Michel Gautier

Ethical issues raised by Genetically modified micro-organisms (G.M.M)

IP bioéthique, Perugia Michel Gautier, 2008

Ethical issues raised by Genetically modified micro-organisms (G.M.M) I-What is a genetically modified micro-organism ? II-In what cases can micro-organisms and GMM be used ? III-What are the risks posed by GMM? V- However… VI) Is it possible to avoid the use of GMM? VII) In conclusion

I- What is an GMM ?

GMM and micro-organisms categories eucarytic cell Protozoa

Unicellular Algae

Moulds

?

GMM

Archaebacteria

Yeast

GMM

GMM

procaryotic cell Eubacteria GMM Giant virus

Virus

GMM Bacteriophage

Prions

Essentially model micro-organisms

E.coli

Gram- Bacteria

Lactococcus lactis

Safe Gram+ Bacteria

Bacillus subtilis

Gram+ Bacteria excretion

S. cerevisiaee

yeast

Procaryotic

Eucaryotic

Aspergillus sp

Mould

I What is a genetically modified micro-organism ? Bacteria to be improved

Other species, other genus ex :cloning of a Bacillus Heterologous gene

thurengiensis gene encoding an insecticide in Pseudomonas sp Animal cell, plan cell

Cloning vector

Foreigner gene Homoologous gene

"ex : - Human gene i,n E.coli: Insulin Growth hormone

however...

In the cases where: "in vitro" construction

The modification is not dangerous

It is an homologous gene The gene is inserted on the chromosome The insertion is known

O.G.M.?

II-In what cases can micro-organisms and GMM be used ?

Using micro-organisms in a fermenter

Confined space

agreement

No lived cells in the environment 109 to 1011 cell/ml

pharmaceutical industry

Naturally synthesized molecules or the micro-organism itself

Antibiotics Vaccines…

Naturally synthesized molecules with a genie genetic improvement

VitB12…

Molecule stemming from a cloned gene

GMM

Vaccine hepatitis B Insulin Growth hormone...

Agri-food industry industry Enzymes Amino acids Organic acids...

Naturally synthesized molecules or the micro-organism itself

yeast proteases amylases…

Naturally synthesized molecules with a genie genetic improvement

Proteases amylases…

Molecule stemming from a cloned gene

Rennin...

GMM

Chemical Industry

Naturally synthesized molecules Naturally synthesized molecules with a genie genetic improvement Molecule stemming from a cloned gene

Enzymes Organic acids Biofuels... GMM Enzymes Organic acids Biofuels...

Using of micro-organisms for the production of fermented foods

Not agreement Travel through the digestive tract environment

Fermented milk products 109 lived micro-organisms/g Fermented meats (dry-cured sausage, salamis) bread, wine ,beer

109 micro-organisms' Which are eliminated or dead

GMM are used for the improvement of fermented products Addition of 106/g at the beginning of the fermentation There is no ideal strain Improvement of the products

MGM

organoleptic qualities hygienic qualities nutritional qualities Reduction of production cost (decrease of the ripening time)

Optimisation of the technological processes

Decrease of production accidents (improvement of the aux bacteriophages resistance )

Use of micro-organisms in the environment

Not agreement (agreement for research : individually)

agriculture fixation of atmospheric N2 bio pesticides production

Cleaning up degradation of pollutant molecules

industry Metal extraction

Development of strains for therapeutic purposes

Modified bacterial strain Effect in the digestive tract or other mucosa

Not agreement Dissemination in the environment

The bacteria presents to the epithelial tissue a vaccinating antigen Ex: Lactobacillus jensenii has been modified to secrete the CD4 protein used by the HIV virus in the vaginal mucosa to penetrate lymphocytes. The secreted protein also traps viruses

Acquisition of fundamental knowledge Experiments in laboratory

Agreement Not dissemination

Research teaching Life science : Genetic, Physiology, Metabolism.... Health Food-nutrition environment...

III Risks posed by the use of GMM.

Great surface of exchange with the environment

Genetic plasticity

Small size (1µm) haploïd

Very quick multiplication

Dispersion

colonisation of the environment

All the mutation are expressed

Gene transfer

adaptation to a new environment

Of what can we be afraid?

Effect on the man and its environment

Perturbation of the ecological balance Undesirable biochemical reactions Production of toxic compounds Domination of the endogenous population Dissemination of heterologous genes

What might happen ?

a) Gene transfer to other organisms b) Development of new micro-organisms in the environment c) The process of use of micro-organisms is not under control

a) Gene transfer to other organisms

Conjugaison Transformation Transduction

Conjugation

Bacteria A Way of genetic information

Plasmids transposons

Bacteria B

Needs the intervention of Specific genes

Tra genes (13 genes)

Transformation B.subtilis Haemophilus influenzae Neisseria gonorrhoeae Streptococcus pneumoniae

Gram + et Gram-

Cyanobacteries Probably many species with a low frequency

B.subtilis (140 genes implicated ) High cellular density Stationnary phase ( risk of cellular dead )

One pheromon Competence factors

Another peptide

Competence genes membrane permeability

Proteins bounded to DNA (all DNA)

One model

Streptococcus pneumoniae 100% of cells Are competent

Haemophilus ADN 1500 sites AAGTGCGGT

Transduction

B Ex : P22 of S.typhimurium (1% of bacterial genome )

Distribution of DNA material obtained by horizontal transfer in some sequenced bacterial genomes La longueur des barres reflète la quantité de séquences codantes. Pour chaque barre, l'ADN natif est en bleu, les EGM identifiés sont en jaunes et l'ADN étranger d'une autre nature est en rouge. Le pourcentage d'ADN étranger total est indiqué à droite de chaque barre. Les archae sont signalées par la lettre A (Ochman et al., 2000)

DNA transfers are happening in nature Their frequency is more or less high and depends of: -genetic support (plasmid, small DNA fragments…) -specific genes (conjugation, genes involved in transformation) -transfer mode -bacterial species -bacterial strains -environmental conditions

They can lead to the emergence of strains allowing new properties:

- A pathogenic potential exacerbated - Able to supplant endogenous strains

-Able to modify the balance of ecological niches Ex: transfers between Agrobacterium tumefaciens and Pseudomonas fluorescens Transfers between bacteria et superior organisms: Wolbachia sp and Callosobruchus chinenesis

b) Development of new micro-organisms in the environment Disruption of the microbial balance and impact on the environment There in no example from MGM but possible situation Examples with micro-organisms which are not GMM Ex: development of the unicellular alga Chryschromulina polylepis in the north sea and the Channel (discharge of nitrogen) Production of toxins which are pathogenic for human

Caulerpa taxifolia (change water of the aquariums of Monaco) Colonisation of the Mediterranean sea bed

c) The process of use of micro-organisms is not under control

Leak or a theft in a research laboratory

Leak or a theft in a collection of micro-organisms

Leak from a fermenter

Some genetic construction obtained in laboratory… Inhibition of a virulent gene of Mycobacterium tuberculosis Creation of a more virulent variant

Modification of the cowpox virus Is able to bypass the immune defences in human due to vaccination

Modification of the cowpox virus Might cross specie barriers Infection of other species

IV Things must be put into perspective

« genetic modifications of micro-organisms also occur in the nature naturally "

Fast multiplication A big population in a few time Gene transfer

Mutations Vertical modifications

Horizontal modifications

Many variations of genetic information

Selection pressure The most adapted survive Modification of the population

Men are using mutagenesis since a long time

Natural mutagenesis

ex : empirical selection of mutants for brewery

Mutagenesis induced using mutagenic agent in laboratory (ex: UV)

ex : Increasing of the production level of molecules such as antibiotics, cephlosporin…

ex : degradation of new chemical compounds

GMM

Recombinant micro-organisms are not well adapted to the environment

Genetically modified strain

E.coli S.cerevisiae b.subtilis

Out of the laboratory

Dead of cells

Difficulties to colonise again the environment

Gene transfers are limited Mechanisms limiting the DNA transfer between specie which are to much different

Conjugation

All the plasmids are not able of replication into the recipient cell

Transformation

Usually, DNA transfers are promoted between closely strains and especially between strains belonging to the same species

Transduction

Usually, transfers are promoted between strains belonging to the same species DNA can be digested by the R/M systems

V) However... Genetic transfer in nature are difficult to assess Little is known about the adaptation potential of “lab crea”

Genetic engineering causes modifications which are not naturally possible

Genetic transfer in nature are difficult to assess It is not easy to determine the importance of gene flow in the environment Numerous species are living in complex communities Most of the species are unknown Ex: less 0,7% of the soil flora is identified In a determined ecosystem the species fluctuate according to environmental factors

Very important studies are necessary GMM of Sinorhizobium melitoti were able to transfer their DNA to the endogenous flora of soil

Little is known about the adaptation potential of “lab crea”

The mechanisms of adaptation of micro-organisms are almost unlimited

« Bacteria are adapted to adaptation » J. Monod

Ex: Survival of Sinorhizobium melitoti GMM in the soil during 6 years, even in absence of the legumes with which they form a symbiotic relationship

Genetic engineering causes modifications which are not naturally possible In the nature spontaneous mutation of gene occur only on existing genes

Many genetic construction created in laboratory are not possible in the nature The production of hormones as insulin using E. Coli was obtained by the way of the cloning of the human gene in the bacteria :this spontaneously occurring by genetic transfer in nature is practically impossible.

VI) Is it possible to avoid the use of GMM?

When a GMM can be used

We have to compare the risk linked to its use with that one linked to its absence

Using micro-organisms in a fermenter

Confined space

agreement

No lived cells in the environment 109 to 1011 cell/ml There is not really an hazard if the process is correctly under control

pharmaceutics industry

Advantage of GMM

Bigger quantities of molecules: possibility of treating more patient possibility of carrying out research into new uses More safe drugs Most of time, not other alternative

No doubt for the advantage of GMM

Food industry Enzymes Amino acids Organic acids... Advantage of GMM

Quality of food products more standardized Safe molecules Reduction in production costs

“economic” advantage of MGM

Chemical Industry Enzymes Organic acids Biofuels... Advantage of GMM

Alternatives way to the chemical production Safe Molecules Improvement of processes: cleaning up…

“economic and environmental” advantages of MGM

Using of micro-organisms for the production of fermented foods

Not agreement Travel through the digestive tract

environment

Dispersion and eventual multiplication

Potentiality of hazard for human and environment

Les MGM pour l’amélioration de la fermentation Advantage of GMM Improvement of the products

Optimisation of the technological processes

organoleptic qualities hygienic qualities nutritional qualities

Reduction of production cost Decrease of production accidents

“economic, sanitary, qualitative” advantages of MGM But There are many alternatives: Selection and mixing of natural strains Control of the fabrication processes

Advantage of GMM : very moderate comparing to the risks

Use of micro-organisms in the environment

Not agreement (agreement for research : individually)

Dispersion and eventual multiplication

Potentiality of hazard for human and environment

agriculture fixation of atmospheric N2

Cleaning up degradation of pollutant molecules

industry Metal extraction

bio pesticides production Advantage of GMM

Humanitarian advantages (increasing of food production) Environmental advantages (cleaning up..) Economical advantages

No doubt for the advantage of GMM according the cases But very important risks (too much?) for human and environment

Development of strains for therapeutic purposes Modified bacterial strain Effect in the digestive tract or other mucosa

Not agreement Dissemination in the environment

Dispersion and eventual multiplication

Potentiality of hazard for human and environment (genetic construction carrying on pathogenic characters)

Advantage of GMM

Possibility of treating very serious diseases Reduction of treatment costs Decreasing of the number of infectious Specific therapy : less offensive

But potentiality of hazard for human and environment

Requires to avoid the dispersion of recombined strains

Acquisition of fundamental knowledge Experiments in laboratory

Agreement

Not dissemination Research teaching

There is not really an hazard if the process is correctly under control

But possibility of very dangerous genetic construction

Advantage of GMM

Accumulation of knowledge

Implications on:

Life science : Genetic, Physiology, Metabolism....

Health Food-nutrition environment...

No doubt for the advantage of GMM But the scientific is it really free? More and more private funds to the detriment of government funds

VII) In conclusion Since the era of Pasteur micro-organisms were studied in vitro, on pure conditions and in controlled environmental conditions Very few knowledge on the “real life” of micro-organisms Numerous environmental factors are implicated Ecological niches are very complex It is very difficult to predict the influence that the dissemination of GMM into our environment may have

Several important studies are will be necessary

And especially on:

Gene transfer in the environment

Genetic construction of strains which are unable to survive in the environment

Improvement of alternatives to the GMM

Studies about the interactions between micro-organisms themselves and with their environment (plant, animal…)