Steidler Lothar

7/29/2019 Steidler Lothar

1/16

12

Genetically engineered probiotics

Lothar SteidlerProfessor

Departments of Medicine and Microbiology, Alimentary Pharmabiotic Center, University College Cork,

Western Road, Cork, Ireland

Probiotic micro-organisms have been used for many years. Originating as food supplements, theyare now most often administered orally and offer an attractive alternative for treating of intestinaldisorders. A better understanding of the mechanisms by which these micro-organisms act hasnow opened up possibilities for designing new probiotic strains. Through genetic engineering, it ispossible not only to strengthen the effects of existing strains, but also to create completely newprobiotics. These need not necessarily be composed only of bacterial products but can also

include elements of regulatory systems or enzymes derived from a foreignhumansource. Ifdesigned carefully and with absolute attention to biological safety in its broadest sense, thedevelopment of genetically modified probiotics has the potential to revolutionize alimentaryhealth.

Key words: genetic engineering; mechanisms of probiotics; Lactobacillus; Lactococcus lactis; in situdelivery; heterologous gene expression; biological containment.

INTRODUCTION

Conducting reliable double-blind experiments allows researches to establish a soundknowledge of the effects of probiotic strains. Speculations on mechanisms of action

have been developed and tested through detailed microbiological, molecular biological,immunological and physiological analyses. In this chapter, genetically engineeredprobiotics will be addressed. The title is of dual connotation: the genetic engineering ofa previously non-probiotic to acquire probiotic properties and the genetic engineeringof a known probiotic to enhance its probiotic properties, both leading to the creationof new, genetically manipulated (GM) probiotics. A knowledge of the mechanism bywhich defined probiotic strains contribute to health allows the rational design ofprobiotic micro-organisms through genetic engineering. Existing mechanisms can bestrengthened, or mechanistic pathways present in different strains can be combined, toyield more potent strains. From the existing literature, it is clear that completely newconcepts can be addressed so one can now create additional mechanisms to enhanceprobiotic activity.

1521-6918/03/$ - see front matter Q 2003 Elsevier Ltd. All rights reserved.

Best Practice & Research Clinical GastroenterologyVol. 17, No. 5, pp. 861876, 2003doi:10.1016/S1521-6918(03)00072-6, www.elsevier.com/locate/jnlabr/ybega

E-mail address: [email protected] (L. Steidler).
http://www.elsevier.com/locate/ybegahttp://www.elsevier.com/locate/ybega


2/16

IS THERE A GENUINE NEED FOR GM PROBIOTICS?

Probiotics used in the treatment of inflammatory bowel disease (IBD) occupy a specialplace. Not only does oral intake provide excellent targetting of the active biologicalagent to the affected site, but the use of probiotic therapies in IBD is also especiallytempting because of the key role of the intestinal microbiota in the onset andmaintenance of mucosal inflammation. Because of the central position of the intestinal

microflora in the development of IBD, manipulating this component provides asintriguing new therapeutic approach.

In this context, a number of examples can be cited. Escherichia coliNissle 19171,2 andthe combined probiotic preparation VSL#3 are, for example used in remissionmaintenance therapy for ulcerative colitis.3 The VSL#3 preparation is also effective inpreventing flare-ups of chronic pouchitis.4 Lactobacillus plantarum decreases pain andflatulence in patients with irritable bowel syndrome5, and Lactobacillus GG improves theclinical status of children with stable Crohns disease.6 In addition, Bifidobacteriumbifidum and Streptococcus thermophilus can reduce the incidence of acute diarrhoea androtavirus shedding in infants.7 Saccharomyces boulardii, a non-pathogenic yeast, can beused in the maintenance treatment of Crohns disease when combined withmesalamine8 and shows activity in the treatment of various diarrhoeal diseases.Lactobacillus GG significantly improved dermatitis in infants with atopic eczema andcows milk allergy. The uptake of the probiotic was further associated with a drop in

fecal tumour necrosis factor and a1-antitrypsin.9

From this, it can be appreciated that,although very promising, probiotic therapies for the treatment of IBD currently appearto be effective only in therapy to in maintain remission.

To allow more active intervention in so-called critical care patients, i.e. patients inneed of acute immunosuppressive intervention, more powerful probiotics need to bedesigned and constructed with the aid of genetic engineering tools. Such new strainsmight utilize mechanisms similar to those used by standard non-engineered strains, butthe way in which they are designed may allow for a stronger impact.

NEW PROBIOTIC MECHANISMS ENGENDERED THROUGHGENETIC ENGINEERING

In order to create novel GM probiotic strains, it is essential to understand the differentpossible mechanisms of action. The effects of probiotics in IBD are manifold and arediscussed in detail in other chapters. They involve most of the interactions between thehost and its commensal microflora. Some strains alter the microbial content anddisplace noxious bacteria and toxic compounds. Others, however, show a directinfluence on immune cells, thereby mainly lowering the immune response andenhancing tolerance, or increasing epithelial barrier integrity.

In the recent literature, a number of studies have been reported that utilize a diversecombination of tools to design probiotics. Heterologous protein expression is anobvious approach. In this, one can make use of expression systems from native cDNAclones as well as of elaborate products of modern DNA technology such as syntheticgenes and designed proteins. One can also expand the range of possible activecomponents beyond protein therapeutics by engineering the metabolism of micro-organisms through the integration of foreign enzymes. This so-called metabolicengineering now allows the use of, for example, modified lipopolysaccharide anddetoxification via the incorporation of new metabolic pathways.

862 L. Steidler


3/16

Sequestration of toxins

Paton co-workers10 have constructed an E. coli strain that could answer the need fortherapeutics to counteract intestinal infection with Shiga toxigenic E. coli (STEC) andother Shiga toxin (Stx)-producing bacteria such as Shigella dysenteriae, the causativeagent of bacillary dysentery. Stx is a compound toxin, composed of a catalytic A subunit,the actual toxin and a pentameric B subunit that is responsible for binding to its

receptor, globotriaosyl ceramide (Gb3, or Gala[1 ! 4]Galb[1 ! 4]Glc-ceramide,found in all human pathogens) or globotetraosyl ceramide depending on the Stx type.Infection and pathology are initiated by a colonization of the gut with STEC thatproduces Stx locally. The toxin is then taken up in the circulation and concentrated atGb3-containing tissues such as the kidneys, possibly causingamong other effectsfatal renal failure.

STEC infection can now be rapidly diagnosed, but therapeutic tools against it are notavailable. Antibiotic treatment leads to a sudden release of surface-associated Stx fromthe pathogen, which only aggravates the situation. By introducing the genes lgtC and lgt Efrom Neisseria meningitidis and N. gonorrhoeae, respectively, encoding glycosyltransferases that account for the incorporation of the oligosacharide structureGala[1 ! 4]Galb[1 ! 4]Glc into E. coli, a strain with an altered LPS structure wasobtained. This strain displayed high binding activity for Stx. When administered twicedaily to mice infected with STEC, thenew strain provided complete protection, the mice

in the control population all dying. This research thus provides a recombinant probioticthat has now acquired the potential to sequester an extremely toxic compound. It fills agap in medicine that has not been met by chemically engineered therapeutics as theauthors have demonstrated the superior capacity of their strain over Gala[1 ! 4]Galb[1 ! 4]Glc covalently linked to silica particles. This is a elegant example of howactive components engineered into existing strains need not be limited to polypeptides.

Replacement therapy

A subset of probiotic therapies can be grouped as replacement therapies, representingan approach whereby a noxious micro-organism is replaced in its ecological niche by amore potent but harmless competitor. Replacement therapy is an innovative approachto preventing microbial diseases. Its origin lies in the complex web of positive andnegative interactions that exist between different species of bacteria within the sameecosystem. Some of these therapeutic approaches have been described above whendiscussing the mechanisms underlying probiotics.

The great potential of genetic engineering to produce such desirable potent yetharmless strains is most elegantly documented in a study conducted by Hillman. Dentalcaries is caused by Streptococcus mutans11, according to the acidogenic theory by theproduction of lactic acid. Therefore, the Streptococcus mutans ldh gene, encoding lactatedehydrogenase, can be considered to be a pathogenic factor. The closely related lactatedehydrogenase-deficient Streptococcus rattus is non-cariogenic in gnotobiotic rats12;Hillman therefore cloned the Streptococcus mutans ldh gene.13 It was, however, shownthat, because ldh is an essential gene in Streptococcus mutans, only temperature-sensitivelactate dehydrogenase-deficient strains could be obtained.14 Aspects of glucosemetabolism appeared to be toxic for growth in non-permissive conditions, but thiscould be overcome by supplemental alcohol dehydrogenase activity.15 A targettedreplacement of the Streptococcus mutans ldh gene in strain JH1140 for the Zymomonasmobilis adh gene yielded the recombinant Streptococcus mutans BCS3-L1, a viable strain

Genetically engineered probiotics 863


4/16

that makes no detectable lactic acid. The mutant therefore typically produces a higherpH after fermentation of various sugars than its parent. Consequently, BCS3-L1 hasdramatically lower cariogenic potential (Figure 1).

Many studies have shown that it is very difficult to displace indigenous Streptococcusmutans with laboratory strains. Hillman, however, selected a natural strain thatproduced a potent lantibiotic bacteriocin, mutacin 1140, which was capable ofaggressively displacing indigenous Streptococcus mutans and colonizing the oral cavity forprolonged periods.16,17 It was this strain, JH1140, that was used for the gene exchangedescribed above. Trials on the effect of this new probiotic for the prevention of dentalcaries in humans will be started in 2003 (J. Hillman, pers. comm. 2002). The preventionof dental caries would be an example of biofilm engineering that offered the potentialfor a highly efficient, cost-effective addition to existing prevention strategies. The use of

this approach might also be applied to the prevention of other bacterial diseases.

Strategies that involve antibody production

Neutralizing antibodies that are directed towards a pathogen, toxin, cytokine or otheragent have proved very valuable and specific tools in medicine. With the emergence ofsingle chain (ScFv) antibody technology, it has now become possible to produceneutralizing antibodies from recombinant bacteria. Most of the work in this area relatesto the production per se of the antibody for downstream processing and use as apurified protein. Owing to their structure, these peptides suffer from very short half-lives in vivo so suitable delivery systems are required to allow their use as therapeutics.A number of applications are now emerging in which the expressor strain itself is usedfor the in situ production of the antibody fragment especially to control colonization bypathogens.

Figure 1. (Top) The high incidence and severity of tooth decay in the molar teeth of a rat that was infectedwith a normal acid-producing strain ofStreptococcus mutans and fed a high-sugar diet for 2 months. (Bottom)

The molar teeth of a rat treated in the same fashion but infected with the modified strain of Streptococcusmutans that is unable to make lactic acid. Courtesy of Dr J. Hillman.

864 L. Steidler


5/16

Next to the earlier discussed replacement therapy, a second strategy to intervenewith Streptococcus mutans has been presented by Kruger et al18 It had been observedthat the oral administration of antibodies against Streptococcus mutans reduced theincidence of caries.19,20 Along the same lines, antibodies that recognize the SAI/IIadhesion molecule ofStreptococcus mutans, so called Guys 13 antibodies21, are effectiveby agglutinating the pathogen. Lb. zeae have therefore been engineered either tosecrete or to surface expose Guys 13 ScFv. All ScFv showed the appropriate reactivity

with SAI/II adhesin. Lb. zeae with surface bound ScFv coagulated with Streptococcusmutans suspensions. The oral inoculation of rats with the surface expresser at 2 dayintervals resulted in a marked decrease in the Streptococcus mutans count and theconcomitant development of dental caries.

An even more elaborate approach has been used to combat Candida albicans, one ofthe most frequent causative agents of mucosal inflamation in humans.22 Infections areseen in the mouth and oesophagus of immunocompromised persons such as HIV-infected subjects. Candida albicans also causes acute vaginitis in otherwise healthywomen. There is a real need for new therapeutic agents in this area because fewadequate agents are known, resistance is increasing, and vaccines are not available.Beninati et al23 have produced a recombinant Streptococcus gordonii, a species with goodvaginal colonization and heterologous expression potential in vivo2426, for theeradication ofC. albicans infections. Anti-idiotypic ScFv were produced, the surface ofwhich resembled the structure of a wide-spectrum killer toxin of Pichia anomala.27,28

Two Streptococcus gordoniistrains were constructed: one that expressed the ScFv at itssurface and a second that secreted the ScFv. Similarity in structure to the killer toxincould be shown by cross-reactivity with specific monoclonal antibodies. Both surface-bound and secreted ScFv showed candidacidal activity over a wide concentration range.Both Streptococcus gordonii strains successfully colonized the vagina and clearedexperimental C. albicans infection in rats, this being dependent on the presence of thisScFv. The secretor, however, showed a 7 days faster reduction of the pathogenic load, aresult comparable with a full course of treatment with the antifungal fluconazole. Thiswork shows that local production of a designer microbicide is a valid approach for thetreatment of a very common mucosal pathology.

Enzyme deficiencies

Non-GM probiotics can contribute to the health of individuals suffering from enzymedeficiencies when the enzymatic conversion that is required is common to themetabolism of the bacterium. The treatment of lactose maldigestion by variouslactobacilli29,30 can serve here as an example. Genetic engineering has, however, thepotential to expand dramatically the range of enzyme deficiencies that can be addressed.

Drouault et al31 have worked along these lines by constructing Lactococcus lactisexpressing the lipase from Staphylococcus hylicus. The aim was to provide a moreadequate treatment of pancreatic insufficiency. The treatment of such diseases suffers atpresent from the poor performance of the pancreatic enzymatic preparations that areavailable. Drouault et al thus built a L. lactis strain that carried the lip gene under thecontrol of the nisin promotor. Upon induction, the lipase accumulated intracellularly upto a level of 15% of the total protein. The pancreatic duct of pigs was then ligated andcut to simulate disease. This resulted in an approximately 20% decrease in fatabsorption (nearly 100% in healthy compared with approximately 80% in the resectedanimals), as determined from the ratio between ingested and excreted fats. Fatabsorption was 10% higher in those animals treated with the lipase expressor strain.



6/16

In this study, use was made of the release of intracellular material from L. lactis after ithad been killed in the upper part of the small bowel.32

Detoxification

Over the past century, human activity has caused a rapid alteration in our environment,accompanied by the often-unwanted accumulation of numerous chemical agents. As

intrinsic inhabitants of this changing environment, we are obliged to face these changes.This means that humans have become confronted, on a totally different time scale fromthat of biological evolution, with a varying spectrum of new and difficult to handleresidues of toxic compounds. We are therefore often not (yet) equipped with adequatecatabolic enzymes to allow for their conversion and elimination. More specifically, suchcompounds are taken up via nutrition, respiration and skin contact, whereby some mayaccumulate, mostly as a consequence of their lipophilic nature. Through the geneticengineering of micro-organisms, one can now build systems for in situ detoxification orbiodetoxification. In this way, exoticeither discovered or designedenzymes thattransform noxious chemicals into harmless ones could be applied in human health-care.

An in road in to this area has now been made by adapting existing detoxificationsystems so that they can be used in a more potent way. Xenobioticschemicalcompounds (drugs, pesticides or carcinogens) that are foreign to living organismsaredetoxified in a two-stage process. This occurs through phase I and II xenobiotic-metabolism enzymes present in the cells of the liver, intestine, kidneys and lung. Phase IXMEs mainly P450 cytochromes, convert the xenobiotics into more reactivecompounds that are further metabolized by phase II xenobiotic-metabolism enzymessuch as glutathione-S-transferase, N-acetyltransferase and aldehyde dehydrogenase.

Blanquet et al33 have chosen to use genetically engineered Sacharomyces cerevisiaethat produces the plant cytochrome P450 73A1 (cinnamate-4-hydroxylase) andoverexpresses yeast NADPH cytochrome P450 reductase.34 This recombinant strainconverts trans-cinnamic acid into p-coumaric acid. To investigate whether such systemwould allow in vivo biodetoxification using P450 cytochromes, they performed a studyusing two-stage in vitro intestinal simulation of the stomach and small intestine (TIM135) and large intestine (TIM 236). The recombinant yeast appeared to be very resistantto the conditions in TIM 1 but more sensitive to TIM 2. In both compartments, theconversion of trans-cinnamic acid into p-coumaric acid, which accumulated to 41% inTIM1, could be observed.

This shows that, using this technology, a plant P450 cytochrome, i.e. an enzyme of awidely disparate evolutionary origin, might be used in a human intestinal environmentfor the detoxification of nutrient-borne chemical contamination. Such an approachwould intrinsically preclude their accumulation in the cells of the body that normallyperformed detoxification and thereby avoid the formation of residues. This is thereforean elegant adaptation of existing strategies of detoxification. There are, however,obvious drawbacks to the use of in vitro intestinal simulation machines. The absence ofan immune system in particular strongly compromises ad hoc extrapolation to usage inhumans.

Metabolic deficiencies resulting from trauma, disease and age may cause theaccumulation of waste products that are otherwise removed from the body to levelsthat are unacceptable for the adequate functioning of the organism. Examples thereofare the accumulation of urea, uric acid or creatine as a result of renal failure, ammon iafrom liver failure and phenylalanine because of phenylketonuria. Prakash and Chang37

have developed E. colithat express Klebsiella aerogenes urease. The oral administration

866 L. Steidler


7/16

of a formulation of these live bacteria, microencapsulated in alginatepolylysine beads,to rats with experimental renal failure led to a significant and substantial reduction inplasma urea, uric acid and, to a lesser extent, of creatine.38,39 In rats, the microcapsulesleave the body with the faeces. The microencapsulation shields the recombinantbacteria from the harsh environment in the digestive tract, and it is likely that thisphysical barrier can also prevent the onset of an immune reaction towards thetransgene. The authors estimate that daily administration of 4 g of this formulation

could normalize the urea concentration in a 70 kg patient suffering from renal failure.

Immune intervention

Immune intervention strategies are those in which the immune system is activelyengaged for the acquisition of health benefits. The immune system supervises all themolecules within an individual, its task being to discriminate friend from foe, protectingand maintaining the former and eliminating the latter. Intervention in terms of theimmune system may help to establish immunity or readjust inaccurate aspects ofimmune discrimination. Immunity against previously unencountered pathogens can beachieved through vaccination. A large body of literature exists on the use of lactic acidbacteria (the majority of current probiotics) as vaccine-delivery vehicles (see Ref. 40 fora recent review). This aspect of the use of GM lactic acid bacteria and other micro-organisms, falls, however, outside the scope of this article.

The immune system constitutes a complex network of cells that are in constantcommunication with one another and with the somatic cells of the body. Thiscommunication occurs via secreted and diffusible mediators and through cell surfaceexposed ligands that encounter their cognate receptors on the surface of the targetcells. An important class of diffusible mediators is that of relatively small proteins calledcytokines, consisting of interleukins (ILs), growth factors, interferons and others. Thesemessengers are often active at a very low concentration and have therefore been usedextensively in various attempts to assist the immune system in acquiring immunity orre-establishing homeostasis in cases of malignant inflammation. When, however, theyare administered as purifiedrecombinantproteins via the systemic route, theydiffuse throughout the body and can cause an effect at sites, where these are unwanted,producing side effects. In some cases, such as immune intervention at the mucosa, onecould, however, speculate that localized delivery via the in situ synthesis of cytokines bygenetically engineered bacteria might prevent side-effects while still allowing thedesired biological effect. We have investigated whether genetically engineered L. lactiscan be used in such a strategy.

Wells et al had reported that, when the C fragment of tetanus toxin (TTFC) wasexpressed intra-cellularly in L. lactis, a protective immunity towards tetanus toxin couldbe provoked by repeated intra-nasal inoculation of that strain.41 In general, both IL-2and IL-6 act as potent stimulators in the onset and maintenance of immune reactions.To investigate whether the immune response to TTFC, responsible for the above-mentioned protection, could be enhanced or modulated via the intra-nasal delivery ofcytokines, we constructed strains of L. lactis that produced TTFC intracellularly andsecreted functional murine IL-2 or IL-6. Immunization was carried out, and the animalswere compared with individuals from the control TTFC-expresser strain. The anti-TTFC serum IgG response was significantly (up to 15 fold) higher in mice immunizedintra-nasally with the viable L. lactis that secreted IL-2 or IL-6. In addition, theconcentration of anti-TTFC serum IgA was considerably higher after immunization withthe IL-6-secreting strain.42 This shows that genetically engineered L. lactis can produce



8/16

IL-2 and IL-6 in the upper airway mucosa and thereby provoke a biological effect thatcan, because IL-6 is a B-cell growth and IgA secretion-stimulating factor, be steeredthrough the appropriate choice of cytokines.

IL-10 is a strong anti-inflammatory cytokine that has shown promise in clinical trialsfor the treatment of IBD.43 The application of this protein suffers, however, from anumber of disadvantages that may be overcome through targeted delivery in theintestine. Side-effects are induced following systemic administration4446, thereby

precluding its prolonged use at an elevated concentration. Moreover, IL-10 is extremelyacid sensitive, hindering any plans to circumvent the s ide-effects by targeted delivery viathe digestive tract.

IBD can be simulated in a number of mouse models. The repeated addition ofdextran sulphate sodium (DSS) to the drinking water of Balb/c mice leads to theinduction of chronic colitis.47 The histology of this condition that of is very similar toulcerative colitis, but the inflammation immunologically tends towards that of Crohnsdisease as it is associated with the upregulation of interferon-gamma, IL-12 and tumournecrosis factor and can be cured by the systemic administration of neutralizingantibodies against these proteins.48 We constructed L. lactis that could secrete bio-active mouse IL-10.49 The daily ingestion for 2 weeks of these L. lactis organismsresulted in 40% of the treated mice reaching a histological score equal to that of healthycontrol mice. The other animals showed only minor patchy remnants of theinflammation (Figure 2). As killing the IL-10-producing bacteria prior to inoculation

abrogated the curative effect, the mechanism of delivery seemed to act via the active invivo synthesis of IL-10. The observed healing was comparable to systemic treatmentwith prominent anti-inflammatory drugs (anti-IL12, dexamethasone and IL-10), but theamount of IL-10 required through L. lactis-mediated delivery was 10 000-fold lower,which is highly encouraging when the aim is to reduce side-effects. IL-102/2 micenormally develop colitis, but daily treatment with IL-10-producing L. lactis led to theprevention of this colitis. Furthermore, it was possible in IL-102/2 mice to demonstratephysically denovo IL-10 synthesis. We thus demonstrated that L. lactis can deliver IL-10,a very sensitive protein, to the intestinal mucosa of mice and thereby prevent or cureexperimental colitis.

Our current work focuses on experiments in humans. For this, we have nowconstructed a biologically contained L. lactis strain that can produce human IL-10. Thiswill enable us to investigate whether L. lactis-mediated IL-10 delivery has the potentialto lead to the development of a new therapeutic for the treatment of IBD in humans.

TOOLS FOR THE DESIGN OF BIOLOGICALLY CONTAINEDGM PROBIOTICS

The final goal of any designer probiotic strain is obviously its use in humans or animals tocounteract or prevent disease. A major focus attention in the discussion of geneticallyengineered probiotics is therefore the fact that, when administering of a liverecombinant micro-organism to a human, one is in fact deliberately releasing agenetically modified organism into the environment. The design of the new probioticshould therefore be such that safety is guaranteed often its usage. This point relatesessentially to the absence of antibiotic selection markers, preventing the of accumulationof the GM micro-organism in the environment and preventing of lateral dissemination ofthe genetic modification to other bacteria. It is best and most elegant if these concernscan be addressed through a biological system that is propagated along with thehost, such

868 L. Steidler


9/16

Figure 2. Haematoxylin and eosin-stained histological slides of colonic tissue: representative images of untreated mice (normal), mice in which colitis was induced by thaddition of dextran sulphate sodium to the drinking water and mice that underwent the same induction followed by treatment with a mouse IL-10-producing Lactobacilactis strain.


10/16

systems being termed biological containment systems. These systems or combinationsthereof should address all of these concerns. As no reports have yet been released onthe use of GM probiotics in human or veterinary medicine, the literature on the specificarea of thebiological containment probiotics is scarce. Thestrategies that arelikely to beadopted will, however, be similar to those used in other disciplines in which proficientuse is made of GM organisms.

Biological containment systems can be subdivided in active and passive. The former

essentially provide control through the conditional production of a bacterial toxinviatightly regulated gene expressionwhich is controlled by an environmentallyresponsive element. Passive systems render growth dependent on the complementa-tion of an auxotrophy or other gene defect, by supplementing either the intact gene orthe essential metabolite.

Active biological containment systems that control GM dissemination

Molina et al50 designed contained GM bacteria that degraded a model pollutant,3-methylbenzoate, using the Pseudomonas putida TOL plasmid for aromatic hydro-carbon metabolism. In Dasd Pseudomonas putida, a background necessary to enforce thesystem51, fusion with the Pm promoter controls lacI. Escherichia coli Gef, a porin-inducing protein, is produced under the control of the P lac promoter. The positiveregulator of the Pm promoter, XylS, is active when 3-methylbenzoate is present.Degradation of the pollutant from the environment leads to a decrease in LacI Gefproduction and killing of the host. This is an elegant system, but its application isobviously very restricted because of the high degree of integration of the variouscomponents. In a streptavidin-based suicide system52, streptavidin expression wasinduced in a similar way by the absence of 3-methyl benzoate, which resulted in a1000-fold reduction of Pseudomonas putida viability within 8 hours.

Escherichia coli K12 relA mutants die faster than wild-type organisms. Whensupplemented with a system in which the alkaline phosphatase gene promoter drivesthe phage T7 lysozyme gene, the rate of killing still increases further followingphosphate depletion.53 Positioning the E. coli alkaline phosphatase gene promoter sothat it controls the parB locus of plasmid R1 leads to stable inheritance of the resultingplasmids and death of the carrier strains as a consequence of phosphate starvation.54

The activity of the E. coli rrnB P1 promoter is completely turned off in the presence ofthe alarmone guanosine tetraphosphate, a compound that is produced followingstarvation. It can thus be used as a biosensor for poor growth conditions such as areencountered upon its release in to the environment.55

The toxin in an active biological containment system can also be an endonuclease,counteracted by a methylase, as in the type II EcoRI restrictionmodification system.The ecoRIR lethal gene is present on a plasmid, and the eco RIM gene encoding thecognate EcoRI methylase is situated on the chromosome.56 Lateral dissemination of theplasmid to the recipient bacteria causes destruction of the DNA and evidently death. Itcan, however, be questioned whether such system is suitable for use in the intestine as alarge part of the microbiota consists ofE. coli. Other species could furthermore acquirethe methylase from the common gene pool.

The bacterial toxin colicin E3 displays an endonucleolytic activity towards the highlyconserved 30 end of the 16S ribosomal RNA. Cleavage inhibits protein synthesis. Acombination of the genes for both colicin production and colicin immunity produces aviable strain. When the E3 gene in such a strain is closely linked to a genetic modification,lateral gene transfer of the latter decreases by several orders of magnitude.57

870 L. Steidler


11/16

A plasmid containment system using the lethal E. coli relF gene has been shown to beeffective in vitro and also in rat intestine.58

Passive systems for biological containment

A number of food-grade selection markers have been described, and these can be usedto eliminate the need for antibiotic selection markers. Similarly, some of the deletionmutants utilized in such system are essentially dependent for their growth on thepresence of a particular compound, which can be used in the development of containedstrains.

D-Alanine is an essential component in the biosynthesis of the cell wall of many lacticacid bacteria. Alanine racemase (Alr) catalyses the interconversion of D-alanine andL-alanine, so L. lactis and Lb. plantarum strains with a detection ofalrshow auxotrophy forD-alanine.59 The alr genes of both species have been used as food-grade selectionmarkers to complement for the deletion of alr. Plasmids carrying a heterologous alrmutation were stably inherited in a alrdeleted background.60 Thedeletion ofalrhas beenused by Hillman et al to contain of the GM Streptococcus mutans designed for theiranticaries therapy, described above (J. Hillman, pers. Comm., 2002). Fu and Xu61

have described a similar containment system for Lb. acidophilus using the thymidilmatesynthase gene (thyA) from Lb. casei as selective marker for plasmid

maintenance. In this cases, a foreign gene is used to avoid the reversion of the mutationby back-recombination of the marker gene.Platteeuw and co-workers62,63 used the soluble carrier enzyme IIALac, encoded by

the 300 bp lacF, as a selection marker in an lacF-deleted L. lactis. This allows stableinheritance of the plasmid when lactose is used as a carbon source.

An amber suppressor, supD, has been utilized as a selectable marker for plasmidmaintenance to complement suppressible pyrimidine auxotrophs.64 This hostplasmidcombination is effective in any pyrimidine-free medium.

Nisin does not have the obvious drawbacks of a classical antibiotic. By placing thenisin immunity gene nisI on a plasmid, its maintenance through bacterial generations canbe assured when nisin is used as a selection antibiotic.65

The integration of expression units into the chromosome of the carrier strain isoften highly desirable because the GM character is then stably inherited and lateral genetransfer is reduced strongly when compared with plasmid-borne systems. Frequently,

however, the level of expression of the transgene decreases significantly as aconsequence of reduced copy number. Leenhouts et al have described a method ofobtaining multiple integrations in a way that is compatible with use in humans. Theintegrated plasmid is devoid ofrepA, an essential replication factor for derivatives of thelactococcal plasmid pWV01, and carries the sucrose utilization genes of the lactic acidbacterium Pediococcus pentosaceus.66,67 Intermediate engineering can be carried outL. lactis strains that produce the pWV01 RepA protein. Single-crossover integration canbe strongly enhanced on medium containing sucrose as the only carbon source.

CONCLUSIONS

Although they have been in use for a long time and described for almost a century, weare only now seeing the onset of a medical use for probiotics. Especially in the area ofintestinal disorders, in which lifelong treatment is often required, they promise elegant



12/16

alternatives to high-impact chemical therapeutics. Mechanisms of action have beengradually described, and reliable clinical studies have demonstrated efficacy. It isconceivable that the creation of new probiotic organisms with the help of geneticengineering will revolutionize this field. With the advent of GM probiotic strategies,mechanistic tools can be applied from foreign sources and combined to make neworganisms. Very much like traditional pharmacology extracts and copies specificcompounds from nature for their interesting properties, active components, even

derived from pathogens, can now be placed in harmless carrier strains through geneticengineering and thus provide a safe vehicle for such elements. Molecular medicalresearch has furnished a large collection of ingenious therapeutic compounds. Theirapplication can, however, be complicated by their sensitivity in vivo and the cost of theirproduction. The use of GM probiotics can circumvent the short half-life and fragility ofthe therapeutics and produce very cost-effective access to such expensive therapeutics.

It is clear that GM probiotics open up a plethora of applications. It is, however, alsoclear that the uninhibited spread of GM micro-organisms in the environment is highlyundesirable. Researchers working in the field must therefore proactively engage indiscussions on the biosafety of the strains they have created. When designing orapplying GM probiotics, one should further realize that they are in many ways similar tonewly synthesized chemical therapeutic agents and require caution while testing themin vivo. Antigenic reactions especially in particular are a key consideration. If a strain isdesigned to introduce in a foreign protein, this may well provoke an immune reaction,

not only jeopardizing the future use of this medication but also possibly even causingbystander recognition and allergy. It is appreciated that man, as a consequence oflifelong consumption, shows tolerance to lactic acid bacteria, S. cerevisiae and othercarriers, but how will the organism react upon extensive exposure to a foreign(bacterial, animal or plant) enzyme? Will this provoke an immune reaction, and if so,could it be cross-reactive to indigenous analogues? Will such hypothetical cross-reaction extend to the carrier? It is clear that these questions may limit the breath ofpotential applications and demand caution when considering the clinical use of thesesystems. This need not, however, be an unsurmountable hurdle as similar problems inother areas have been overcome through the humanization of the foreign proteins.

The GM probiotics that have so far been designed are the first attempts in what hasthe potential to become a new avenue of pharmacology. Although the goals are clearlyambitious, the problems encountered in real-life application are often immense. But thisis also true of any cutting-edge development. The challenge is now to creative

molecular biologists and pioneering physicians to conceive a new generation ofpowerful tools to combat what are among the most malicious of diseases.

SUMMARY

Probiotic microorganisms can be effective in IBD therapy via a number of mechanisticpathways. Some strains can displace noxious bacteria by competitive binding. This canalso be achieved through death or growth inhibition by the production of antibacterialcompounds or a lowering of the pH. Some probiotics have a defined influence on thehost immune cells, for example the induction of cytokine production or an increase inlocal IgA secretion. Some can sequester toxins and remove these from the intestine orcounteract their biological effects. Probiotics can influence epithelial and tissue integrityby low-dose nitric oxide synthesis, stimulating mucus production, enhancing

872 L. Steidler


13/16

proliferation of the gut epithelium, inhibiting endogenous carcinogen production andproviding nutrients by short chain fatty acid production.

The current probiotic treatment of IBD is still, however, limited to the maintenanceof remission. To obtain more powerful strains, genetic engineering to adapt current orestablish new mechanistic pathways may open up new perspectives. Examples of suchGM probiotics include strains that sequester toxins through altered lipopolysaccharidestructure or compete with and displace pathogens. Further strategies involve the

production of antibodies and antiidiotypic ScFv that resemble a toxin, the production ofenzymes for detoxification and the complementation of enzyme deficiencies, andproduction of cytokines for immune intervention.

The biological safety of GM probiotics must be assured through systems forbiological containment that are either activeproviding control through theconditional production of a bacterial toxinor passivein which growth dependson the complementation of an auxotrophy or other gene defect.

ACKNOWLEDGEMENTS

L.S. research group received support from the Vlaams Interuniversitair Instituut voorBiotechniologie, Ghent University (GOA project nos. 12050700 and 12051501) and thecommission of the European Communities, specifically the RTD programme Quality oflife and Management of Living Resources QLK1-2000-00146, Probiotic strains withdesigned health properties. L.S. is currently affiliated as an investigator to the ScienceFoundation of Ireland (SFI/01/F.1/B036).

REFERENCES

1. Rembacken BJ, Snelling AM, Hawkey PM et al. Non-pathogenic Escherichia coliversus mesalazine for thetreatment of ulcerative colitis: a randomised trial [see comments]. Lancet 1999; 354: 635639.

2. Kruis W, Schutz E, Fric P et al. Double-blind comparison of an oral Escherichia coli preparation and

mesalazine in maintaining remission of ulcerative colitis. Alimentary Pharmacology and Therapeutics 1997;11: 853858.

Practice points

some selected probiotics can be used in the remission maintenance of IBD no GM probiotics are currently use in medicine

Research agenda

a molecular description of probiotic activity should be provided mechanistic pathways need to be applied to create new strains the construction of biologically contained GM probiotics is of vital importance new strains must be evaluated in animal models clinical experimentation will allow an assessment of the efficacy of GM

probiotics



14/16

3. Venturi A, Gionchetti P, Rizzello F et al. Impact on the composition of the faecal flora by a new probioticpreparation: preliminary data on maintenance treatment of patients with ulcerative colitis. AlimentaryPharmacology and Therapeutics 1999; 13: 11031108.

4. Gionchetti P, Rizzello F, Venturi A et al. Oral bacteriotherapy as maintenance treatment in patients withchronic pouchitis: a double-blind, placebo-controlled trial [see comments]. Gastroenterology 2000; 119:305309.

5. Nobaek S, Johansson ML, Molin G et al. Alteration of intestinal microflora is associated with reduction inabdominal bloating and pain in patients with irritable bowel syndrome. American Journal of Gastroenterology2000; 95: 12311238.

6. Gupta P, Andrew H, Kirschner BS et al. Is lactobacillus GG helpful in children with Crohns disease?Results of a preliminary, open-label study [In process citation]. Journal of Pediatric Gastroenterology andNutrition 2000; 31: 453457.

* 7. Saavedra JM, Bauman NA, Oung I et al. Feeding ofBifidobacteriumbifidum and Streptococcusthermophilustoinfants in hospital for prevention of diarrhoea and shedding of rotavirus. Lancet 1994; 344: 10461049.

8. Guslandi M, Mezzi G, Sorghi M et al. Saccharomyces boulardiiin maintenance treatment of Crohns disease.Digestive Diseases and Sciences 2000; 45: 14621464.

9. Majamaa H & Isolauri E. Probiotics: a novel approach in the management of food allergy. Journal of Allergyand Clinical Immunology 1997; 99: 179185.

10. Paton AW, Morona R & Paton JC. Neutralization of Shiga toxins Stx1, Stx2c, and Stx2e by recombinantbacteria expressing mimics of globotriose and globotetraose. Infection and Immunity 2001; 69:19671970.

11. Anderson MH. Changing paradigms in caries management. Current Opinion in Dentistry 1992; 2: 157162.12. Johnson CP, Gross SM & Hillman JD. Cariogenic potential in vitro in man and in vivo in the rat of lactate

dehydrogenase mutants of Streptococcus mutans. Archives of Oral Biology 1980; 25: 707713.13. Hillman JD, Duncan MJ & Stashenko KP. Cloning and expression of the gene encoding the fructose-1,6-

diphosphate-dependent L-()-lactate dehydrogenase of Streptococcus mutans. Infection and Immunity1990; 58: 12901295.

14. Chen A, Hillman JD & Duncan M. L-()-lactate dehydrogenase deficiency is lethal in Streptococcus mutans.Journal of Bacteriology1994; 176: 15421545.

* 15. Hillman JD, Chen A & Snoep JL. Genetic and physiological analysis of the lethal effect of L-()-lactatedehydrogenase deficiency in Streptococcus mutans: complementation by alcohol dehydrogenase from

Zymomonas mobilis. Infection and Immunity 1996; 64: 43194323.16. Hillman JD, Yaphe BI & Johnson KP. Colonization of the human oral cavity by a strain of Streptococcus

mutans. Journal of Dental Research 1985; 64: 12721274.17. Hillman JD, Dzuback AL & Andrews SW. Colonization of the human oral cavity by a Streptococcus mutans

mutant producing increased bacteriocin. Journal of Dental Research 1987; 66: 10921094.* 18. Kruger C, Hu Y, Pan Q et al. In situ delivery of passive immunity by lactobacilli producing single-chain

antibodies. Nature Biotechnology 2002; 20: 702706.* 19. Michalek SM, Gregory RL, Harmon CC et al. Protection of gnotobiotic rats against dental caries by

passive immunization with bovine milk antibodies to Streptococcus mutans. Infection and Immunity1987; 55:23412347.

20. Hamada S, Horikoshi T, Minami T et al. Oral passive immunization against dental caries in rats by use ofhen egg yolk antibodies specific for cell-associated glucosyltransferase of Streptococcus mutans. Infectionand Immunity 1991; 59: 41614167.

21. Lehner T, Caldwell J & Smith R. Local passive immunization by monoclonal antibodies againststreptococcal antigen I/II in the prevention of dental caries. Infection and Immunity 1985; 50: 796799.

22. Fidel Jr. PL & Sobel JD. Immunopathogenesis of recurrent vulvovaginal candidiasis. Clinical MicrobiologyReview 1996; 9: 335348.

* 23. Beninati C, Oggioni MR & Boccanera M. Therapy of mucosal candidiasis by expression of an anti-idiotypein human commensal bacteria [in process citation]. Nature Biotechnology 2000; 18: 10601064.

24. Medaglini D, RushCM, Sestini P et al. Commensal bacteria as vectors for mucosal vaccines against sexuallytransmitted diseases: vaginal colonization with recombinant streptococci induces local and systemicantibodies in mice. Vaccine 1997; 15: 13301337.

25. Medaglini D, Oggioni MR & Pozzi G. Vaginal immunization with recombinant gram-positive bacteria.American Journal of Reproductive Immunology1998; 39: 199208.

26. Di Fabio S, Medaglini D, Rush CM et al. Vaginal immunization of Cynomolgus monkeys with Streptococcusgordoniiexpressing HIV-1 and HPV 16 antigens. Vaccine 1998; 16: 485492.

27. Magliani W, Conti S, De Bernardis F et al. Therapeutic potential of antiidiotypic single chain antibodieswith yeast killer toxin activity. Nature Biotechnology 1997; 15: 155158.

28. Polonelli L & Morace G. Production and characterization of yeast killer toxin monoclonal antibodies.Journal of Clinical Microbiology 1987; 25: 460462.

874 L. Steidler


15/16

29. Rizkalla SW, Luo J, Kabir M et al. Chronic consumption of fresh but not heated yogurt improves breath-hydrogen status and short-chain fatty acid profiles: a controlled study in healthy men with or withoutlactose maldigestion. American Journal of Clinical Nutrition 2000; 72: 14741479.

30. Lin MY, Yen CL & Chen SH. Management of lactose maldigestion by consuming milk containing lactobacilli.Digestive Diseases and Sciences 1998; 43: 133137.

31. Drouault S, Juste C, Marteau P et al. Oral treatment with Lactococcus lactis expressing Staphylococcus hyicuslipase enhances lipid digestion in pigs with induced pancreatic insufficiency. Applied and EnvironmentalMicrobiology 2002; 68: 31663168.

32. Drouault S, Corthier G, Ehrlich SD et al. Survival, physiology, and lysis ofLactococcus lactis in the digestive

tract. Applied and Environmental Microbiology 1999; 65: 48814886.33. Blanquet S, Marol-Bonnin S, Beyssac E et al. The biodrug concept: an innovative approach to therapy.

Trends in Biotechnology 2001; 19: 393400.34. Urban P, Truan G, Bellamine A et al. Engineered yeasts simulating P450-dependent metabolisms: tricks,

myths and reality. Drug Metabolism and Drug Interactions 1994; 11: 169200.35. Marteau P, Minekus M, Havenaar R et al. Survival of lactic acid bacteria in a dynamic model of the stomach

and small intestine: validation and the effects of bile. Journal of Dairy Science 1997; 80: 10311037.36. Minekus M, Smeets-Peeters M, Bernalier A et al. A computer-controlled system to simulate conditions of

the large intestine with peristaltic mixing, water absorption and absorption of fermentation products.Applied Microbiology and Biotechnology1999; 53: 108114.

* 37. Prakash S & Chang TM. Microencapsulated genetically engineered live E. coliDH5 cells administered orallyto maintain normal plasma urea level in uremic rats. Nature Medicine 1996; 2: 883887.

38. Prakash S & Chang TM. In vitro and in vivo uric acid lowering by artificial cells containingmicroencapsulated genetically engineered E. coli DH5 cells. International Journal of Artificial Organs 2000;23: 429435.

39. Prakash S & Chang TM. Artificial cells microencapsulated genetically engineered E. coliDH5 cells for thelowering of plasma creatinine in vitro and in vivo. Artificial Cells, Blood Substitutes and ImmobilizationBiotechnology2000; 28: 397408.

40. Seegers JF. Lactobacilli as live vaccine delivery vectors: progress and prospects. Trends in Biotechnology2002; 20: 508515.

41. Wells JM, Wilson PW, Norton PM et al. Lactococcus lactis: high-level expression of tetanus toxin fragmentC and protection against lethal challenge. Molecular Microbiology 1993; 8: 11551162.

* 42. Steidler L, Robinson K, Chamberlain L et al. Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 byrecombinant strains of Lactococcus lactis coexpressing antigen and cytokine. Infection and Immunity 1998;66: 31833189.

43. van Deventer SJ, Elson CO & Fedorak RN. Multiple doses of intravenous interleukin 10 in steroid-refractory Crohns disease. Crohns disease Study Group. Gastroenterology 1997; 113: 383389.

44. Schreiber S, Fedorak RN, Nielsen OH et al. Safety and efficacy of recombinant human interleukin 10 inchronic active Crohns disease. Crohns disease IL-10 cooperative study group. Gastroenterology 2000;119: 14611472.

45. Fedorak RN, Gangl A, Elson CO et al. Recombinant human interleukin 10 in the treatment of patientswith mild to moderately active Crohns disease. The interleukin 10 inflammatory bowel diseasecooperative study group. Gastroenterology 2000; 119: 14731482.

46. Tilg H, Ulmer H, Kaser A et al. Role of IL-10 for induction of anemia during inflammation. Journal ofImmunology2002; 169: 22042209.

47. Okayasu I, Hatakeyama S, Yamada M et al. A novel method in the induction of reliable experimental acuteand chronic ulcerative colitis in mice. Gastroenterology 1990; 98: 694702.

48. Kojouharoff G, Hans W, Obermeier F et al. Neutralization of tumour necrosis factor (TNF) but not of IL-1 reduces inflammation in chronic dextran sulphate sodium-induced colitis in mice. Clinical andExperimental Immunology 1997; 107: 353358.

* 49. Steidler L, Hans W, Schotte L et al. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10 [see comments]. Science 2000; 289: 13521355.

50. Molina L, Ramos C, Ronchel MC et al. Construction of an efficient biologically contained Pseudomonasputida strain and its survival in outdoor assays.Applied and Environmental Microbiology1998; 64: 20722078.

51. Ronchel MC & Ramos JL. Dual system to reinforce biological containment of recombinant bacteriadesigned for rhizoremediation. Applied and Environmental Microbiology 2001; 67: 26492656.

52. Kaplan DL, Mello C, Sano T et al. Streptavidin-based containment systems for genetically engineeredmicroorganisms. Biomolecular Engineering 1999; 16: 135140.

53. Schweder T, Hofmann K & Hecker M. Escherichia coli K12 relA strains as safe hosts for expression ofrecombinant DNA. Applied Microbiology and Biotechnology 1995; 42: 718723.

54. Schweder T, Schmidt I, Herrmann H et al. An expression vector system providing plasmid stability andconditional suicide of plasmid-containing cells. Applied Microbiology and Biotechnology 1992; 38: 9193.



16/16

55. Tedin K, Witte A, Reisinger G et al. Evaluation of the E. coli ribosomal rrnB P1 promoter and phage-derived lysis genes for the use in a biological containment system: a concept study. Journal of Biotechnology1995; 39: 137148.

56. Torres B, Jaenecke S, Timmis KN et al. A gene containment strategy based on a restriction-modificationsystem. Environmental Microbiology 2000; 2: 555563.

* 57. Diaz E, Munthali M, de Lorenzo V et al. Universal barrier to lateral spread of specific genes amongmicroorganisms. Molecular Microbiology 1994; 13: 855861.

58. Knudsen S, Saadbye P, Hansen LH et al. Development and testing of improved suicide functions forbiological containment of bacteria. Applied and Environmental Microbiology 1995; 61: 985991.

* 59. Hols P, Defrenne C, Ferain T et al. The alanine racemase gene is essential for growth of Lactobacillusplantarum. Journal of Bacteriology 1997; 179: 38043807.

60. Bron PA, Benchimol MG, Lambert J et al. Use of the alrgene as a food-grade selection marker in lactic acidbacteria. Applied and Environmental Microbiology 2002; 68: 56635670.

61. Fu X & Xu JG. Development of a chromosome plasmid balanced lethal system for Lactobacillus acidophiluswith thyA gene as selective marker. Microbiology Immunology 2000; 44: 551556.

62. MacCormick CA, Griffin HG & Gasson MJ. Construction of a food-grade host/vector system forLactococcus lactis based on the lactose operon. FEMS Microbiology Letters 1995; 127: 105109.

63. Platteeuw C, van Alen-Boerrigter I, van Schalkwijk S et al. Food-grade cloning and expression system forLactococcus lactis. Applied and Environmental Microbiology 1996; 62: 10081013.

64. Sorensen KI, Larsen R, Kibenich A et al. A food-grade cloning system for industrial strains ofLactococcuslactis. Applied and Environmental Microbiology 2000; 66: 12531258.

65. Takala TM & Saris PE. A food-grade cloning vector for lactic acid bacteria based on the nisin immunitygene nis I. Applied Microbiology and Biotechnology 2002; 59: 467471.

66. Leenhouts K, Bolhuis A, Venema G et al. Construction of a food-grade multiple-copy integration systemfor Lactococcus lactis. Applied Microbiology and Biotechnology 1998; 49: 417423.

67. Leenhouts K, Bolhuis A, Boot J et al. Cloning, expression, and chromosomal stabilization of the

Propionibacterium shermanii proline iminopeptidase gene ( pip) for food-grade application in Lactococcuslactis. Applied and Environmental Microbiology 1998; 64: 47364742.

876 L. Steidler

Steidler Lothar

Documents

Transcript of Steidler Lothar