Neglected tropical diseases
Neglected tropical diseases (NTD) as defined by the WHO, is a diverse group of 20 diseases which are
underdiagnosed in poorer communities, unproportionally affecting women and children. Thus, NTDs are diseases
where little work can give a great benefit. One of the most prevalent of these diseases is Schistosomiasis; a
condition caused by the parasitic flatworm Schistosoma.
Schistosomiasis is both an acute and chronic disease, mostly found in subtropical and tropical regions of South America, the Caribbean, the Middle East, and Asia [1]. Infection occurs when humans are in contact with contaminated water through fishing, bathing, or drinking. The worms penetrate the skin by secreting proteolytic enzymes which widen the skin pores. The adult worm lives in blood vessels, where the females lay eggs. The eggs travel through the body, some of them being excreted continuing transmission, and some becoming trapped in body tissues, causing damage and immune reactions [1,2].
Figure 1. Schistosoma Japonicum [3].
Schistosomiasis is considered by WHO the second-most socioeconomically devastating parasitic disease after
malaria, with an infection rate of around 240 million and about 700 million people living in endemic areas.
Untreated, the disease can among other complications, cause diarrhoea, abdominal pain, increased risk of
bladder cancer, and liver damage. The WHO has estimated the annual death rate of Schistosomiasis to be between
24 000 and 200 000 globally. [1,2,4].
The main current mode of diagnosis revolves around the detection of eggs. For urogenital schistosomiasis caused by
S.haematobium, the eggs are studied in urine samples through filtration techniques. The filtrate is
then examined under light microscopy in a lab. People infected with S.haematobium almost always have
microscopic hematuria (blood in urine), which can be detected by chemical reagent strips or with microscopes
[5].
The detection of eggs in intestinal schistosomiasis caused by
S.mansoni, S.japonicum, S.mekongi, S.malayensis, or S.intercalatum; instead uses a faeces sample in the
Kato-Katz technique. The results, however, are not always reliable due to the egg secretion being low or
intermittent, leading to possible negative test results. [1,2,4].
These methods, however, require specialised equipment and trained personnel. This makes them non-ideal in endemic areas with poor infrastructure. The Kato-Katz method is also hindered by the fact that the eggs are first detectable in stool samples late in the life cycle of Schistosoma, that is to say when symptoms are already present and the disease has already been able to spread. These techniques are therefore not sufficient in meeting the diagnostic needs.
The WHO has laid out a road map for the control of NTDs from 2021-2030 [6]. The goal for Schistosomiasis is the total elimination of transmission, and as a public health problem. The current and main strategy is the mass administration of the drug Praziquantel, mostly focused on school-age children (SAC), due to their prevalence of intense infections. Although to reach the goal set, mass administration reach needs to be extended from being heavily focused on SAC, to focus more on adults and adolescents. The current heavy focus on SAC has resulted in only 44.5% of adults (2019) requiring treatment to receive it, compared to 67.2% for SAC [1]. It is evident that more diagnostic tools are needed, to allow a more targeted administration of Praziquantel.
To increase the diagnosis rate, and thus reduce Praziquantel use, a more targeted tool would need to be implemented. This tool would ideally be cheap to be economically feasible for large scale use, and have a high specificity to retain efficacy. The tool should also be easy to use, requiring neither specialized equipment nor trained personnel; the hallmarks of point of care.
Our Project
To contribute to the WHO’s 2030 goal, we decided to develop a modular diagnostic tool, based upon detecting
fragments of Schistosoma DNA shown a few days after infection of Schistosoma [7]. This concept
would circumvent the six week waiting time needed for Schistosoma eggs to appear, required for the
Kato-katz technique.
The tool would ideally be self-contained, requiring no equipment to read the results. As such, we decided that a visual readout would be the optimal choice. Storage and transport were also two important factors considered when designing our project. Since endemic areas are mostly prevalent in rural areas with poor infrastructure, the diagnostic tool would need to have a long shelf life, and be stable at many different conditions. Due to the varying degrees of medical training among infected regions, the tool should also be easy to use.
To achieve all of these requirements, we developed two approaches or techniques for our project. The first one is cell based and uses zinc-fingers on
S.cerevisiae to detect DNA and induce an expression of violacein to produce a visual signal. The cell’s
mating system is also used for signal amplification. The second approach is cell free, and uses dCas9 for DNA
binding, and our designed Inhibited Beta-galactosidase (iGal), with X-gal to give a visual signal.
With a cell-free system, there would be no living organisms in the final system, thus increasing the shelf life. The cell-based system would rely on
S.cerevisiae or baker’s yeast, due to its suitability for low cost intracellular expression [8]. Both
systems produce a visual pigment in response to detected DNA, thus making the results easy to interpret.
Previous studies have shown that PCR-tests, both on water and patient samples, show promise as diagnostics for Schistosomiasis [9]. This would incur that our DNA detection method would also work for detecting
Schistosoma in freshwater. The scope of disease mapping would be greatly improved, as larger screening
of Schistosoma breeding grounds would be allowed, ultimately contributing to the WHOs 2030 goals.
Cell-Free System
The first system starts its reaction chain with two dCas9 molecules; CRISPR Cas9 molecules which are altered to not cut at their target site. The dCas9 protein is guided towards its target DNA sequence by its associated single guide RNA (sgRNA) [10]. This is accomplished through the dCas9 opening up the DNA strand, allowing for base pair complementation between the sgRNA and target DNA sequence. Flanking the target sequence are protospacer adjacent motifs (PAMs) which are recognized by a component of the sgRNA called CRISPR RNA (crRNA), allowing for further identification of the target DNA.
Figure 2. dCas9 recognition and binding to DNA. The His-tags are added for easy protein extraction.
Tobacco etch virus protease (TEVp) is a protease which cleaves the amino acid sequence ENLYFQG [11]. This protease is able to be split into the two parts N-terminal TEVp (nTEV), and C-terminal TEVp (cTEV), becoming non-functional units [11]. When combined, the two halves will spontaneously form a whole functional TEV protease. For the cell-free system, an nTEV is attached to the left dCas9, and a cTEV is attached to the right dCas9 using amino acid linkers, an approach which was also used by the iGEM Peking 2015 team but with luciferase instead of TEVp [12]. The sgRNAs have been designed to make the two dCas9 proteins bind close to each other on the
Schistosoma DNA. This induced proximity increases the likelihood of the TEVp halves combining, and
becoming a functional unit.
Figure 3. dCas9 binding to free floating DNA, with accompanying TEVp halves.
Free floating in the same solution is our designed protein, iGal, that consists of two mutated parts of a
Beta-galactosidase monomer [13,14] and X-gal. The two parts of the monomer, alpha and omega, are connected in
reverse order to how they naturally connect, using an amino acid linker containing a TEVp cut site. When a
functional TEVp is formed, it will cut the linker between the alpha and omega subunits. These are then able to
undergo alpha-complementation to form an iGal monomer. Four of these monomers then further combine to form a
functional iGal tetramer. A functional iGal is then able to cleave X-gal, producing a blue pigment, giving a
visual readout.
Figure 4. Schematic of the iGal system. TEVp is shown as the white ball.
Cell-Based System
((Strains: E.coli DH5alpha and the S. cerevisiae is a CENPK derivative))
The goal of the cell-based system is for the cell to produce a visible purple pigment when coming in contact with DNA from the parasite. Due to low levels of target DNA in patient samples, a signal amplification system was also integrated [15].
The cell-based system builds upon the S. cerevisiae strain yWS677 D4, a S288C derivative [16]. The recognition
of the DNA fragment is carried out by two different zinc-finger complexes. A zinc-finger complex consists of a
zinc-finger protein motif with a DNA recognition site, a transmembrane protein in the cellular membrane and a
split ubiquitin half [17,18,19,20]. All parts of the complex are connected by linkers and the complex is
illustrated in figure 5 below. The two zinc-finger complexes have affinity to different recognition sites on
the target sequence and are each connected to one half of a ubiquitin protein, NubG and Cub. Cub is attached
to a LexA-VP16 transcription factor. When the target DNA fragment comes in contact with the zinc-finger’s
recognition site, the zinc-fingers will bind to their corresponding binding site on the fragment.
Figure 5. Illustration of the two zinc-finger complexes. The "fingers" represents the zinc-fingers, the ovals represents the transmembrane protein, the "puzzle circles" represents NubG respective Cub. The white lines connecting each part are linkers. The transcription factor LexA-VP16 is connected to Cub by a linker.
The two binding sites are located closely to each other on the DNA fragment so that when the zinc-fingers bind,
they will get into close proximity. This will bring NubG and Cub together, activating the ubiquitin [18,19]. The
ubiquitin will mark the linker that connects the transcription factor for degradation. Ubiquitin protease,
present in the cell, will cut the linker, releasing the transcription factor LexA-VP16. The transcription factor
contains a nuclear localization signal, allowing it to be transported into the nucleus. LexA-VP16 binds to the
promoter LexO, allowing the transcription of two target genes, one essential gene in the biosynthesis pathway of
the purple pigment Violacein and mating factor alpha 1 (MF⍺1).
The transcription of Violacein is a five-step enzymatic reaction pathway illustrated in figure 6 below [21,22]. The pathway starts with the amino acid L-tryptophan which is naturally present in the cell and does not require any additional substrate. In order to control the expression of Violacein, the gene for the enzyme VioE is placed under the control of the LexO promoter. Vio A, B, C and D are integrated into the genome under the native promoters to be constantly expressed.
Figure 6. The biosynthesis pathway of violacein and its chemical structures.
The amplification system is based on the natural yeast mating system. MF⍺1, which codes for the alpha-peptide pheromone, is introduced to the yeast genome and is placed under the control of the LexO promoter [23]. The yeast strain expresses the alpha-peptide receptor STE2 which, when bound to the peptide, releases the transcription factor Ste12-LexA, targeting the LexO promoter. When the target DNA is recognized by the zinc-finger complex and the transcription factor LexA-VP16 is released, the transcription of alpha-peptide is initiated. Alpha-peptide is transported to the cellular membrane and excreted, allowing it to spread to neighboring cells. The receptors on the neighboring cells then initiate the transcription of both Violacein and the alpha-peptide, producing a visual output as well as continuing the signal amplification.
The complete system is visualized in Figure 7 below.
Figure 7. Overview of the yeast system.
Modularity
Both the cell-based and cell-free methods are based upon producing visual readouts in response to detected
Schistosoma DNA. Their specificity to Schistosoma DNA is however designed, meaning that it can
be changed.
This opens up many more applications for both techniques, as they could be designed to detect any free floating DNA sequence. Their high specificity and low cost of production would allow them to be used both in the field to diagnose NTDs, and in the home to diagnose infections without risking the infection of others.
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