Anatomy and actions of microscopic agents of terror

AnthraxBacillus anthracis, the bacterium causing anthrax is an obvious choice for a biological weapon because it is relatively easy to culture, and, under appropriate conditions, will form spores.For maximum effect, these spores need to be delivered in an aerosol so that they are inhaled. The smaller the spores, the more dangerous the weapon, because they penetrate more deeply into a person's lungs.B. anthracis is a gram-positive, soil-dwelling bacterium that can be picked up by grazing animals, and most natural infections occur through contact with animals or contaminated products. In addition to inhalation, B. anthracis spores can penetrate the skin through cuts and abrasions, or be ingested in food.Once in the body, spores may germinate at the site of entry or may be engulfed by macrophages and carried to lymph nodes. There they germinate, multiply and enter the blood stream.The bacterium has three virulence factors that enable it to survive and cause damage — an antiphagocytic capsule and two protein exotoxins termed 'lethal' and 'oedema'.The bacterial capsule is made of a polymer of poly-D-glutamic acid that, in contrast to its spore, enables the bacteria to resist phagacytosis as well as protecting it from being broken apart by serum cationic proteins. Genes coding for the capsule (capA, capB and capC) are contained on the 95.3kb plasmid, pXO2.The toxin genes are found on the 184.5kb plasmid, pXO1s, and generate toxins that are composed of a receptor-binding moiety called protective antigen (PA) and two enzymatic moieties – oedema factor (OF) and lethal factor (LF). While the individual proteins are non-toxic when released from the bacteria, human cell-surface proteases split PA creating one fragment that binds to the cell in clumps of seven. This heptamerised PA then binds OF and LF and the complex is drawn into the cell by endocytosis forming a vesicle — composed of a piece of cell wall, the receptor and the toxin — an endosome. Within the endosome OF and LF become liberated into the interior of the cell, the cytosol.LF is a zinc-dependant metallo-protease that stimulates macrophages to release tumor necrosis factor alpha, interleukin-1beta and other cytokines, which together play a role in the sudden death of anthrax victims.OF (a calmodulin-dependent adenylate cyclase) disturbs the system of intracellular messengers, upsetting water homeostasis and resulting in oedema. It also inhibits phagocytes that would otherwise engulf and remove infecting cells.Asymptomatic patients undergo a 60-day course of ciprofloxacin tablets, though there must be concern that excess prescribing of this drug could increase fluoroquinolone resistance. Intravenous antibiotics are given if symptoms are present.BotulismFirst recognised as "sausage poisoning" in the 18th century, Botulism is normally associated with poor food hygiene, where food has either been not heated or incompletely heated. Initial symptoms usually include blurred vision, and difficulty swallowing and walking. The botulism toxin is the most potent toxin known, and, while anti-toxins exist, a victim will need a high level medical care until they take effect.The root cause of the disease is the anaerobic, spore-forming, motile, gram-positive bacterium Clostridium botulinum, but it is the microbe's toxin rather than an infection per se that causes the symptoms. Temperatures greater than 85oC denature the proteins, thus inactivating the toxin.But, if the toxin remains active it inhibits release of acetylcholine at presynaptic nerve endings, paralysing the nervous system with symptoms appearing first in the thorax and arms, but spreading rapidly down the body.There are seven distinct forms of the toxin, named A through G. Each is composed of a 100-kDa heavy chain joined by a disulphide bond to a 50-kDa light chain. A carboxyl-terminal domain of the light chain binds to the nerve ending of peripheral nerves.The toxin is internalised by endocytosis, and a region of the light chain containing a zinc dependant metallo-protease then cleaves the proteins of the nerve-cell apparatus that would normally enable the nerve terminal to release acetylcholine. With the inability to release this neurotransmitter, the muscles are paralysed. Death is usually caused by respiratory failure from paralysis of the diaphragm.PlagueYersinia pestis, the microbe causing plague, killed more than a third of the European population in the 1346 pandemic. While advances in public health and antibiotics reduce the chance of a similar pandemic, plague could still cause infection and subsequent panic. That there is no room for complacency became starkly apparent in 1995 when a case of multi-drug resistant Y. pestis occurred in Madagascar. The bacteria had somehow managed to acquire a plasmid conferring multi-drug resistance, probably after coming into contact with Escherichia coli.There are three different types of this gram-negative, non-motile, facultative anaerobic coccobacillus — Antiqua, Orientalis and Mediaevalis — that differ in details of their metabolism.The bacteria live in rodents and are transmitted via fleas, which move to human hosts if the rat population suddenly declines, or if people live in close proximity to the rats. Releasing infected rats into a city would be a very effective way of initiating an outbreak.The flea that is normally blamed for plague is Xenopsylla cheopis, but Yersinia pestis can be transmitted by over 1,500 flea species. The most significant vector in the U.S. is Diamanus montanus, the flea that lives on rock squirrels and Californian ground squirrels.When a flea feeds on an infected rat, Y. pestis enters its gut. At temperatures below 27oC the bacteria grow, but clump together and block the flea's gut. With its gut blocked the flea becomes hungry and the bacteria are regurgitated at the next feed. Above 27oC the bacteria do not aggregate, the gut remains clear and the flea feeds less. Consequently transmission is less common in the summer than the winter.Although not fully understood, Y. pestis virulence comes packaged on a 70-kb plasmid (sometimes called pYV) that enables it to survive and multiply in the host's lymphoid tissues. The plasmid carries codes for 29 proteins that form a dedicated type of secretion apparatus (Ysc) and a set of secreted proteins collectively known as Yops.When living in a nutrient rich environment at 37oC, the bacteria install Yscs and place a series of binding proteins on their surface. The bacteria build a stock of effector Yops, and bide their time.Upon contact with a cell from a multi-cellular organism — humans, for example — the bacteria lock onto the cell, open the Ysc and use three more proteins to build a 16-23Å pore in the plasma membrane of the cell. This allows the effector Yops to gain access to the cells under the guiding influence of translocators YopB, YopD and LcrV.Of the many Yops that pass through, YopE and YopD appear to have the most profound effect. YopE, a GTPase activating protein (GAP), is the only one that can kill a cell if injected on its own. YopD seems to be involved in getting YopE into the cell where YopE disrupts the eukaryotic cell cytoskeleton, an action that contributes to the bacterium's ability to resist phagacytosis.Other effector proteins down-regulate the host's inflammatory response, induce death of macrophages and inhibit antigen-specific T and B lymphocyte responses.There is still much to be discovered about this ancient killer.Smallpox and camelpoxA worldwide campaign of eradication allowed the World Health Organisation to declare in 1980 that the smallpox virus, variola, had been banished from the wild and was now confined to two laboratories, one in the USA and one in the Soviet Union. In the days before the Cold War ended, the Soviet laboratory had a programme designed to develop large quantities of the virus and to build ballistic weapons that could transport the pathogen to a target population. There are now fears that since the demise of the Soviet Union these stocks may not be fully contained.Initial symptoms of smallpox infection are high fever, malaise and severe headache and backache. This is followed by a raised rash that starts on the mucosa of the mouth, pharynx, face and forearms, and spreads to the trunk and legs. The rash forms painful blisters that fill with puss. Crusts begin to form after eight days, and, when the scabs fall off, they leave pigment-free pitted scars.Smallpox is highly infective, spreading easily from person to person and can remain stable in the environment for long periods. It kills about one in three of those infected.Sequence analysis has revealed the 168,000 bp genome code contains information for some 187 genes, making smallpox one of the most complex viruses known.Part of the secret of its action lies in the fact that 100 of these genes build proteins that are found within the human immune system. Part of the body's defence system is a suite of complement proteins that help scavenging phagocytes to detect and destroy invading foreign microbes. The virus vaccinia complement-control protein (VCP) inactivates the human complement system, making it easier for the virus to invade and survive in human cells.While there is a vaccine, few people in the world have been given it because the disease has been eradicated. Since the 11 September 2001 attacks the US government has started stockpiling vaccines.There were rumours that Iraq was trying to develop weapons based on camelpox, a similar virus. The concept was that people indigenous to the region might be naturally immune to the disease, while foreigners would be susceptible to infection. This would be a form of ethnically sensitive weapon.

Anatomy and actions of microscopic agents of terror

A primer describing the fundamental biology and basis of toxicity of five of the organisms most likely to be used as bioweapons.

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