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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

Validation of E. coli whole-cell biosensors.(a) Biosensor circuit design and localisation in the outer membrane of the cell. (b) Induction of biosensor expression. Representative cell pellets (OD600 4.0) of either induced 100 mM xylose (+) or uninduced (−) biosensor-expressing cells were labelled with 1.25 μg streptavidin-R-phycoerythrin (SAPE)-conjugated antibody. Cell labelling (Red) indicates appropriate expression and localisation of the whole-cell biosensor. (c) Flow cytometry analysis of whole-cell biosensors. E. coli expressing either TEV (mCPX-TEV), elastase (mCPX-ELA) or control (mCPX-CON) biosensors were treated with the indicated proteases: AcTEV protease (TEV) or control proteases - PreScission protease (PRE) or Enterokinase (ENT). Treated cells were labelled with SAPE-conjugated antibody and analysed by flow cytometry. Labelled, non-protease treated cells and E. coli transformed with an empty vector plasmid (EV) served as experimental controls. (d) Summary of flow cytometry data. E. coli expressing either mCPX-TEV, mCPX-ELA or mCPX-CON biosensors were treated with the indicated proteases. The fluorescence (Geometric mean FL5) of protease treated cells were normalised against labelled, non-protease treated cells (No Prot). These data were analysed using FlowJo (vX 10.0.7r2) software and are representative of three independent biological repeats. (e) Sensitivity of mCPX-TEV biosensor. E. coli expressing mCPX-TEV biosensor were treated with 0–10 Units of AcTEV and analysed via flow cytometry. These data are normalised against untreated (0 U) labelled cells and represent the mean geometric mean ± the standard deviation of three independent experiments. Student t-test ****P < 0.0001.
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f3: Validation of E. coli whole-cell biosensors.(a) Biosensor circuit design and localisation in the outer membrane of the cell. (b) Induction of biosensor expression. Representative cell pellets (OD600 4.0) of either induced 100 mM xylose (+) or uninduced (−) biosensor-expressing cells were labelled with 1.25 μg streptavidin-R-phycoerythrin (SAPE)-conjugated antibody. Cell labelling (Red) indicates appropriate expression and localisation of the whole-cell biosensor. (c) Flow cytometry analysis of whole-cell biosensors. E. coli expressing either TEV (mCPX-TEV), elastase (mCPX-ELA) or control (mCPX-CON) biosensors were treated with the indicated proteases: AcTEV protease (TEV) or control proteases - PreScission protease (PRE) or Enterokinase (ENT). Treated cells were labelled with SAPE-conjugated antibody and analysed by flow cytometry. Labelled, non-protease treated cells and E. coli transformed with an empty vector plasmid (EV) served as experimental controls. (d) Summary of flow cytometry data. E. coli expressing either mCPX-TEV, mCPX-ELA or mCPX-CON biosensors were treated with the indicated proteases. The fluorescence (Geometric mean FL5) of protease treated cells were normalised against labelled, non-protease treated cells (No Prot). These data were analysed using FlowJo (vX 10.0.7r2) software and are representative of three independent biological repeats. (e) Sensitivity of mCPX-TEV biosensor. E. coli expressing mCPX-TEV biosensor were treated with 0–10 Units of AcTEV and analysed via flow cytometry. These data are normalised against untreated (0 U) labelled cells and represent the mean geometric mean ± the standard deviation of three independent experiments. Student t-test ****P < 0.0001.

Mentions: The design of the E. coli housed whole-cell biosensor is based on that previously used in a study to identify protease recognition specificities using cellular libraries of peptide substrates (CLiPS)1314. The CLiPS system uses the circularly permuted version of the E. coli outer membrane protein OmpX, CPX, as the anchor module1314. CPX has been engineered such that both its N- and C-termini are exposed to the exterior environment14. A poly-histidine tag is located on the C-terminal whilst the protease detection module with the streptavidin-binding peptide (WCHPMWEVMCLR)13 epitope tag is present on the N-terminus (Fig. 3a). The three E. coli housed biosensors employed in this study (mCPX-TEV, mCPX-ELA and mCPX-CON) were created by replacing the recognition motif present in the CLiPS system with the relevant peptide sequence as described in the materials and methods section. The biosensor fusion genes were placed under the control of the xylose inducible promoter xylF (BBa_I741018) and the moderately strong RBS B0032 (BBa_B0032)15 as shown in the circuit visualised using Pigeon16 (Fig. 3a). This was to avoid any potential deleterious effects caused by overexpression of this outer-membrane protein by the cells. Furthermore, it was decided that the E. coli cloning strain NEB-10beta would be the host of choice for the biosensors, as this is not classically an expression strain and so, theoretically, would not overexpress the biosensors and would therefore minimise the risk of any detrimental burden-based responses.


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)

Validation of E. coli whole-cell biosensors.(a) Biosensor circuit design and localisation in the outer membrane of the cell. (b) Induction of biosensor expression. Representative cell pellets (OD600 4.0) of either induced 100 mM xylose (+) or uninduced (−) biosensor-expressing cells were labelled with 1.25 μg streptavidin-R-phycoerythrin (SAPE)-conjugated antibody. Cell labelling (Red) indicates appropriate expression and localisation of the whole-cell biosensor. (c) Flow cytometry analysis of whole-cell biosensors. E. coli expressing either TEV (mCPX-TEV), elastase (mCPX-ELA) or control (mCPX-CON) biosensors were treated with the indicated proteases: AcTEV protease (TEV) or control proteases - PreScission protease (PRE) or Enterokinase (ENT). Treated cells were labelled with SAPE-conjugated antibody and analysed by flow cytometry. Labelled, non-protease treated cells and E. coli transformed with an empty vector plasmid (EV) served as experimental controls. (d) Summary of flow cytometry data. E. coli expressing either mCPX-TEV, mCPX-ELA or mCPX-CON biosensors were treated with the indicated proteases. The fluorescence (Geometric mean FL5) of protease treated cells were normalised against labelled, non-protease treated cells (No Prot). These data were analysed using FlowJo (vX 10.0.7r2) software and are representative of three independent biological repeats. (e) Sensitivity of mCPX-TEV biosensor. E. coli expressing mCPX-TEV biosensor were treated with 0–10 Units of AcTEV and analysed via flow cytometry. These data are normalised against untreated (0 U) labelled cells and represent the mean geometric mean ± the standard deviation of three independent experiments. Student t-test ****P < 0.0001.
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Related In: Results  -  Collection

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f3: Validation of E. coli whole-cell biosensors.(a) Biosensor circuit design and localisation in the outer membrane of the cell. (b) Induction of biosensor expression. Representative cell pellets (OD600 4.0) of either induced 100 mM xylose (+) or uninduced (−) biosensor-expressing cells were labelled with 1.25 μg streptavidin-R-phycoerythrin (SAPE)-conjugated antibody. Cell labelling (Red) indicates appropriate expression and localisation of the whole-cell biosensor. (c) Flow cytometry analysis of whole-cell biosensors. E. coli expressing either TEV (mCPX-TEV), elastase (mCPX-ELA) or control (mCPX-CON) biosensors were treated with the indicated proteases: AcTEV protease (TEV) or control proteases - PreScission protease (PRE) or Enterokinase (ENT). Treated cells were labelled with SAPE-conjugated antibody and analysed by flow cytometry. Labelled, non-protease treated cells and E. coli transformed with an empty vector plasmid (EV) served as experimental controls. (d) Summary of flow cytometry data. E. coli expressing either mCPX-TEV, mCPX-ELA or mCPX-CON biosensors were treated with the indicated proteases. The fluorescence (Geometric mean FL5) of protease treated cells were normalised against labelled, non-protease treated cells (No Prot). These data were analysed using FlowJo (vX 10.0.7r2) software and are representative of three independent biological repeats. (e) Sensitivity of mCPX-TEV biosensor. E. coli expressing mCPX-TEV biosensor were treated with 0–10 Units of AcTEV and analysed via flow cytometry. These data are normalised against untreated (0 U) labelled cells and represent the mean geometric mean ± the standard deviation of three independent experiments. Student t-test ****P < 0.0001.
Mentions: The design of the E. coli housed whole-cell biosensor is based on that previously used in a study to identify protease recognition specificities using cellular libraries of peptide substrates (CLiPS)1314. The CLiPS system uses the circularly permuted version of the E. coli outer membrane protein OmpX, CPX, as the anchor module1314. CPX has been engineered such that both its N- and C-termini are exposed to the exterior environment14. A poly-histidine tag is located on the C-terminal whilst the protease detection module with the streptavidin-binding peptide (WCHPMWEVMCLR)13 epitope tag is present on the N-terminus (Fig. 3a). The three E. coli housed biosensors employed in this study (mCPX-TEV, mCPX-ELA and mCPX-CON) were created by replacing the recognition motif present in the CLiPS system with the relevant peptide sequence as described in the materials and methods section. The biosensor fusion genes were placed under the control of the xylose inducible promoter xylF (BBa_I741018) and the moderately strong RBS B0032 (BBa_B0032)15 as shown in the circuit visualised using Pigeon16 (Fig. 3a). This was to avoid any potential deleterious effects caused by overexpression of this outer-membrane protein by the cells. Furthermore, it was decided that the E. coli cloning strain NEB-10beta would be the host of choice for the biosensors, as this is not classically an expression strain and so, theoretically, would not overexpress the biosensors and would therefore minimise the risk of any detrimental burden-based responses.

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