Tuesday, February 14, 2006

Evolution vs. Design: Man made biological weapons part I

Biological agents that cause disease have a long and terrible history in human warfare. During the crusades, a common tactic to break a seige was to toss the rotten corpses of animals into the starving populace to spread disease. Much of the army that Napoleon marched into Russia was struck down by typhus, which ground his offensive to a halt and caused him to retreat. Even more recently in World War 2, the Japanese were very interested in the use of biological and chemical agents. Projects conducted by the Japanese included dropping plague infected fleas onto Chinese cities and experiments with anthrax. Anthrax was considered as a last resort biological weapon by the British military, with an island off the coast of Scotland being rendered completely uninhabitable by various tests.

Today, unlike in the previous examples the threat of biological weapons is much greater, because technology has advanced to the point where we can simply design our own pathogens to be more effective weapons. Indeed, there has been a lot of concern over the potentials of newly altered biological weapons as potential WMDs (Weapons of Mass Destruction). The candidate list for organisms is also pretty long including such former greasts as smallpox as well as anthrax, plague, bacterial toxins like botulism, ebola and even influenza. As a result, being able to determine the hallmarks that would give away a potentially human designed organism could be essential in stopping it.

For example, an engineered strain of a virus could have entirely different functions compared to the normal wild-type. An organism that typically doesn't spread very easily person to person, could be engineered so that it is able to be aerosolised and spread through the air. This would greatly increase its ability to cause general havok by more rapidly spreading throughout the target population. The question that arises is how to determine if an organism is designed by human techniques, or if the pathogen is just another example of a newly emerged pathogen that has arisen by evolutionary processes.

This distinction is in fact fairly difficult to make in reality. Virulent microorganisms are highly capable of switching their genetic material around between one another by a process called Horizontal Gene Transfer (HGT). HGT involved numerous different mechanisms, but microorganisms are capable of simply grabbing DNA that is in the environment (say from other dead cells releasing it), circularised stretches of DNA often containing unique genes called plasmids, viruses can transfer genes from one organism to another (including between entirely different kingdoms) and even mobile stretches of DNA called transposons. A number of pathogens today, like several of the dreaded 'superbugs' that are found in hospitals such as MRSA (Methicillin Resistant Staphylococcus aureus) are the direct result of past HGT events.

So what sort of features would give away a pathogen that was designed by humans? Well this determination can only be made not by some irrelevant 'probability' calculation but by understanding the methods, motives and limitations that humans have when engineering new organisms.

Painting a scenario of a potential biological attack:

In this hypothetical scenario, patients manifesting signs of a rapid respiratory disease have been found in New York city with a nasty cough, quickly followed by a rapid scepticemia after a few days and almost inevitable death. Doctors observe to their horror that patients fail to respond to all front line antibiotics and the CDC (Centers for Disease Control) are rapidly called in to investigate. Their investigations simply confirm what health authorities already suspect, the disease in question is pneumonic plague caused by one of mankinds oldest enemies, Yersinia pestis. However, investigators are particularly worried by the complete unresponsiveness to the key antibiotics used to typically treat plague streptomycin, chloramphenicol and tetracycline. Most clinical strains of Y. pestis are nearly universally susceptible to antibiotics it raises a disturbing prospect: Is this a newly emerged and potentially epidemic quality kind of plague?

Even more agitating to investigators is another possibility that this is not a natural organism, but is instead the product of tampering by individuals attempting to utilise it as a weapon. Investigators have good reason to think this is a possibility, as in 2001 letters containing anthrax spores were distributed to several senators. Although only 5 people were killed and 13 infected, it was a pertinent reminder of the willingness and ability for potential terrorist organisations to employ biological agents. The source of these attacks is still unknown even to the present day and the perpetrator could still be at large. That investigators could be looking at a biological weapon with more on the way becomes a very real prospect.

The first step in combating the new strain after appropriate quarantine measures are taken, is to sequence the genome of Y. pestis strains collected from infected patients, as well as any extrachromosomal elements such as plasmids. Once done, investigators begin their analysis and their attention is immediately drawn to a large plasmid not normally associated with strains of plague found in America. Sequencing reveals the presence of a region of the plasmid with almost 4 different antibiotic resistance genes. Resistance plasmids are not particularly new in terms of Y. pestis, as highly multidrug resistant strains have already been found existing naturally in nature. Investigators analysing the strain immediately recognise something unusual, each of the genes is linked together by sequences exactly matching those recognised by restriction enzymes.

Restriction enzymes are the workhorses of clinical microbiology, biotechnology and genetics. Restriction enzymes are produced by bacteria to defend themselves from attacks from viruses called bacteriophages. By recognising specific repetitive sequences in the attacking viruses DNA, they prevent an infection by essentially chopping the viral genome into bits. One of the unusual properties of some of these enzymes is that when they 'cut' the target DNA they leave overhanging stretches of DNA. These overhangs can then be used to string together different genes that have been cut by the same restriction enzyme. Potentially as well, these enzymes could be used to string together antibiotic resistance genes and virulence factors needed to make a new biological weapon.

The investigators soon discover that this region of the plasmid containing the antibiotic genes are indeed linked by restriction enzymes, specifically using two that are very commonly available, EcoRI and BamHI. While this leads weight to the conclusion that the organism could have been designed by humans, it does not immediately indicate that this is the case. Genes for resistance have been used in biotechnology for numerous years, including in applications on plants and other transgenics including modified bacteria. In many cases, the antibiotic resistance genes are used as markers to select for bacteria that support the plasmid for replication purposes. After insertion into a plant or similar, the gene is useless for the plant but is still kept as a passenger into the environment.

As a result, it is theoretically possible that the genes on this plasmid are possibly derived from antibiotic markers used in standard genetic engineering. This could potentially explain the structure of this antibiotic group as a simple leftover from their previous application. Ruling this hypothesis out will require a deeper investigation into what antibiotic markers are commonly used in genetic engineering and particularly the question "how could Y pestis have aquired these genes?". Further, numerous important clues to the origin of this organism await to be found on the chromosome of this new strain and the plasmid it carries.


Anisimov A.P., L.E. Lindler and G.B. Pier (2004). Intraspecific diversity of Yersinia pestis. Clinical microbiology reviews, 434-464

Galimand M., A. Guiyoule, G. Gerbaud, B. Rasoamanana, S. Chanteau, E. Carniel and P. Courvalin (1997). Multidrug resistance in Yersinia pestis mediated by a transferable plasmid. The New England Journal of Medicine, 337:677-680.

Roberts R.J. (2005). How restriction enzymes became the workhorses of molecular biology. PNAS, 102:5905-5908.

Salyers A.A. and D.D. Whitt (2002). Bacterial Pathogenesis: A molecular approach 2nd edition. ASM press, Chapter 13:203-215.