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 an expert in Biomedical Parasitology currently working on the "Control of schistosomiasis and molecular epidemiology of Schistosoma mansoni in Ugandan infants and pre-school children".
The life cycle of schistosoma and why we want to target them:
For more information on the life cycle of schistosoma and the impact this parasite has on humans this site provides a great introduction.
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:
- Induce them to release proteases
- Express a protein on the bacteria's surface which can be specifically cleaved by one of the proteases
- Detect one of the resultant cleavage peptides
- 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.
Products of Linoleic acid metabolism by cercaria
Following detection and uptake of linoleic acid in water, the cercaria rapidly metabolize the fatty acid and produce a number of quite distinct products called eicosanoids including prostaglandins, hydroperoxyeicosatetraenoic caid, leukotriens and many others. For full overview of the products of linoleic acid catabolism see Fusco et al. 1985.
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.
Detection via Parasite proteases
Upon detection of human (or mice) skin lipids and temperatures close to 37°C, the invasive behaviour is triggered. Several proteases are released from pre- and postacetabular glands or the cercaria at the leading edge of the invading parasite. While multiple enzymes are released, only one protease activity was found to be present in the gland-excretions and to be necessary for invasion in S. mansoni, haematobium and douthitti (Kasny et al 2009): Schistosoma elastase 2a and b (SmCE-1a/b) (Salter et al. 2002). SmCE are trypsin family serine proteases the specificity of which has been investigated by various studies. It appears that the following sites are preferred:
- P4 - S,T
- P3 - S,W,Y
- P2 - P
- P1 - L
Subtle differences exist between SmCE-2a and b as far as their P4 and P3 sites are concerned. Cleavage kinetics were determined for four different sites, P4-P1: SWPL, TWPL, RWPL, RRPL with R previously determined as unfavourable at P4 and P3. For P4 an 11-fold difference in activity was determined between favourable S/T and R, while a 3 fold difference was determined for P3 W to R (Salter et al. 2002) (see figure 1). The most favourable sequence would therefore be SWPL.
|Uniprot accession||MEROPS ID||Clan||Family||pH optimum||Molecular weight (practical/theoretical)|
Previous trap designs:
„This inability of study precisely where cercaria occur in the body of water has precluded observation of actual transmission potential and the study of cercarial distribution in the habitat itself.“ Glass slide 25mm x 75mm with stimulant matrix applied close to 1 end. Matrixes tested included agar but most satisfactory matrix was comprised of clear nail varnish base and linoleic acid. Preparation: one drop of nail varnish on slide and 5 µl linoleic acid added. Mixed and spread over 4cm2. Dried at room temperature under vacuum to remove organic solvents (storage in dark for 24 hours does not alter efficacy). If submerged in water for 2-3 hours and then taken out, cercaria (if present) will have attached to the slide and can be easily counted (no stain used). Was tested in lab and field. Allowed (reproducible) retrival of cercaria at high than expected level. In fiel around 30% of expected population in the volume of water was trapped and worked well at both high and low density of cercaria in water. There is evidence from directed movement towards the stimulus and the more slides (up to 50) were used the more cercaria were trapped.
In this study the same trap design was used as by Shiff et al. 1993. But rather than studying the three anthropophilic species of schistosome cercariae, Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum, which can produce a local skin reaction and dermatitis schistosomica, they focused on detecting avian schistosoma which amongst others are present in the United States. The avian schistosome cercariae also produce a local skin reaction known as freshwater cercarial dermatitis, i.e., ‘‘swimmer’s itch’’, or marine cercarial dermatitis, e.g., ‘‘sea-bather’s eruption’’ or ‘‘clam-digger’s disease’’. Freshwater or marine cercarial dermatitis is manifested by macular eruptions, diffuse erythema, persistent itching, and purpuric lesions. The similarity of skin lipid composition between aquatic birds and humans may contribute to the erroneous penetration of avian schistosomes. Schistosoma mansoni cercariae are sensitive to stimuli from unsaturated fatty acids present in lipids on human skin surface.
Despite the fact that cercarial dermatitis is reported across the United States and around the world, the diagnosis is always presumptive as it is based on self-reported symptoms and exposure to water. This parasitic infection is now considered an emerging disease in Europe due to the increased public and economic impact of recreational and occupational outbreaks. Progressively more evidence has shown that bird-specific schistosomes can cause serious problems, i.e., neuropathology and neurologic and neuromotoric disorders.
The trap worked very effectively for human schistosoma as well as avian parasites.
This trap follows the original design by Shiff et al. 1993, but it aims at taking advantage of locally available resources to make its use more feasible. In particular naturally occurring oils were examined including sesame, olive, corn germ, sunflower, pea nut, safflower, palm, cotton seed, plum plain seed, soya, coconut and linseed oils (see link for table with linoleic acid content of these oils). In the end a sesame-olive oil mixture seems to have proven most effective.
This approach proved problematic because the natural oils did not mix readily with the nail varnish base but rather formed a separate layer on top of it which was less able to trap cercaria. Furthermore this posed a problem in moving water, but not in standing water such as irrigation systems.