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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

Detection of cercarial elastase activity from SmCTF biological samples.(a) Reconstituted SmCTF samples were incubated with either PBS (1X) or Suc-AAPF-pNA (200 mM) substrate. Proteolytic activity against Suc-AAPF-pNA altered the sample absorbance (400 nm) which was measured every 5 minutes for 5 hours. These data represent the mean absorbance ± the standard deviation of three experimental repeats. (b) E. coli expressing either mCPX-TEV, mCPX-ELA or mCPX-CON biosensors were treated with three independently generated SmCTF (1–3) biological samples. Treated cells were labelled with (SAPE)-conjugated antibody and analysed via flow cytometry with FlowJo (vX 10.0.7r2) software. A representative panel of flow cytometry data is shown and three independent biological repeats are summarised in the accompanying graph. Labelled, non-protease treated cells and E. coli transformed with an empty vector plasmid (EV) served as experimental controls. The fluorescence (Geometric mean FL5) of protease treated cells were normalised against labelled, non-protease treated cells (No Prot). (c) B. subtilis expressing either LytCCWD-TEV, LytCCWD-ELA or LytCCWD-CON biosensors were treated with three independently generated SmCTF (1–3) biological samples. Treated cells were labelled with (His-PE)-conjugated antibody and analysed via flow cytometry with FlowJo (vX 10.0.7r2) software. A representative panel of flow cytometry data is shown and three independent biological repeats are summarised in the accompanying graph. Labelled, non-protease treated cells and B. subtilis transformed with an empty vector plasmid (EV) served as experimental controls. The fluorescence (Geometric mean FL5) of protease treated cells were normalised against labelled, non-protease treated cells (No Prot). Student t-test ***P < 0.001 and ****P < 0.0001.
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f5: Detection of cercarial elastase activity from SmCTF biological samples.(a) Reconstituted SmCTF samples were incubated with either PBS (1X) or Suc-AAPF-pNA (200 mM) substrate. Proteolytic activity against Suc-AAPF-pNA altered the sample absorbance (400 nm) which was measured every 5 minutes for 5 hours. These data represent the mean absorbance ± the standard deviation of three experimental repeats. (b) E. coli expressing either mCPX-TEV, mCPX-ELA or mCPX-CON biosensors were treated with three independently generated SmCTF (1–3) biological samples. Treated cells were labelled with (SAPE)-conjugated antibody and analysed via flow cytometry with FlowJo (vX 10.0.7r2) software. A representative panel of flow cytometry data is shown and three independent biological repeats are summarised in the accompanying graph. Labelled, non-protease treated cells and E. coli transformed with an empty vector plasmid (EV) served as experimental controls. The fluorescence (Geometric mean FL5) of protease treated cells were normalised against labelled, non-protease treated cells (No Prot). (c) B. subtilis expressing either LytCCWD-TEV, LytCCWD-ELA or LytCCWD-CON biosensors were treated with three independently generated SmCTF (1–3) biological samples. Treated cells were labelled with (His-PE)-conjugated antibody and analysed via flow cytometry with FlowJo (vX 10.0.7r2) software. A representative panel of flow cytometry data is shown and three independent biological repeats are summarised in the accompanying graph. Labelled, non-protease treated cells and B. subtilis transformed with an empty vector plasmid (EV) served as experimental controls. The fluorescence (Geometric mean FL5) of protease treated cells were normalised against labelled, non-protease treated cells (No Prot). Student t-test ***P < 0.001 and ****P < 0.0001.

Mentions: To analyse the relative amounts of cercarial elastase activity present in the three samples we decided to use a previously described assay used to detect for the activity of S. mansoni elastase recovered from snail shedding experiments7. In this assay, elastase activity was measured as activity against the substrate succinyl-ala-ala-pro-phe-p-nitroanilide (Suc-AAPF-pNA)7. Cleavage of Suc-AAPF-pNA releases 4-nitroaniline, which is yellow in colour and can be measured spectrophotometrically. Aliquots (10 μl) of SmCTF samples were incubated at 30 °C with 200 μM Suc-AAPF-pNA and the activity of cercarial elastase measured as abosorbance at 400 nm. SmCTF2 was found to have a very high enzymatic activity, whilst SmCTF1 and SmCTF3 had relatively lower activities (Fig. 5a).


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)

Detection of cercarial elastase activity from SmCTF biological samples.(a) Reconstituted SmCTF samples were incubated with either PBS (1X) or Suc-AAPF-pNA (200 mM) substrate. Proteolytic activity against Suc-AAPF-pNA altered the sample absorbance (400 nm) which was measured every 5 minutes for 5 hours. These data represent the mean absorbance ± the standard deviation of three experimental repeats. (b) E. coli expressing either mCPX-TEV, mCPX-ELA or mCPX-CON biosensors were treated with three independently generated SmCTF (1–3) biological samples. Treated cells were labelled with (SAPE)-conjugated antibody and analysed via flow cytometry with FlowJo (vX 10.0.7r2) software. A representative panel of flow cytometry data is shown and three independent biological repeats are summarised in the accompanying graph. Labelled, non-protease treated cells and E. coli transformed with an empty vector plasmid (EV) served as experimental controls. The fluorescence (Geometric mean FL5) of protease treated cells were normalised against labelled, non-protease treated cells (No Prot). (c) B. subtilis expressing either LytCCWD-TEV, LytCCWD-ELA or LytCCWD-CON biosensors were treated with three independently generated SmCTF (1–3) biological samples. Treated cells were labelled with (His-PE)-conjugated antibody and analysed via flow cytometry with FlowJo (vX 10.0.7r2) software. A representative panel of flow cytometry data is shown and three independent biological repeats are summarised in the accompanying graph. Labelled, non-protease treated cells and B. subtilis transformed with an empty vector plasmid (EV) served as experimental controls. The fluorescence (Geometric mean FL5) of protease treated cells were normalised against labelled, non-protease treated cells (No Prot). Student t-test ***P < 0.001 and ****P < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Detection of cercarial elastase activity from SmCTF biological samples.(a) Reconstituted SmCTF samples were incubated with either PBS (1X) or Suc-AAPF-pNA (200 mM) substrate. Proteolytic activity against Suc-AAPF-pNA altered the sample absorbance (400 nm) which was measured every 5 minutes for 5 hours. These data represent the mean absorbance ± the standard deviation of three experimental repeats. (b) E. coli expressing either mCPX-TEV, mCPX-ELA or mCPX-CON biosensors were treated with three independently generated SmCTF (1–3) biological samples. Treated cells were labelled with (SAPE)-conjugated antibody and analysed via flow cytometry with FlowJo (vX 10.0.7r2) software. A representative panel of flow cytometry data is shown and three independent biological repeats are summarised in the accompanying graph. Labelled, non-protease treated cells and E. coli transformed with an empty vector plasmid (EV) served as experimental controls. The fluorescence (Geometric mean FL5) of protease treated cells were normalised against labelled, non-protease treated cells (No Prot). (c) B. subtilis expressing either LytCCWD-TEV, LytCCWD-ELA or LytCCWD-CON biosensors were treated with three independently generated SmCTF (1–3) biological samples. Treated cells were labelled with (His-PE)-conjugated antibody and analysed via flow cytometry with FlowJo (vX 10.0.7r2) software. A representative panel of flow cytometry data is shown and three independent biological repeats are summarised in the accompanying graph. Labelled, non-protease treated cells and B. subtilis transformed with an empty vector plasmid (EV) served as experimental controls. The fluorescence (Geometric mean FL5) of protease treated cells were normalised against labelled, non-protease treated cells (No Prot). Student t-test ***P < 0.001 and ****P < 0.0001.
Mentions: To analyse the relative amounts of cercarial elastase activity present in the three samples we decided to use a previously described assay used to detect for the activity of S. mansoni elastase recovered from snail shedding experiments7. In this assay, elastase activity was measured as activity against the substrate succinyl-ala-ala-pro-phe-p-nitroanilide (Suc-AAPF-pNA)7. Cleavage of Suc-AAPF-pNA releases 4-nitroaniline, which is yellow in colour and can be measured spectrophotometrically. Aliquots (10 μl) of SmCTF samples were incubated at 30 °C with 200 μM Suc-AAPF-pNA and the activity of cercarial elastase measured as abosorbance at 400 nm. SmCTF2 was found to have a very high enzymatic activity, whilst SmCTF1 and SmCTF3 had relatively lower activities (Fig. 5a).

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