IGEM:Imperial/2010/Detection module/Parasites

We came to think that parasites can be an issue in the developing world. However, not knowing much about the situation out there we have decided to invite Dr Martha Betson who is specialist who is an expert in Biomedical Parasitology who is currently working on the "Control of schistosomiasis and molecular epidemiology of Schistosoma mansoni in Ugandan infants and pre-school children".

Martha Betson explaining the life cycle of Schistosoma

Schistosomiasis

We would like to detect schistoma cercariae. This could be done by inducing them to produce proteases using lipids such as linoleic acid and linolenic acid. We would express a protein on the surface of a Gram positive bacterium, which would be cleaved by one of the schistoma proteases. The resulting peptides would then be sensed by quorum sensing receptors on the surface of our bacterium, resulting in a downstream signaling cascade.

So, to detect cercariae, we need to:

1. Induce them to release proteases
2. Express a protein on the bacteria's surface which can be specifically cleaved by one of the proteases
3. Detect one of the resultant cleavage peptides
4. Transduce this signal to produce a response

Induction of invasive behaviour in cercaria:

Why is induction of invasive behaviour needed for parasite development?

The schistosoma parasite has the amazing ability to penetrate the skin (McKerrow and Salter 2002) of its host rather than relying on an insect vector or a wound to overcome the skin barrier. In order to enter the skin the parasite releases proteases that degrade keratin, collagen and other extracellular matrix (ECM) proteins. Without invasive behaviour the parasite could not enter its definitive host and would die, especially since survival of cercaria is limited to under 30 hours (100% infective for 3 hours, 50% infective after 8 hours) (Oliver 1966) in water. Rather than producing the proteases upon induction, they are premade and stored in a specialized gland - called acetabular gland complex – at the posterior region of the head (Fishelson et al. 1992) of the parasite, avoiding time delay caused by transcription/translation mechanism. This also implies though that the parasite cannot release proteases constitutively but needs a mechanism to sense the presence of a nearby host.

Why do we want to induce invasive behaviour?

We want to exploit the proteases produced as part of invasive behaviour to activate our quorum-sensing based system. The proteases provide a unique way to specifically detect schistosoma in water sample and are an integral part to this system. As explained before proteases are only released as part of the invasive behaviour that has to induced.

How is invasive behaviour induced naturally?

As the cercaria want to penetrate skin, they have specialized sensory systems able to detect skin fat in particular fatty acids. In schistosoma species that infect humans, cercariae are attracted to light (positively phototropic)and thus congregate near the surface of shallow water in order to maximise human contact. Additionally Cercariae follow a thermal gradient towards the host body. Upon contact with human skin, cercariae respond to chemical signals, particularly medium-chain free fatty acids, as a signal for skin invasion (McKerrow and Salter 2002): Of the C18 acids examined, stearic (18:0) is inactive, oleic (18:1) slightly active, linoleic (18:2) and linolenic (18:3) acids highly active (Austin et al. 1974). These stimuli lead to signal-dependent breakdown of inositol phospholipids which is directly linked to activation of protein kinase C (via elevated diacylglycerol level) and mobilization of calicium (via elevated levels of inositol triphosphate) which in turn evokes subsequent cellular response such as release reactions (Matsumura et al. 1991).

How can we induce invasive behaviour?

In order to induce invasive behaviour in the lab we should aim at providing as many natural stimuli as possible: Temperature can be adjusted by transferring the cercaria to warmer water or raising the temperature of the water. In order to trigger protease release, the presence of fatty acids will be essential and Austin et al. (1974) used crude egg lecithin, where oleic acid was shown to be the most abundant fatty acid and probably the biggest stimulant as well. Alternatively chemically synthesised fatty acids could be used, skin fat extracts or fatty acids synthesised by our GM organisms itself. All of the later possibilities seems more expensive or labour intensive than the commercially available egg lecithin.

Outline of the way we could deal with inducing invasive behaviour

If the GM bacteria are stored as spores we can add the fatty acids maybe in form of a small tablet or capsule. This prevents activated bacteria from metabolizing the stimulus. When the water sample is added, the tablet will dissolve and the releases acids will activate invasive behaviour in the parasite if present, triggering the protease detection system.