It's in the genes

For years, Nathalie De Jonge, Remy Loris and their colleagues of the VIB Department of Molecular and Cellular Interactions at VUB, have applied their relentless dedication to the study of the precise structure and function of the toxin-antitoxin complex, a system that had not been the focus of much interest in the past. Only in the last couple of years the rest of the scientific world come to realize its importance and as a result the number of papers in this field has exploded.

All living creatures, people as well as bacteria, store their genetic information in the same way, i.e. in the DNA. Every human cell contains 46 neatly folded DNA strands that together measure two meters, while bacteria have to make do with around one millimetre of DNA. A piece of DNA containing the recipe for one characteristic, such as "how to make citric acid" or "how to make hair curl," is called a gene. Humans have several tens of thousands of genes.

Toxin and antitoxin

If your genetic information becomes damaged, you have a good chance of becoming ill or even dying. This is also true for bacteria, which over time developed a handy way of providing extra protection to important genes – the toxin-antitoxin (T-A) system. These T-A genes are tucked in near the genes to be protected. T-A genes contain instructions for both a toxin and its antitoxin. As long as the cell is producing both, all is well. However, if for some reason the piece of DNA where the T-A gene is located gets damaged or lost, the production of toxin and antitoxin comes to a halt and a time bomb starts ticking. Because the toxin is more stable than the antitoxin, it is broken down more slowly by the cell's clean-up mechanisms. Once the antitoxin is all gone, there is still enough toxin left to kill the bacterium. The upshot for the species is that bacteria that loses their T-A gene – and probably have sustained damage to the important genes just next to it – can no longer reproduce.

Our best-known intestinal residents, Escherichia coli bacteria, more commonly known as E.coli, have such a T-A system in five different locations in their DNA, while Mycobacterium tuberculosis bacteria even have them in 60 locations.

A difficult feat

The T-A mechanism has been known for a while, but nobody clearly understood the workings of the proteins carrying out the instructions of the T-A gene. The VIB researchers clarified in detail both the appearance of the toxin and antitoxin, the mechanism of their interaction and the forms they take while in action – a difficult feat to pull off, requiring the simultaneous use of a whole range of different technologies. One of the difficulties for instance lay in the fact that part of the antitoxin lacks a fixed structure. This formlessness keeps it from being brought into view.

Future

Now that we finally know how the time bomb functions (or more exactly, one of the time bombs, as there are several closely related T-A systems), biomedical scientists can start looking for substances to start the time bomb of pathogens ticking, i.e. substances that imitate the toxin protein, block the antitoxin protein, or disrupt the interaction between the toxin and antitoxin. In time, a new class of antibiotics might come out of it – though Nature mostly has a countermove up its sleeve against any move scientists do.

The researchers examined tissue samples from 58 people with squamous cell carcinoma (SCC), a highly curable form of skin cancer that is expected to affect more than 200,000 Americans this year.

They identified the virus in more than a third of the patients and in 15 percent of the tumors tested. In addition, all of the virus found in tumor cells had a mutation that could enable the viral DNA to integrate into the DNA of the host cell.

“This is indirect evidence that the virus might play a role in causing some cases of squamous cell carcinoma,” says principal investigator Amanda E. Toland, assistant professor of molecular virology, immunology and medical genetics and a researcher with the Ohio State University Comprehensive Cancer Center - James Cancer Hospital and Solove Research Institute.

The findings are published in a recent issue of the Journal of Investigative Dermatology.

The virus was first discovered in patients with Merkel cell carcinoma, a rare, aggressive skin cancer that occurs mainly in the elderly and people with a suppressed immune system. The people in the new study all had a healthy immune system.

“Originally it was thought that this virus caused only this rare skin cancer, but our findings indicate that it is a lot more prevalent than we initially thought.”

To learn if people with SCC harbored the virus, Toland, working closely with first author and graduate research associate Amy Dworkin and Ohio State pathologists O. Hans Iwenofu and Sara B. Peters, examined DNA samples from SCC tumors, from normal-appearing skin adjacent to the tumor, when available; from white blood cells, and from cells washed from the mouth.

The investigators detected the virus in 26 of 177 SCC samples, 11 of 63 adjacent-skin samples, and one sample from a mouthwash. They found no viral DNA in any of the blood samples from 57 patients. In all, 21 of 58 SCC patients, or 36 percent, tested positive for the virus.

By sequencing the viral DNA from 31 normal and tumor samples, the researchers showed that the same mutation was present in all the viruses tested from tumors, and in 60 percent of the viruses tested from adjacent healthy-looking tissue.

“That suggests that the virus may develop a mutation that causes it to integrate into host-cell DNA, and, therefore, may play a role in causing the cancer,” Toland says.

Next, Toland wants to test normal skin in healthy individuals to learn how common this virus is in people generally and to learn whether the virus actually integrates with the host DNA.

“If it proves to be a cancer-causing virus, and if it proves to be common in the general population, it might be something we should begin screening people for,” she says.

Funding from the American Cancer Society supported this research. Ohio State researchers Stephanie Y. Tseng and Dawn C. Allain were also involved in this study.