The Beetle to Beat TB

Over at Infectious Thoughts, Siouxsie Wiles has a video up about her research, which makes a great introduction to a paper that I've wanted to blog about for some time.

 

The goal of this research was to create a glowing strain of mycobacteria, and looking at this video, one may be fooled into thinking that this research was "simple". This is far from the truth. In fact , the route to getting a bioluminescent strain of mycobacteria was fraught with difficulty.

Getting any bacteria to express genes from other organisms is a difficult process. In nature, bacteria are often subject to attack from foreign genes from viruses. Some bacteria have defenses against this, others simply don't react well at all. many a synthetic biologist has created a "perfect" construct, only to find that it immediately kills all bacteria that express it.

The way in which genes are introduced into bacteria using a plasmid. Plasmids are circular pieces of DNA that can replicate in their host. With the inclusion of special sequences, they can integrate into the genome of the host. different plasmids can do ifferent things. Some of them can replicate a lot in their host, so each bacterium can have lots of plasmids, and therefore lots of copies of a gene. Conversely, you can have plasmids that copy themselves less often. The most extreme form is actually getting the plasmid to integrate within the chromosome, so that there is only one copy, that only replicates when the host cell replicates.

The number of plasmids containing your gene can affect the level to which it is expressed. More plasmids = more gene copies, and therefore more expression of that gene.

And then one needs to think about the gene to be used.

Firstly, whilst this video focuses on the firefly, this video could easily have featured a number of different organisms in its place. In nature, bioluminescence is surprisingly common, and a number of different organisms use different methods to glow. So when setting out to make mycobacteria luminescent, they had three candidates:

Firefly: The beetle that is shown in this video. Firefly bioluminescence is known to require a Luciferase enzyme (called Luc), and a compound known as D-Luciferin. Whilst we know that D-luciferin is needed for bioluminescence, the actual genetic system that creates this compound is not fully understood.

Gaussia Princeps: This tiny marine copepod glows in the dark of the deep sea. It's bioluminescence system requires a compound known as coelentarazine, and is one of the few systems that doesn't require oxygen as a substrate. The copepod itself doesn't make it's own coelentarazine. Instead, it often hunts other marine organisms to obtain it, and it is not known specifically what organisms are producing this compound. So the genes encoding it are not known.

Photorhabdus Luminescens : A nematode/ caterpillar pathogen. When it feeds on a caterpillar, it causes it's carcass to glow to attract more nemotode hosts. The luminescence system for this bacteria is simpler than those found for the eukaryotic organisms, with the genes encoding both the luciferase enzyme, and it's substrate are both known.

These facts will come in useful when thinking about taking these genes out, and actually using them in a practical situation.

One of the problems is the mycobacterium itself. The main purpose of this research was to develop bioluminescent strains of the bacterium Mycobacterium tuberculosis. However, it is probably not a good idea to expose themselves to TB on a daily basis, and moreover, it takes a long time to grow the bacteria. So instead of moving straight into TB, the researchers tested out their bioluminescent constructs in Mycobacterium smegmatis. M. smegmatis is generally non-pathogenic, and grows a lot faster than other strains of Mycobacterium. So it can be more easily used to test out these bioluminescent constructs.

So the author took three plasmids, one which has a high copy number, one with a low copy number, and one which integrates, and then partnered them up with the three different luciferase systems, shown above.

So you'd expect that with more copies of the gene, you would see more light production. What was found was in fact quite different.

 In fact the bacteria with just one copy of the luciferase genes glowed better than the bacteria with multiple copies. This same effect was found for the firefly luciferases , and for the bacterial luciferases. 

Whilst this initially surprising to me, my synthetic biologist friend simply made some derisive noises along the lines of "well duh". Bioluminescence is an energy intensive reaction. So a bacteria using up a lot of energy for bioluminescence will not have much energy for other important processes, like nutrient acquisition. And so the system feeds back on itself, setting up the paradoxical situation where less really is more.

But this was not the end of it. The amount a certain gene is expressed is also dependant on something known as a promoter. This is a section of gene sequence which controls how much a gene is expressed, by attracting proteins which "open up" the DNA strand, allowing the sequence to be read. They tried a number of different promoters, and found the best one to express the luminescent signal.

Using these techniques, the maximum amount of messenger RNA is made in the cell. But at some level, the decoding of these genes into actual proteins is limited. In order for protein to be made from messenger RNA, each segment of the DNA sequence needs to be partnered up with an amino acids. This decoding wis performed by transfer RNA, which binds to an amino acid, and to a three base pair sequence on the messenger RNA

There are 21 different amino acids which can be used to make up proteins, but 64 different combinations of codons. So some tRNAs that bind to different DNA sequences can bind to the same amino acid.

However, different organisms have different amounts of tRNA. So a firefly cell may have more of one type of tRNA than another, but a bacterial cell may have different number of tRNA.

If you take a gene directly from one and put it into the other, you may find that the protein takes longer to form, and therefore you get less of it.

However, if you take the amino acid sequences for the firefly luciferase, and then translate it into the DNA code according to the most abundant tRNA present in a cell, you can increase the chances of the right tRNA binding to the right area, and thus increase the rate of transcription. This technique is known as "codon optimisation". So the researchers performed this "codon optimisation" and found that it did indeed make the bacteria glow brighter.

So now that they had made sure that their genes worked in Mycobacterium smegmatis, they put them into Mycobacterium tuberculosis and found that they glowed just as brightly. The brightest signal came from the bacteria with the firefly luciferase.

Now that we have this glowing bacteria, we can see where it goes and what it does during an infection, without the need to kill animals at every single time point. And using this technique, the bacteria can show us where they go during infection. This is an unprecedented opportunity to find out, not only how this bacteria causes infection, but also to reveal new treatments to combat it. And all because of one beetle.

Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, Ripoll J, Parish T, Bancroft GJ, Schaible U, Robertson BD, & Wiles S (2010). Optimisation of bioluminescent reporters for use with mycobacteria. PloS one, 5 (5) PMID: 20520722