is on the
also studied. TFF are a class of non-mitogenic peptides upswing. Scientists are bringing this traditional therapy
that helps in the protection and repair of the intestinal into the 21st century by genetically engineering the
epithelium [37]. These peptides have strong protective microbes to enhance their effect on the immune system.
effects for repairing mucosa after damage. They are They hope that the new bugs will ultimately help in the
usually administered by an oral route where they are treatment of inflammatory bowel diseases such as Crohn’s
absorbed at the intestinal level. Intra-gastric disease and ulcerative colitis, as well as other disorders
administration of recombinant L. lactis secreting TFF that result from an overactive immune system.
leads to the expression of the active peptides in the Current probiotic research encourages the search and
colon and prevents and repairs damage due to acute characterization of gut bacteria as a model for finding new
colitis [38]. natural and/or engineered probiotic strains that can be
Recombinant L. lactis was also used to treat colitis used to restore the normal balance of the human gut
that secretes Lcr V antigen, anti-inflammatory protein ecosystem. The complete knowledge of molecular
produced by Yersinia pseudotuberculosis to escape the mechanisms and active compounds in commensal and
immune response of the host. probiotic strains is still lacking. Taking into account
Lactococcus lactis is the most widely used LAB in the
African J. Basic & Appl. Sci., 6 (3): 57-64, 2014
62
that the human GIT is composed of 10 -10 anti-inflammatory commensal bacterium identified
13 14
microorganisms, it is important to focus on the exact role by gut microbiota analysis of Crohn disease patients.
of bacterial active compounds in homeostasis and immune Proceedings of National Academy of Sciences USA,
response. 105: 16731-16736.
ACKNOWLEDGEMENTS Boedeker, N. Harpaz, N.R. Pace, 2007. Molecular-
Authors are grateful to Dr. Ashok K Chauhan, imbalances in human inflammatory bowel diseases.
Founder President and Mr. Atul Chauhan, Chancellor, Proceedings of National Academy of Sciences USA,
Amity University UP, Noida, India for the encouragement, 104: 13780-13785.
research facilities and financial support. 8. Bermúdez-Humarán, L.G., C. Aubry, J.P. Motta, C.
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... In a recent study reported by Kawashima et al. (2018), it is shown that the production of IgA antibody induced via IL-10 in mucosal sites of host is a host defence mechanism against the pathogens. The genetically engineered Lactococcus lactis strainproduced anti-inflammatory mediator such as IL-10 and IL-12 showed reduction of dextran sodium sulfate-induced colitis in mice models (Gupta et al. 2014). In another study reported by Ozdemir (2010), it was found that there is a reduction of IgE levels in allergic diseases. ...
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