Antibiotics and Synthetic Biology

The model for bacterial death by antibiotics was fairly simply until recently. Antibiotics work by targeting a certain area of the bacteria; beta-lactams target the cell wall, Rifamycins target RNA synthesis, tetracyclins inhibit protein synthesis etc. The theory was that by inhibiting these processes, a certain vital function within the bacteria would be stopped, leading to its death.

However due to research done by Kohanski (references below) the story is looking a bit more complicated. Looking at three different classes of antibiotics they found that no matter what the site of action, all the antibiotics induced hydroxyl radicals. This was in bactericidal drugs, which actually kill bacteria, rather than bacteristatic ones (which just prevent cell growth). They also demonstrated that this mechanism of hydroxyl radical production was the end product of a chain of reactions involving damage to the TCA cycle (aka the Krebs cycle - which is a major part of respiration) which lead to damage to iron-sulphur clusters and subsequent production of the DNA-damaging hydroxyl radicals. This is shown diagramatically below, and this first paper was covered by Jim at Mental Indigestion with some great follow-up comments and discussion.

They've recently put out a review (second reference below) of which I find the most exciting parts are the two little extra-information boxes. One of them covers drug synergy and the second covers synthetic biology, both of which I'm getting increasingly more interested in.

Drug synergy

One of the most useful things about modelling drug actions is it can help to show which drugs would work most effectively in pairs. Using two drugs together can have many potential effects; it can make the treatment more effective, sometimes is can make the treatment less effective and of course some can be dangerous for the patient. Work on drug synergy showed that aminoglycoside antibiotics (which affect RNA synthesis) become more affective when given simultaneously with B-lactam antibiotics (which lead to cell wall breakdown) as the increased cell wall breakdown helps the aminoglycosides to get inside the cell. Conversely, drugs that inhibit protein synthesis are less effective when given at the same time as drugs which inhibit DNA synthesis as making it harder to synthesise proteins from sub-optimal DNA actually makes the cell more able to survive.

These interactions will affect the dosage of drugs used during synergistic treatments, and it is hoped that using two different types of antibiotics at low doses might be more healthy for the patient, and might help to combat against antibacterial resistance to one of the drugs.

Synthetic Biology

Another interesting concept the paper brings attention too is the potential use of synthetic biology to aid in both the study and application of antibiotic-related death systems. By using synthetic genes to disrupt or alter the proposed antibiotic network novel drug targets could be discovered. If turned into a high-throughput system this would be far more useful than the current screening system which tests for a potential drugs interaction with a target, rather than the ability of this interaction to lead to cell death.

Synthetic genes can be delivered into the bacterial cell via bacteriophages. Adding a synthetic gene into a bacteriophage for bacteria cell delivery has been attempted successfully before when they were used to enhance E. coli cell death by delivering genes for proteins that disrupted the DNA-repair system within the bacteria. This allowed faster and more effective killing of the bacteria at lower doses of antibiotic.

At a time when bacteria are fast becoming resistant to even the front line drugs, research that suggests novel ways of killing bacteria can produce some very useful outcomes. Using combinations of drugs at lower concentrations, or aiding antibiotics by introducing them along with synthetic genes in bacteriophages allows an increased shelf-life of the drugs that we currently possess as well as providing potential systems to aid the discovery of new antibiotics.

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Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, & Collins JJ (2007). A common mechanism of cellular death induced by bactericidal antibiotics. Cell, 130 (5), 797-810 PMID: 17803904

Kohanski MA, Dwyer DJ, & Collins JJ (2010). How antibiotics kill bacteria: from targets to networks. Nature reviews. Microbiology, 8 (6), 423-35 PMID: 20440275

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The Lab Rat guide to DNA Synthesis

The structure of DNA is a double helix of two sugar-phosphate backbones joined by hydrogen-bonds between nitrogenous bases, as shown below:

Image from Bioinformaticsweb.org

The letters of the DNA code come from the bases; adenine (A), thymine (T), guanine (G) and cytosine (C). They code for amino-acids, which make up proteins, in groups of threes, i.e GCC codes for alanine, GGA codes for glycine etc.

Each base, along with the associated sugar and phosphate, forms its own little subunit. Joining these together in the correct order can code for any protein you want. As a Lab Rat I don't know very much about this process, except that I send the sequence off and get back a little vial full of DNA (or a stab containing the bacteria that have my DNA held on a separate plasmid). So what I'm writing here is just what I've managed to find out about the process - it might not reflect the most up-to-date method used in the top sequencing companies, but it's a plausible way to make DNA.

There are two main types of DNA synthesis. Firstly there's small sequence oligonucleotide (aka small-bit-of-DNA) synthesis, to make primers and things. Secondly there's whole gene synthesis, which deals with larger sections of DNA. As whole gene synthesis mostly involves sticking together little bits of DNA, I'm mostly going to focus on small oligonucleotide synthesis.

The basic process involves sticking the growing DNA strand to a solid support and then just washing the next DNA base through, over and over again. This is pretty much automated nowadays, so you just program a robot to do it. The supports used are mostly either Controlled Pore Glass or macroporous polystyrene (plastic with small holes to select for size, allowing the salts and bases to be washed away before the larger DNA molecule is eluted). Both of them covalently attach to the end of the DNA chain, holding it in place as the nucleotides are washed through.

In their natural state, however, nucleotides are not very reactive, so special modified versions are used. Large bulky groups such as DMT and cyanoethyl are used to block the ends of the bases and the phosphorous linkages, to stop them reacting or participating in reactions.

The first base is then attached to the support and the DMT group (attached to the bottom of the base - the five sided ring) cleaved off with an alkaline wash. The next subunit is then activated before being added to the support. This involves adding tetrazole, which cleaves off the three big rings shown on the left, making the subunit more reactive. The activated subunit is then washed through the column, where it can react with, and bind too, the preceding base.

Once all the bases have been added in the correct order the mixture is purified, to isolate the required sequence. This is done by desalting, usually with chromatography, to produce the final product.

In case anyone was wondering, the robot/machine/computer used for synthesis looks like this:

Image taken from monash university website.