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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 B. subtilis whole-cell biosensors.(a) Biosensor circuit design and localisation in the cell wall. (b) Induction of biosensor expression. Representative cell pellets (OD600 4.0) of either induced 1 mM IPTG (+) or uninduced (−) biosensor-expressing cells were labelled with 2.5 μg His-phycoerythrin (His-PE)-conjugated antibody. Cell labelling (Red) indicates appropriate expression and localisation of the whole-cell biosensor. (c) Flow cytometry analysis of whole-cell biosensors. B. subtilis expressing either TEV (LytCCWD-TEV), elastase (LytCCWD-ELA) or control (LytCCWD-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 His-PE-conjugated antibody and analysed by flow cytometry. Labelled, non-protease treated cells and B. subtilis transformed with an empty vector plasmid (EV) served as experimental controls. (d) Summary of flow cytometry data. B. subtilis expressing either LytCCWD-TEV, LytCCWD-ELA or LytCCWD-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 LytCCWD-TEV biosensor. B. subtilis expressing LytCCWD-TEV biosensor were treated with 0-40 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 < 0.001.
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f4: Validation of B. subtilis whole-cell biosensors.(a) Biosensor circuit design and localisation in the cell wall. (b) Induction of biosensor expression. Representative cell pellets (OD600 4.0) of either induced 1 mM IPTG (+) or uninduced (−) biosensor-expressing cells were labelled with 2.5 μg His-phycoerythrin (His-PE)-conjugated antibody. Cell labelling (Red) indicates appropriate expression and localisation of the whole-cell biosensor. (c) Flow cytometry analysis of whole-cell biosensors. B. subtilis expressing either TEV (LytCCWD-TEV), elastase (LytCCWD-ELA) or control (LytCCWD-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 His-PE-conjugated antibody and analysed by flow cytometry. Labelled, non-protease treated cells and B. subtilis transformed with an empty vector plasmid (EV) served as experimental controls. (d) Summary of flow cytometry data. B. subtilis expressing either LytCCWD-TEV, LytCCWD-ELA or LytCCWD-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 LytCCWD-TEV biosensor. B. subtilis expressing LytCCWD-TEV biosensor were treated with 0-40 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 < 0.001.

Mentions: We also chose B. subtilis as a host for our biosensors as it is a well-studied and widely utilised organism that has successfully been used to overexpress many useful compounds and antibodies171819, is generally recognised as safe by the FDA8 and has been used as a host for the surface localisation of enzymes including recombinant lipases such as the B. subtilis lipase B (LipB)20 and the Aspergillus oryzae lipolytic enzyme CutL21. Indeed, the binding of LipB to the surface of this bacterium was accomplished by fusing it to the N-terminal cell wall-binding domain of the native B. subtilis autolysin CwlB (LytC)2022. To this end, we decided to incorporate the cell wall-binding domain of B. subtilis LytC into our biosensor design, and use it as the cell anchor module (Fig. 4a). This is not the only difference between the B. subtilis biosensor design and that of the E. coli biosensors. Unlike the E. coli based biosensor, the B. subtilis based biosensors only have their C-termini exposed to the external environment. Furthermore, the detection module for the B. subtilis based biosensors differs slightly in that the epitope employed is a poly-histidine tag. However, the three recognition motifs were identical and this resulted in the construction of the three biosensors LytCCWD-TEV, LytCCWD-ELA and LytCCWD-CON.


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 B. subtilis whole-cell biosensors.(a) Biosensor circuit design and localisation in the cell wall. (b) Induction of biosensor expression. Representative cell pellets (OD600 4.0) of either induced 1 mM IPTG (+) or uninduced (−) biosensor-expressing cells were labelled with 2.5 μg His-phycoerythrin (His-PE)-conjugated antibody. Cell labelling (Red) indicates appropriate expression and localisation of the whole-cell biosensor. (c) Flow cytometry analysis of whole-cell biosensors. B. subtilis expressing either TEV (LytCCWD-TEV), elastase (LytCCWD-ELA) or control (LytCCWD-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 His-PE-conjugated antibody and analysed by flow cytometry. Labelled, non-protease treated cells and B. subtilis transformed with an empty vector plasmid (EV) served as experimental controls. (d) Summary of flow cytometry data. B. subtilis expressing either LytCCWD-TEV, LytCCWD-ELA or LytCCWD-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 LytCCWD-TEV biosensor. B. subtilis expressing LytCCWD-TEV biosensor were treated with 0-40 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 < 0.001.
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f4: Validation of B. subtilis whole-cell biosensors.(a) Biosensor circuit design and localisation in the cell wall. (b) Induction of biosensor expression. Representative cell pellets (OD600 4.0) of either induced 1 mM IPTG (+) or uninduced (−) biosensor-expressing cells were labelled with 2.5 μg His-phycoerythrin (His-PE)-conjugated antibody. Cell labelling (Red) indicates appropriate expression and localisation of the whole-cell biosensor. (c) Flow cytometry analysis of whole-cell biosensors. B. subtilis expressing either TEV (LytCCWD-TEV), elastase (LytCCWD-ELA) or control (LytCCWD-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 His-PE-conjugated antibody and analysed by flow cytometry. Labelled, non-protease treated cells and B. subtilis transformed with an empty vector plasmid (EV) served as experimental controls. (d) Summary of flow cytometry data. B. subtilis expressing either LytCCWD-TEV, LytCCWD-ELA or LytCCWD-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 LytCCWD-TEV biosensor. B. subtilis expressing LytCCWD-TEV biosensor were treated with 0-40 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 < 0.001.
Mentions: We also chose B. subtilis as a host for our biosensors as it is a well-studied and widely utilised organism that has successfully been used to overexpress many useful compounds and antibodies171819, is generally recognised as safe by the FDA8 and has been used as a host for the surface localisation of enzymes including recombinant lipases such as the B. subtilis lipase B (LipB)20 and the Aspergillus oryzae lipolytic enzyme CutL21. Indeed, the binding of LipB to the surface of this bacterium was accomplished by fusing it to the N-terminal cell wall-binding domain of the native B. subtilis autolysin CwlB (LytC)2022. To this end, we decided to incorporate the cell wall-binding domain of B. subtilis LytC into our biosensor design, and use it as the cell anchor module (Fig. 4a). This is not the only difference between the B. subtilis biosensor design and that of the E. coli biosensors. Unlike the E. coli based biosensor, the B. subtilis based biosensors only have their C-termini exposed to the external environment. Furthermore, the detection module for the B. subtilis based biosensors differs slightly in that the epitope employed is a poly-histidine tag. However, the three recognition motifs were identical and this resulted in the construction of the three biosensors LytCCWD-TEV, LytCCWD-ELA and LytCCWD-CON.

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