BILE SALTS BACTOSENSOR AND USE THEREOF FOR DIAGNOSTIC AND THERAPEUTIC PURPOSES

Abstract

Bile salts are steroid acids derived from cholesterol in the liver, are released into the gastrointestinal tract to aid in digestion and are thoroughly modified by the resident gut microbiota. Bile acids act as versatile signaling molecules with a variety of endocrine functions and are linked to several diseases. In particular, serum and urinary bile salts represent biomarkers for early diagnostics of liver dysfunction, yet their current detection methods are impractical and hard to scale. Here the inventors engineered engineered synthetic bile salt receptors using TcpP as sensing domains connected to E. coli CadC system which activates transcription upon dimerization. The performance of the system was assayed for various selection of promoters and they can show that fine tunable response that may be reached by changing expression levels of the bile salt receptor. By performing multiple rounds of directed evolution of the TcpP sensor the inventors obtained a collection of variants with a lower limit of detection and a higher sensitivity. Finally, they show that their bactosensor can detect pathological bile-salt concentrations in samples from patients with liver dysfunction. The present invention thus relates to bile salts bactosensor and use thereof for diagnostic and therapeutic purposes.

Claims

1. A bile salts sensing domain having an amino acid sequence as set forth in SEQ ID NO:3 wherein: X.sub.47 represents N, D, W, Y, T, V or F X.sub.48 represents Y or F X.sub.49 represents E, G, V, I, L, S or K X.sub.50 represents Q, V, H, A, T, D, L or S and provided that the bile salt domain does not consist of the amino acid sequence as set forth in SEQ ID NO:24.

2. The bile salts sensing domain of claim 1 that comprises the amino acid sequence as set forth in at least one of SEQ ID NOS: 25-33.

3. A toxin coregulated pilus biosynthesis protein P of Vibrio cholerae (TcpP) polypeptide having a sequence as set forth in SEQ ID NO:34 wherein: X.sub.47 represents N, D, W, Y, T, V or F X.sub.48 represents Y or F X.sub.49 represents E, G, V, I, L, S or K X.sub.50 represents Q, V, H, A, T, D, L or S and provided that the TcpP polypeptide does not consist of the amino acid sequence as set forth in SEQ ID: 35.

4. The TcpP polypeptide of claim 3 that comprises the amino acid sequence as set forth in at least one of SEQ ID NOS: 36-44.

5. A fusion protein wherein a TcpP polypeptide is fused to a heterologous polypeptide, and wherein the TcpP polypeptide has a sequence as set forth in SEQ ID NO:34 wherein: X.sub.47 represents N, D, W, Y, T, V or F X.sub.48 represents Y or F X.sub.49 represents E, G, V, I, L, S or K X.sub.50 represents Q, V, H, A, T, D, L or S.

6. The fusion protein of claim 5 wherein the heterologous polypeptide is a DNA binding domain.

7. The fusion protein of claim 5 wherein the heterologous polypeptide is a an E coli CadC transcriptional activator DNA binding domain .

8. The fusion protein of claim 5 wherein the TcpP polypeptide is fused either directly or via a linker to the heterologous polypeptide.

9. The fusion protein of claim 8 wherein the linker consists of the amino acid sequence as set forth in SEQ ID NO:46.

10. The fusion protein of claim 8 that comprises the amino acid sequence as set forth in SEQ ID NO:47 wherein: X.sub.47 represents N, D, W, Y, T, V or F X.sub.48 represents Y or F X.sub.49 represents E, G, V, I, L, S or K X.sub.50 represents Q, V, H, A, T, D, L or S.

11. The fusion protein of claim 10 that comprises the amino acid sequence set forth in one of SEQ ID NOS:48-57.

12. A polynucleotide that encodes the fusion protein of claim 5.

13. An expression cassette comprising the polynucleotide of claim 5 operably linked to control sequences allowing expression in a prokaryotic host cell.

14. The expression cassette of claim 13 wherein the promoter is selected from the group consisting of p14, p10, and p9 promoter having respectively a nucleic acid sequence as set forth in SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:60.

15. A prokaryotic host cell genetically engineered with the polynucleotide of claim 12 or the an expression cassette comprising the polynucleotide.

16. The prokaryotic host cell of claim 15 that is selected from a bacterium from the genera Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium, Escherichia and Lactobacillus.

17. The prokaryotic host cell of claim 15 that is an Escherichia coli bacterium.

18. The prokaryotic host cell of claim 15 that comprises a polynucleotide that encodes for the TcpH polypeptide having an amino acid sequence as set forth in SEQ ID NO:2, wherein optionally said polynucleotide is operatively linked to the promoter p5 having the nucleic acid sequence as set forth in SEQ ID NO:61.

19. The prokaryotic host cell of claim 15 that comprises at least one further polynucleotide encoding for an output molecule for which the expression is under the control of the fusion protein in which a TcpP polypeptide is fused to a polypeptide, and wherein the TcpP polypeptide has a sequence as set forth in SEQ ID NO:34 wherein: X.sub.47 represents N, D, W, Y, T, V or F X.sub.48 represents Y or F X.sub.49 represents E, G, V, I, L, S or K X.sub.50 represents Q, V, H, A, T, D, L or S.

20. The prokaryotic host cell of claim 15 that further comprises a polynucleotide encoding for an output molecule operatively linked to the CadBA promoter of SEQ ID NO:62.

21. The prokaryotic host cell of claim 20, wherein the output molecule is a detection protein.

22. The prokaryotic host cell of claim 20, wherein the output molecule is a therapeutic polypeptide.

23. A method for detecting the presence of bile salts in a sample suspected of containing said bile salts, comprising i) providing at least one prokaryotic host cell of claim 21; b) contacting said at least one prokaryotic host cell with the sample suspected of containing said bile salts for a time sufficient to allow the oligomerization of the fusion proteins encoded by the at least one prokaryotic host cell to bind and then to express the detection protein; and c) detecting the expression level of the detection protein, wherein the expression level correlate with the amount of the bile salts present in the sample.

24. A method for determining whether a subject has or is at risk of having a liver dysfunction and treating the subject comprising i) providing at one least prokaryotic host cell of the claim 21; b) contacting said at one least prokaryotic host cell with a sample obtained from the subject for a time sufficient to allow the oligomerization of the fusion proteins encoded by the at least one prokaryotic host cell to bind and then to express the detection protein; c) detecting the expression level of the detection protein, wherein the expression level correlateds with the amount of the bile salts present in the sample, and d) treating the subject determined to have an elevated amount of bile salts with a liver dysfunction treatment.

25. A method of treating obesity, inflammatory bowel disease, colorectal cancer, liver disease or hepatobiliary disease in a subject in need thereof comprising administering to the subject an effective amount of the prokaryotic host cell of claim 22.

26. (canceled)

27. A method of screening a plurality of test substances comprising i) contacting a population of prokaryotic host cells of claim 21 with said plurality of test substances in the presence of an amount of bile salts, and ii) selecting the test substances capable of modulating the expression of the output molecule.

28. The fusion protein of claim 7 wherein the E coli CadC transcriptional activator DNA binding domain comprises an amino acid sequence having at least 90% of identity with SEQ ID NO:45.

29. The prokaryotic host cell of claim 21, wherein the detection protein is a fluorescent protein.

Description

FIGURES

[0134] FIG. 1. Rewiring virulence sensing into modularized synthetic receptor platform. Schematic diagram of isolation and rewiring the bile salt sensing module - transmembrane and periplasmic domain of TcpP and TcpH from pathogen V.cholerae into modularized E. coli synthetic receptor platform. The sensing domain TcpP detecting bile salts is plugged into a synthetic receptor which activates GFP expression when bile salts are present.

[0135] FIG. 2. System performance of constitutively expressed synthetic CadC-TcpP_TcpH receptor platform. (A) architecture of the expression system. encoding CadC-TcpP and TcpH controlled by constitutive promoters (P14, P10, P9 for TcpP and P5 for TcpH). (B) response of the different construcs to increasing concentration of bile salts (taurocholic acid). The area labeled with symbols (Δ) indicates the serum bile salt concentration for healthy people (4.8 ± 0.6 .Math.M,Δ), patients with chronic liver disease (51.3 ± 3.8 .Math.M, ΔΔ), and patients with drug induced liver injuries (110 ± 3.4 .Math.M, ΔΔΔ).(C) The activation fold of P14, P10 and P9 controlled CadC-TcpP are measured under the presence of 120 .Math.M Taurocholic acid.

[0136] FIG. 3. Specificity testing of the rewired TcpP-Cadc System across different bile salts.

[0137] FIG. 4. Sensitivity engineering of TcpP bile salt sensing domain Taurocholic acid titration curve of selected TcpP functional variants.

[0138] FIGS. 5. Response of bile salt bactosensor in clinical samples. (A) Response of bacterial cells harboring synthetic P9-CadC-TcpP_P5-TcpH receptor in clinical serum samples. (B) Response of bacterial cells harboring synthetic P9-CadC-TcpP_P5-TcpH receptor in clinical urine samples.

[0139] FIG. 6. Colorimetric assay for bactosensor mediated bile salts detection. Bile salt specificity profile of the TcpP SV_3-3-18 variant characterized using SV_3-3-18-LacZ sensor. Response of SV_3-3-18-LacZ was quantified as ΔA580 (the difference in absorbance at 580 nm (A580) with or without ligand bile salts). The bar graph and error corresponds to the mean value of three experiments performed in triplicate. Cells growing in exponential phase were incubated with bile salts for 4 hours before flow cytometry analysis.

[0140] FIG. 7. Bactosensor-based pathological bile salt detection in clinical samples. Comparing the analysis result of 21 clinical serum samples between TcpP18LacZ and a bile salt assay kit. The response of TcpP SV_3-3-18-LacZ is shown in black bars, left axis. The serum total bile salt concentration measured by a bile salt enzymatic assay kit is labeled in green asterisks, right axis.

EXAMPLE 1: TCPP SENSOR

[0141] The liver is a vital organ coordinating metabolic, detoxification, and immunological processes. Liver diseases including hepatitis, cirrhosis, fatty liver disease and cancer are major public health problems and require large-scale screening methods for prevention, diagnosis, and therapeutic monitoring. Liver function is usually monitored by quantifying serum enzymatic activities and bilirubin, but these markers are detectable when damage has already progressed, and are not entirely specific. Liver function is usually monitored by quantifying several enzymatic activities simultaneously due to their lack of specificity. Serum and urinary bile salts are alternative biomarkers for early diagnostics of liver dysfunction, yet their current detection methods are impractical and hard to scale.

[0142] Here we engineered a bacterial biosensor based on non-pathogenic E. coli detecting pathological concentrations of bile salts in clinical samples. We repurposed the bacterial one-component TcpP bile salts sensing domain from Vibrio cholerae which controls activation of virulence operons when the pathogen enters the gut. We engineered synthetic bile salt receptors using TcpP as sensing domains connected to E. coli CadC system which activates transcription upon dimerization (FIG. 1). The performance of the system was assayed for various selection of promoters (FIGS. 2A, B) and we can show that fine tunable response may be reach by changing expression levels of the bile salt receptor. We also test the system across different bile salts (FIG. 3). We measured the response of the bactosensor to a panel of twelve different bile salts, including both primary and secondary types. Interestingly, while no sensing module was specific for a single bile salt species, the CadC-TcpP system was highly specific for primary conjugated bile salts and did not respond to secondary bile salts.

[0143] We aimed to identify key residues determining the sensitivity of the TcpP sensing module, and targeted those to improve synthetic receptor sensitivity and LOD. To do so, we coupled comprehensive mutagenesis with functional screening and Next-Generation Sequencing (NGS), an approach which supports the identification of functional variants together with the sequence determinants within the local structural motifs. Transition from intramolecular to intermolecular disulfide bonds between TcpP monomers is a key determinant of TcpP response to bile salts and is mediated by two cysteine residues, Cys207 and Cys218. By performing multiple sequence alignments of different TcpP bacterial homologs (data not shown), we found a significant conservation of the amino-acids flanked by these two cysteines (data not shown). Secondary structure prediction and ab initio 3D prediction using the Rosetta modelling suite (data not shown) suggested that each cysteine was located in rigid beta-sheets separated by a flexible loop region between Asn211 and Gln214. This loop propensity to form a turn would allow the two beta sheets and the cysteines to come in close proximity and form an intramolecular disulfide bond. We hypothesized that the flexibility of the turn region between Cys207 and Cys218 was a key parameter controlling the transition rate between the two states, and that altering its amino-acid composition could change the system’s sensitivity to bile salts.

[0144] We thus built a comprehensive mutational library (NNK x 4, theoretical library complexity ≅ 1.05 × 10.sup.6 variants) targeting the NYEK residues inside the turn, and cloned it into the plasmid constitutively expressing CadC-TcpP and producing GFP in response to bile salts (data not shown). The resulting library was induced with TCA, and GFP-positive variants were isolated by fluorescence-activated cell sorting (FACS). We performed three rounds of enrichment (200 .Math.M of TCA as ligand in 1st and 2nd rounds of selection, and 20 .Math.M for the 3rd round) and observed an increasing fraction of the cell population responding to different ligand concentrations (20 to 80 .Math.M) (data not shown). We collected, cultured, and sequenced single variants and tested their response to TCA (data not shown). We found that comprehensive mutagenesis of residues Asn210 to Gln213 could alter the limit of detection, the sensitivity, and the fold activation of our biosensor. The 3.3-fold difference in LOD between SV_3-3-18 and SV_3-3-22 (EC50 from 28.3 to 92.5 .Math.M) indicated the broad range of sensitivity engineering obtained by mutating the loop region of the TcpP sensing module. Further kinetic analysis revealed that the sequence variation of this loop region changes reaction speed and system interaction of bile salts with the synthetic receptor, and variant SV_3-3-18 has 13-fold increase in ligand affinity and faster response at low ligand concentration compared to wt TcpP (data not shown).

[0145] To better understand the sequence features influencing the response of TcpP to bile salts, we sequenced the whole pool of enriched variants by next-generation sequencing (NGS). Surprisingly, the sequence features of functional variants were different from those expected from natural TcpP homologs (data not shown). First, and in contrast with wt TcpP homologs, we observed a strong depletion of long-chain, negatively charged amino-acids (Asp and Glu) along with long-chain polar amino-acids (Asn and Gln) at position 211. Lysine at position 211 also appeared to be depleted in functional variants (despite being commonly found at this position in other TcpP homologous proteins). Second, amino acids with bulky aromatic side chain such as Phe and Tyr, and hydrophobic side chain such as Leuwere highly conserved in selected functional variants, strongly indicating the important role of hydrophobic residues at position 211 in the C-terminal loop region for the function of V. cholerae TcpP. The best engineered variant was SV_3-3-18.

[0146] By performing multiple rounds of directed evolution of the TcpP sensor we obtained a collection of variants with a lower limit of detection and a higher sensitivity (Table 1 and FIG. 4). Finally, we show that our bactosensor can detect pathological bile-salt concentrations in samples from patients with liver dysfunction (FIGS. 5A, B).

TABLE-US-00016 list of different variants and their characteristics # Variant Sequence EC50 (.Math.M) Response EC50 (RPU) Max fold change SV_3-3-22 YYVL (SEQ ID NO: 12) 92.531 56.19 69.06 TcpPwt NYEQ (SEQ ID NO: 4) 89.405 56.5 164.74 SV_3-3-7 WYVH (SEQ ID NO: 6) 86.029 53.37 51.31 SV_3-1-78 FYES (SEQ ID NO: 13) 84.668 59.62 69.06 SV_3-3-11 YYIV (SEQ ID NO: 7) 82.167 53.83 31.76 SV_3-3-14 TFLA (SEQ ID NO: 8) 81.636 62.97 222.67 SV_3-3-3 DFGV (SEQ ID NO: 5) 61.48 61.18 142.21 SV_3-3-19 FFKA (SEQ ID NO: 11) 59.186 61.29 127.75 SV_3-3-16 DFLT (SEQ ID NO: 9) 42.058 59.34 75.73 SV_3-3-18 VFSD (SEQ ID NO: 10) 28.344 58.99 84.92

[0147] Our work paves the way to a sensitive, scalable, and affordable screening platform for liver dysfunction, that could be deployed in point-of-care or at-home settings and enable large scale monitoring of liver associated diseases. This work also shows how synthetic biology can help address global healthcare challenges while providing tools to decipher and target basic cellular mechanisms, in this case pathogens signaling.

EXAMPLE 2: COLORIMETRIC ASSAY

[0148] Colorimetric assay provides a simple and intuitive method for simple and direct estimation of test results by the naked eye. In addition, colorimetric assays support straightforward development of quantitative assays using smartphone-based platforms for POC or home-based diagnosis. We used SV_3-3-18 variant coupled with the reporter beta-galactosidase LacZ (termed SV_3-3-18-LacZ) and its substrate chlorophenol red-β-D-galactopyranoside (CPRG) to provide a colorimetric output (data not shown). Similarly to the biosensor equipped with a GFP output, the bile salt specificity profile of the SV_3-3-18-LacZ system was slightly shifted from TCA to GCDCA (FIG. 6). We thus evaluated the LOD and signal output threshold of SV_3-3-18-LacZ in response to increasing concentrations of GCDCA. We also explored the influence of varying cell density and incubation time (data not shown). Increasing cell density or incubation time both improved the dynamic and operating ranges of SV_3-3-18-LacZ; however, background signal also increased. After optimization, the SV_3-3-18-LacZ demonstrated linear response to GCDCA within the concentration from 0 to 40 .Math.M in one hour (data not shown). In addition, adjusting cell density and incubation times allows its threshold activation level to match various clinical levels associated with specific liver-related medical conditions.

EXAMPLE 3: BACTOSENSOR-MEDIATED DETECTION OF ELEVATED BILE SALTS LEVELS IN SERUM FROM PATIENTS WITH LIVER TRANSPLANT

[0149] We tested the sensor on samples from patients having undergone liver transplantation. After liver transplant, the main complications are bile ducts stenosis and acute cellular rejection.

[0150] We tested our bactosensor in clinical 21 serum samples from liver transplantation patients (data not shown). The patients were followed at the Montpellier hospital after their liver transplant, most of them having been performed in the last 2 years. These patients had received a liver transplant for end-stage liver disease as a result of alcoholic related liver disease or non-alcoholic fatty liver disease, chronic cholangitis or liver cancer. A complete hepatic check-up was performed, and serum bile salts were also measured using an enzymatic assay (data not shown). We found that patients who had a high potential of acute cellular rejection (ACR) after liver transplantation (serum bile acid > 37 .Math.M) had significant and visible colorimetric signal changes (data not shown) in bactosensor assays. Three patients with elevated serum bile salts concentration raised our attention. Two of them presented abnormalities in their hepatic enzymatic values (ASAT, ALAT, GGT, Pal, and bilirubin). For these patients, the bile salt bactosensor produced the strongest colorimetric change easily detectable with the naked eye (FIG. 7). These results indicate that our bactosensor is able to provide a simple, reliable, and cost-effective method for monitoring patient condition after liver transplantation.

REFERENCES

[0151] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.