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A protease-based biosensor for the detection of schistosome cercariae.

Webb AJ, Kelwick R, Doenhoff MJ, Kylilis N, MacDonald JT, Wen KY, McKeown C, Baldwin G, Ellis T, Jensen K, Freemont PS - Sci Rep (2016)

Bottom Line: Rapid and cost-effective approaches to detect parasites are needed, especially in resource-limited settings.Collectively, S. mansoni and several other schistosomes are responsible for the infection of an estimated 200 million people worldwide.Since our biosensors are maintained in lyophilised cells, they could be applied for the detection of S. mansoni and other parasites in settings without reliable cold chain access.

View Article: PubMed Central - PubMed

Affiliation: Centre for Synthetic Biology and Innovation, Imperial College London, London, UK.

ABSTRACT
Parasitic diseases affect millions of people worldwide, causing debilitating illnesses and death. Rapid and cost-effective approaches to detect parasites are needed, especially in resource-limited settings. A common signature of parasitic diseases is the release of specific proteases by the parasites at multiple stages during their life cycles. To this end, we engineered several modular Escherichia coli and Bacillus subtilis whole-cell-based biosensors which incorporate an interchangeable protease recognition motif into their designs. Herein, we describe how several of our engineered biosensors have been applied to detect the presence and activity of elastase, an enzyme released by the cercarial larvae stage of Schistosoma mansoni. Collectively, S. mansoni and several other schistosomes are responsible for the infection of an estimated 200 million people worldwide. Since our biosensors are maintained in lyophilised cells, they could be applied for the detection of S. mansoni and other parasites in settings without reliable cold chain access.

No MeSH data available.


Related in: MedlinePlus

Schistosoma biosensor work-flow.S. mansoni cercariae are shed from infected snails and mechanically-transformed to produce S. mansoni cercarial transformation fluid (SmCTF) samples, which were lyophilised. Whole-cell biosensors are treated with either reconstituted SmCTF biological samples or a control protease from a commercially sourced panel. Whole-cell biosensors are washed and labelled. Labelled cells are analysed via flow cytometry. Proteolytic cleavage of the biosensor via the activity of SmCTF-derived cercarial elastase prevents cell labelling, thus resulting in a ‘loss of colour’ biosensor output (+detection of S. mansoni). Labelled cells indicate that the biosensor was not proteolytically cleaved thus resulting in a ‘gain of colour’ biosensor output (−detection of S. mansoni).
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f2: Schistosoma biosensor work-flow.S. mansoni cercariae are shed from infected snails and mechanically-transformed to produce S. mansoni cercarial transformation fluid (SmCTF) samples, which were lyophilised. Whole-cell biosensors are treated with either reconstituted SmCTF biological samples or a control protease from a commercially sourced panel. Whole-cell biosensors are washed and labelled. Labelled cells are analysed via flow cytometry. Proteolytic cleavage of the biosensor via the activity of SmCTF-derived cercarial elastase prevents cell labelling, thus resulting in a ‘loss of colour’ biosensor output (+detection of S. mansoni). Labelled cells indicate that the biosensor was not proteolytically cleaved thus resulting in a ‘gain of colour’ biosensor output (−detection of S. mansoni).

Mentions: For a whole-cell-based biosensor to be functional and detect its specific target in the external environment, the biosensor component itself needs to be localised on the cell surface such that it is exposed to the target. Our biosensors have been designed to target the elastase activity released by S. mansoni cercariae to facilitate their invasion into their primary host: humans (Fig. 1). The biosensors have two general modular components: 1) an anchor module to localise and bind the sensor on the cell and 2) a detection module that has specificity for the target of the sensor (Fig. 1). The detection module comprises flexible linkers, the specific recognition motif of the protease that the sensor targets and an epitope tag for detection. When the biosensor is expressed in the host cell and localised to the outer membrane (E. coli) or cell wall (B. subtilis) in the correct orientation, the detection module is exposed to the external environment. Exposure of the biosensor to the protease specific for the recognition motif results in the removal of the epitope tag (Fig. 1). The fluorescent label that recognises and interacts with the epitope tag is therefore unable to bind to the biosensor and the cells appear colourless, thereby giving us a positive detection output (Figs 1 and 2). If the biosensor is exposed to a protease that does not recognise the detection module the motif is not cleaved, leaving the epitope tag still attached (Fig. 1). The fluorescent label is thus able to bind to the biosensor and the cells appear red in colour resulting in a negative detection output (Figs 1 and 2).


A protease-based biosensor for the detection of schistosome cercariae.

Webb AJ, Kelwick R, Doenhoff MJ, Kylilis N, MacDonald JT, Wen KY, McKeown C, Baldwin G, Ellis T, Jensen K, Freemont PS - Sci Rep (2016)

Schistosoma biosensor work-flow.S. mansoni cercariae are shed from infected snails and mechanically-transformed to produce S. mansoni cercarial transformation fluid (SmCTF) samples, which were lyophilised. Whole-cell biosensors are treated with either reconstituted SmCTF biological samples or a control protease from a commercially sourced panel. Whole-cell biosensors are washed and labelled. Labelled cells are analysed via flow cytometry. Proteolytic cleavage of the biosensor via the activity of SmCTF-derived cercarial elastase prevents cell labelling, thus resulting in a ‘loss of colour’ biosensor output (+detection of S. mansoni). Labelled cells indicate that the biosensor was not proteolytically cleaved thus resulting in a ‘gain of colour’ biosensor output (−detection of S. mansoni).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835807&req=5

f2: Schistosoma biosensor work-flow.S. mansoni cercariae are shed from infected snails and mechanically-transformed to produce S. mansoni cercarial transformation fluid (SmCTF) samples, which were lyophilised. Whole-cell biosensors are treated with either reconstituted SmCTF biological samples or a control protease from a commercially sourced panel. Whole-cell biosensors are washed and labelled. Labelled cells are analysed via flow cytometry. Proteolytic cleavage of the biosensor via the activity of SmCTF-derived cercarial elastase prevents cell labelling, thus resulting in a ‘loss of colour’ biosensor output (+detection of S. mansoni). Labelled cells indicate that the biosensor was not proteolytically cleaved thus resulting in a ‘gain of colour’ biosensor output (−detection of S. mansoni).
Mentions: For a whole-cell-based biosensor to be functional and detect its specific target in the external environment, the biosensor component itself needs to be localised on the cell surface such that it is exposed to the target. Our biosensors have been designed to target the elastase activity released by S. mansoni cercariae to facilitate their invasion into their primary host: humans (Fig. 1). The biosensors have two general modular components: 1) an anchor module to localise and bind the sensor on the cell and 2) a detection module that has specificity for the target of the sensor (Fig. 1). The detection module comprises flexible linkers, the specific recognition motif of the protease that the sensor targets and an epitope tag for detection. When the biosensor is expressed in the host cell and localised to the outer membrane (E. coli) or cell wall (B. subtilis) in the correct orientation, the detection module is exposed to the external environment. Exposure of the biosensor to the protease specific for the recognition motif results in the removal of the epitope tag (Fig. 1). The fluorescent label that recognises and interacts with the epitope tag is therefore unable to bind to the biosensor and the cells appear colourless, thereby giving us a positive detection output (Figs 1 and 2). If the biosensor is exposed to a protease that does not recognise the detection module the motif is not cleaved, leaving the epitope tag still attached (Fig. 1). The fluorescent label is thus able to bind to the biosensor and the cells appear red in colour resulting in a negative detection output (Figs 1 and 2).

Bottom Line: Rapid and cost-effective approaches to detect parasites are needed, especially in resource-limited settings.Collectively, S. mansoni and several other schistosomes are responsible for the infection of an estimated 200 million people worldwide.Since our biosensors are maintained in lyophilised cells, they could be applied for the detection of S. mansoni and other parasites in settings without reliable cold chain access.

View Article: PubMed Central - PubMed

Affiliation: Centre for Synthetic Biology and Innovation, Imperial College London, London, UK.

ABSTRACT
Parasitic diseases affect millions of people worldwide, causing debilitating illnesses and death. Rapid and cost-effective approaches to detect parasites are needed, especially in resource-limited settings. A common signature of parasitic diseases is the release of specific proteases by the parasites at multiple stages during their life cycles. To this end, we engineered several modular Escherichia coli and Bacillus subtilis whole-cell-based biosensors which incorporate an interchangeable protease recognition motif into their designs. Herein, we describe how several of our engineered biosensors have been applied to detect the presence and activity of elastase, an enzyme released by the cercarial larvae stage of Schistosoma mansoni. Collectively, S. mansoni and several other schistosomes are responsible for the infection of an estimated 200 million people worldwide. Since our biosensors are maintained in lyophilised cells, they could be applied for the detection of S. mansoni and other parasites in settings without reliable cold chain access.

No MeSH data available.


Related in: MedlinePlus