Genetic Modification of Pathogens

Forward Genetics = Strives to identify the basic genetic sequences which bring about a specific phenotype/trait. Used to identify novel genes involved in a phenotype.

E.g. The identification of which genetic sequences were required to be present in E.coli for it to confer an ability to infect lambda phages. This was conduction by collecting E.coli strains, each with one of the 3985 non-essential genes knocked out, and analysing their ability to infect lambda phages.

Reverse Genetics = Strives to identify phenotypes which occur as a direct result of a series of known genetic sequences. This involves the mutation of said genes before analysing the results of this modification. This methodology is usually utilised for identifying the function of unknown genes which are accidentally found in genome projects.

An example of reverse genetics in use is in the identification of metacaspase enzymes which are overexpressed in Leishmania Major

Chemical Genetics = This approach utilises molecules to interrupt the functionality of proteins. So...

Forward Chemical Genetics = Strives to identify the proteins which bring about a certain phenotype
Reverse Chemical Genetics = Strives to identify the phenotype which arises a result of a series of known proteins

Koch's Postulates

This criteria was set out by the German physician as a checklist to determine whether a bacteria is the direct cause of a certain disease/condition. They are as follows:

It is essential that the micro-organism exists in every documented case of the condition
The micro-organism be retrieved from a host exhibiting the disease process and subsequently grown in culture.
The disease itself must be replicable when the micro-organism is transferred from a pure culture into a relevant host
The micro-organism must then be able to be isolated from this newly infected host and once again grown in pure culture.

These postulates may not hold up if:

The bacteria in question is unable to be replicated in pure culture (e.g. Mycobacterium leprae which causes leprocy)
It has infected deeper tissues within the infected host
It is causing a disease within an immunocompromised individual.

In 1988, Falkow wrote a set of 'Molecular Koch's Postulates' which stated that, in order to prove that a specific gene brings about disease virulence:

The deficiency of the gene in question brings about an attenuated phenotype
The reintroduction of said gene at the required level brings about virulence

An example of the above is shown in Leishmania footpad infection in mice; when the gene was knocked out, there was no virulence in infected mice, however when the gene was reintroduced the virulence, once again, was restored within the infected mice.

These 'molecular' postulates are important to establish whether it was the gene itself which caused the loss of virulence or whether it was an external factor, such as

The pathogen being held in culture for too long (thus becoming attenuated)
The pathogen randomly mutating to cause a loss of virulence

Reporter Genes

A reporter gene is one that is able to specifically interact with a genetic sequence of interest within an organism (whether it's bacteria, plants, animals, etc). These genes express markers (usually luminescent proteins) which are easily measurable and thus directly 'report' to a research group the activity of the gene in question within the target organism. This is typically introduced to the target organism via integration within a plasmid. Reporter genes are typically used to assess the effectiveness of gene delivery processes, determine the functionality of a gene product within a cell and/or determine the result of attempted cloning (among a variety of other uses).

Common examples include:

Jellyfish Green Fluorescent Protein (GFP). This reporter gene, when exposed to blue light, brings about a green fluorescence within the cells expressing the target gene.
Luciferase. This reporter gene (taken from fireflies) encodes Luciferase. This enzyme reacts with D-luciferin which is known to luminesce as it is converted to Oxyluciferin. Oxyluciferin releases a photon of light as it sts from an electrically excited state to a resting state. Signal detected via a luminometer, expression can be tracked in real time.
E.coli LacZ Gene. This gene brings about the synthesis of beta galactosidase, which brings about a blue appearance of bacteria when exposed to X-gal and IPTG containing media.
Chloramphenicol Acetyltransferase (CAT). This enzyme brings about resistance to the antibiotic chloramphenicol by catalysing the transfer of the acetyl group of acetyl-coenzyme A to chloramphenicol which prevents interaction with ribosomes. The chloramphenicol is radioactively labelled with 14C. It is typically used as a reporter gene in eukaryotes. The acetylated chloramphenicol is identifiable from the non-acetylated chloramphenicol via thin layer chromatography.
Epitope tagging. An epitope which is known to interact with a certain monoclonal antibody is bound with a protein to be investigated. This method is typically utilised to identify proteins for which no antibodies are known. It can be used to investigate function, interactions with other molecules and to identify the exact position of the protein within a structure. This method can also be used to investigate a protein which has a antigenicity homologous to another protein.

The following methods answer the following question:

"How could you investigate the function of an enzyme in a pathogen and determine whether it is a virulence determinant?"

Method 1: Targeted Gene Knockout

This methodology involves selectively knocking out certain genes within the enzyme to determine whether they were or were not directly linked to virulence.

The most long-standing method by which a gene knockout is conducted is via homologous recombination. This process involves designing a DNA construct (e.g. plasmid) containing the mutation that is to be integrated into the target organism's genome. This usually consists of the desired gene being replaced by a drug resistance marker. The construct is then inserted into stem cells, which then integrate the construct into their genome via homologous recombination. This is, however, an inefficient way of doing so, as homologous recombination only facilitates a small number of DNA integrations. If the recombination is successful, the stem cells are then used in vivo (e.g. in mice) to confer the mutation in the target region.

Method 2: RNA Interface (RNAi)

This process utilises dsRNA to inhibit gene expression of specifically targeted mRNA. It is used to identify and suppress specific genes within a target organism. The creators of this won a nobel prize in 2006 for their work on RNAi in C.elegans published in 1998.

The enzyme 'Dicer' initiates the process by cleaving inserted dsRNA into multiple fragments of Small Interfering RNA (siRNA), which are then utilised to instruct enzymes to degrade specific strands of mRNA. One of the strands of the double stranded siRNA is broken down and the remaining strand is then inserted into the RNA-induced Silencing Complex (RISC). The catalytic 'Argonaute' section of RISC then brings about post-translational silencing of mRNA within the target cell.

Method 3: Cre-Lox & diCre-Lox

This process relies on the Cre recombinase enzyme to genetically modify target sections of DNA within host cells. This process can be used to specifically target one cell type and can also be initiated by stimuli exterior to the cell. This methodology inserts specific sequences, known as LoxP sequences (isolated from bacteriophage P1), either side of a genetic sequence to be genetically modified (e.g. by, deletion, translocation, etc). These LoxP sites have specific binding sites for Cre recombinase which allow splicing to occur via its binding to the initial and final 13 base pair segments of the LoxP sequences. This complex then binds with another, similar complex, thus forming a tetramer. The orientation of LoxP sequences determines the type of genetic modification. For example:

Inverted LoxP sites on the same chromosome brings about an inversion of the spliced DNA sequence between the sites
Repeated LoxP sites on the same chromosome brings about deletion of the segment of DNA between the sites
For LoxP sites on 2 different chromosomes, a translocation of the DNA sequence between the sites will occur

Example of Cre-Lox in action:

Deletion of the CRK3 gene in Leishmania Mexicana
The diCre enzyme interrupts the 1st allele of the CRK3 gene before manipulating the 2nd allele with spliced CRK3-GFP. None of this affects function.
Rapamycin excises the CRK3-GFP complex which brings about significant problems with the cell cycle resulting in growth issues.

Method 4: CRISPR-Cas9

Stands for 'Clustered Regularly Interspaced Short Palindromic Repeats'.

The CRISPR DNA sequences are naturally found in bacteria and archaea and comprise small fragments of remnant DNA from bacteriophage infection. These sequences can be used to identify DNA of homologous phages during infections, thus allowing infected cells to be targeted and killed. These small segments of DNA make up a large part of the 'immune system' of the prokaryotic cell which specifically defends against viral infection.

The Cas9 enzyme is an endonuclease enzyme which is able to recognise the CRISPR segments and cleave complementary strands which are produced from them, therefore controlling any bacteriophage infection which may arise in the cell. As this enzyme is RNA-guided, by providing it with a synthetic RNA strand scientists are able to target and cleave specific strands of DNA with high efficacy. Thus, specific genes can be selectively removed or inserted in vivo.

The Cas9 enzyme can either insert genetic material via homology directed repair, or remove genetic material by bringing about non-homologous end joining of DNA strands. This typically results in random mutations within the strand, however, which may in turn result in dysfunctional genes. Cas9 enzymes isolated from Streptococcus Pyogenes has allowed for specific gene editing in eukaryotic cells.

Methods including the RNAi method do not entirely suppress the functionality of a gene, whereas the CRISPR-Cas9 method irreversibly knocks out the gene in question. It is thus a far more reliable method that RNAi. Also, in comparison, CRISPR-Cas9 technology is much cheaper than that required for RNAi.

There are, however, concerns over the ethical responsibilities posed to those utilising CRISPR-Cas9 systems. A potential avenue for scientific endeavour is that of germline editing, with the possibility to specific engineer human embryos to confer selected phenotypes. This raises concern surrounding the possibility of 'designer babies', whereby those with the money to do so could choose the traits of their prospective children, thus giving them selective advantages over children from poorer backgrounds, thus dividing the class system further.

End of answer to question

Genetically Modified Organisms

An organism which has had its genetic makeup altered by recombination. This includes genetically modified plants/animals as well as research microbes, but not organisms with traits conferred by selective breeding.

UK allows GM crop research but does not allow commercial sale of GM crops. All foods in the UK which contain GM ingredients must have this visibly labelled on the container.

Advantages:

Food production

Increased shelf life (insertion of ACC oxidase gene in tomatos slows the rate of ripening)
Crops have increased yield (LeETR4 insertion in tomatos brings about tomatos which ripen earlier than wild type tomatoes)
Allergens can be removed from certain foods (Arah2 insertion into the genome of peanuts downregulates the expression of peanut allergens)
Crops can be made to be resistant to certain pests. Crops are typically modified to have siRNAs of insects known to attack them (e.g. genetically insert NADPH-cytochrome P450 into cotton plants to reduce the ability of bollworms to grow and feed upon the cotton plant - as this reverses its resistance to gossypol expressed by the cotton plant).
Can facilitate new products (e.g. decaffeinated coffee plants are made from CaMXMT1 gene insertion bringing about theobromine synthase expression)

Pharmaceuticals

The vaccine for Hepatitis B was produced by genetically engineering yeast
The fungus Penicillium chrysogenum was genetically modified to drastically increase its yield of penicillin
Insulin (previously only extractable from animal pancreas') and Human Growth Hormone (previously only isolated from pituitary glands of cadavers) are now synthesised by processes involving genetic modification, thus facilitating a more accessible and affordable treatment of diabetes and dwarfism (respectively).

Disadvantages:

Risk of gene spread bringing about an increased rate of antibiotic resistance. Leak into the environment may also cause pests to develop resistance, thus rendering conferred genes for proteins such as cytochrome P450 useless thus causing a global shortage of cotton.
Animal toxicity studies have suggested that genetically modified crops may be associated with toxicity within the hepatic, renal, pancreatic and reproductive systems of humans.
- For example, it has been shown that synthetic growth hormone can increase the levels of IGF-1 in the individual, which may in fact increase that individual's risk of developing cancer.

	Created by Matthew Coulson over 4 years ago

Honours Degree Microbiology (Core Skills in Microbiology) Note on Genetic Modification of Pathogens, created by Matthew Coulson on 30/03/2020.