Patent Grant
11899014
A Sequence Listing conforming to the rules of WIPO Standard ST.25 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter in ASCII formatted text. The electronic document, created on Sep. 1, 2023, is entitled “070050_6405_ST25.txt”, and is 281,368 bytes in size.
The present invention relates to methods and compositions for detecting the presence of an agent in a test sample using a whole cell reporter. In certain embodiments, detection can be performed without the aid of instrumentation, for example outside of a laboratory setting, permitting home and field tests for interrogating the status of biological systems. The present invention may be used, for example, to identify pathogens and thereby limit the dissemination of disease.
2.1. Whole-Cell Biosensors
Microbial whole-cell bio-reporters present unique advantages for environmental sensing, such as the probing of complex biochemical processes, compatibility with aqueous media, self-renewal by replication, portability by freeze-drying, availability of numerous natural sensing pathways, and ease of engineering new functions (e.g., by directed evolution).1,2 Bacterial whole cell sensors have previously been demonstrated for detection of DNA damage,3 heat shock,4 oxidative stress,5 heavy metals,6-8 viruses,9 and light.10 Yeast and mammalian whole cell sensors have also been reported. For yeast whole cell sensors, see Hollis (2000) and Radhika (2007). For mammalian whole cell sensors, see Rider, (2003).
2.2. Peptides as Analytes
While natural receptors can be utilized for detection of a broad range of analytes, proteins and their peptide epitopes present a ubiquitous pool of natural biomarkers which are highly characteristic of the organisms that produce them. Peptides can thus be used as targets for detection of pathogenic organisms, food born toxins, immunogens and bioterrorism agents. For example, see the recent development of mass spectrometry of proteolized samples as a diagnostic tool for various diseases.11,12
2.3. Using GPCRs for Detection
G-protein coupled receptors (GPCR) constitute a large family of seven-transmembrane receptors for hormones, neurotransmitters, chemokines, calcium, odorants, taste molecules and even light.19 GPCR signaling pathways are highly conserved among diverse species. Furthermore, GPCR-activation of the Mitogen-activated protein kinase (MAPK) phosphorylation cascade is conserved from yeast to mammals,19 with different MAPK families activated by multiple different GPCRs.
It was shown that yeast pheromone receptors can be functionally replaced by expressing mammalian GPCRs that couple to the endogenous MAPK signaling pathway, so that the corresponding mammalian agonist activates the yeast pheromone response using different reporter genes21-23 beta-galactosidase24-26 or auxotrophic markers.27-29
G-protein coupled receptors (GPCRs) have previously been implemented in yeast to develop high-throughput drug discovery assays based around mammalian receptors by using a growth based reporter.13,14 Additionally, yeast has also been used to functionally express native fungal receptors to study the biology of the respective fungi.15-18 These previous studies coupled the GPCRs to the endogenous pheromone response pathway by using laboratory assays requiring instrumentation.
The present invention relates to methods and compositions for detecting the presence of an agent, for example, but not limited to, a human disease agent (e.g., a pathogenic agent), an agricultural agent, an industrial and model organism agent, a bioterrorism agent, or a heavy metal contaminant, by detecting the presence of an analyte indicative of the presence of the agent in a test sample. In certain embodiments, the analyte is the agent itself, a portion of the agent (e.g., a portion generated by proteolysis), or a product of the agent. The methods utilize a sensor cell bearing a receptor that is specific for the analyte, where binding of the receptor to the analyte triggers a detection event that is indicative of the presence of the agent. The reporter can be coupled to the receptor. In certain embodiments, the sensor cell is a microbe that is easy and quick to propagate, for example a yeast cell, and the reporter gene product is detectable to the naked eye, for example a pigmented compound such as (red) lycopene. In certain non-limiting embodiments, the present disclosure provides an engineered baker's yeast that uses G-protein coupled receptors (GPCRs) to detect a range of peptide ligands associated with specific target agents and uses the red plant pigment lycopene as a fast, non-technical, visual readout. In certain non-limiting embodiments, the present disclosure provides methods of engineering peptide-activated GPCRs to detect non-cognate agent-specific peptides and to improve performance (e.g., sensitivity and/or specificity) against peptide ligands, using directed evolution.
The present invention provides methods of detecting the presence of an agent of interest in a sample. In certain embodiments, the method comprises: contacting the sample with a sensor cell comprising a non-native G-protein coupled receptor (GPCR) that binds to an analyte indicative of the presence of the agent, wherein binding of the analyte to the receptor triggers appearance of a reporter detectable by the naked eye, wherein the increased expression is indicative of the presence of the agent. The agent can be selected from the group consisting of human disease agents, agricultural agents, industrial and model organism agents, bioterrorism agents, and heavy metal contaminants. In certain embodiments, the non-native GPCR receptor is engineered to bind to the analyte. In certain embodiments, the non-native GPCR receptor is engineered by directed evolution. In certain embodiments, the non-native GPCR receptor is a fungal pheromone GPCR. In certain embodiments, the non-native GPCR receptor is selected from the group consisting of the GPCRs listed in Tables 2 and 6.
In certain embodiments, the sensor cell is a microbe. In certain embodiments, the sensor cell is a fungal cell. In certain embodiments, the sensor cell is a yeast cell. In certain embodiments, the sensor cell is S. cerevisiae. In certain embodiments, the sensor cell comprises a nucleic acid encoding the receptor. In certain embodiments, the nucleic acid is linked to a promoter.
In certain embodiments, the analyte is a cognate ligand for the non-native GPCR receptor. In certain embodiments, the analyte is a non-cognate ligand for the non-native GPCR receptor.
In certain embodiments, the analyte is a peptide. In certain embodiments, the peptide is a fungal mating pheromone. The fungal mating pheromone can be selected from the group consisting of human fungal mating pheromones (meaning mating pheromones of fungi that can colonize or infect humans), non-human animal fungal mating pheromones (meaning mating pheromones of fungi that colonize or infect a non-human animal), plant fungal mating pheromones (meaning mating pheromones of fungi that colonize or infect a plant), food fungal mating pheromones (meaning mating pheromones of fungi that colonize or infect human or non-human animal food items), and industrial/model fungal mating pheromone. In non-limiting examples, the human fungal mating pheromone can be selected form the group consisting of the mating pheromones of C. albicans, C. glabrata, P. brasiliensis, L. elongisporous, P. rubens, C. guillermondi, C. tropicalis, C. parapsilosis, C. lusitaniae, S. scheckii, and Candida krusei. An example of a non-human animal fungal mating pheromone is the mating pheromone of P. destructans. In non-limiting examples, the plant fungal mating pheromone can be selected from the group consisting of the mating pheromones of F. graminearum, M. oryzea, B. cinerea, G. candidum, and C. purpurea. In non-limiting examples, the food fungal mating pheromone can be selected from the group consisting of the mating pheromones of Zygosaccharomyces bailii, Zygosaccharomyces rouxii, and N. fischeri. In non-limiting examples, the industrial/model fungal mating pheromone can be selected from the group consisting of the mating pheromones of S. cerevisiae, K. lactis, S. pombe, V. polyspora (receptor 1), V. polyspora (receptor 2), S. stipitis, S. japonicas, S. castellii, and S. octosporus, A. oryzae, T melanosporum, D. haptotyla, C. tenuis, Y. lipolytica, T. delbrueckii, B. bassiana, K. pastoris, A. nidulans, N. crassa, and H. jecorina.
In non-limiting examples, the peptide can be selected from the group consisting of the peptides listed in Table 5. In certain embodiments, the peptide has a length of about 5-25 residues. In certain embodiments, the peptide has a length of about 9-23 residues.
In certain embodiments, the peptide is associated with a bacterial infection. In certain embodiments, the peptide is associated with Vibrio cholera. In non-limiting examples, the peptide associated with Vibrio cholerae can be selected from the group consisting of a peptide having an amino acid sequence set forth in VEVPGSQHIDSQKKA (SEQ ID NO: 26), a peptide having an amino acid sequence that is at least 80%, at least 90% or at least 95% about homologous to SEQ ID NO: 26, a peptide having an amino acid sequence set forth in VPGSQHIDS (SEQ ID NO: 27), and a peptide having an amino acid sequence that is at least about 80%, at least 90% or at least 95% homologous to SEQ ID NO: 27. In certain embodiments, the peptide is derived from cholera toxin. The peptide derived from cholera toxin can be selected from the group consisting of the peptides listed in Table 7.
In certain embodiments, the non-native GPCR receptor is coupled to the reporter. In certain embodiments, the method further comprises culturing the sensor cell for an effective period of time; and determining expression of the reporter gene. In certain embodiments, determining expression of the reporter gene does not comprise instrumentation. In certain embodiments, the reporter is a biosynthesized visible-light pigment. In certain embodiments, the reporter is lycopene. In certain embodiments, the sensor cell is engineered to express the receptor.
In certain embodiments, the sample is selected from the group consisting of water samples and body fluid samples. The water sample can be selected from the group consisting of fresh water, sea water, and sewage samples. The body fluid sample can be selected from the group consisting of intestinal fluids, diarrhea, mucus, blood, cerebrospinal fluid, lymph, pus, saliva, vomit, urine, bile, and sweat.
Additionally, the present invention provides a sensor cell comprising a non-GPCR receptor that binds to an analyte indicative of the presence of the agent, wherein binding of the analyte to the receptor triggers appearance of a reporter detectable by the naked eye, wherein the increased expression is indicative of the presence of the agent.
Furthermore, the present invention provides a kit for detecting the presence of an agent of interest, comprising a sensor cell as described above. In certain embodiments, the kit further comprises a negative control. In certain embodiments, the kit further comprises a substrate that comprises the sensor cell. In certain embodiments, the substrate is comprised in a dipstick. In certain embodiments, the kit further comprises a nutrient source.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The present invention relates to methods and compositions for detecting the presence of an agent of interest in a test sample.
For clarity and not by way of limitation, the detailed description is divided into the following subsections:
Presently disclosed sensor calls can be used to detect the presence of a variety of agents. Non-limiting examples of suitable agents include human disease agents (human pathogenic agents), agricultural agents, industrial and model organism agents, bioterrorism agents, and heavy metal contaminants.
Human disease agents include, but are not limited to infectious disease agents, oncological disease agents, neurodegenerative disease agents, kidney disease agents, cardiovascular disease agents, clinical chemistry assay agents, and allergen and toxin agents.
Infectious disease agents include, but are not limited to, fungal pathogens, bacterial pathogens, viral pathogens, and protozoan pathogens, as well as toxins produced by same. Non-limiting examples of fungal pathogens include C. albicans, C. glabrata, P. brasiliensis, L. elongisporous, P. rubens, C. guillermondi, C. tropicalis, C. parapsilosis, C. lusitaniae, S. scheckii, and Candida krusei.
Non-limiting examples of bacterial pathogens include Vibrio cholerae, Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus (MRSA) strains, Bacillus subtilis, Streptococcus pneumonia, Group B Streptococcus, Salmonella sp., Listeria monocytogenes, Chlamydia trachomatis, Neisseria gonorrhoeae, Clostridium difficile, Yersinia enterocolitica, Legionella sp., Mycobacterium tuberculosis, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Neisseria meningitis, Streptococcus pneumoniae, Pseudomonas aeruginosa, Streptococcus pyogenes, botulinum toxin of Clostridium botulinum, Shigella/Enteroinvasive E. coli, Shiga toxin from the Shiga toxin-producing Escherichia coli (STEC), and Verotoxin derived from Shigella dysenteriae. Analytes that are indicative of the presence of bacterial pathogens include, but are not limited to, quorum sensing small molecules such as the Vibrio cholera CAI-1,69 inter-species bacterial quorum sensing AL-2,70 or components of the bacterial LPS.
Non-limiting examples of viral pathogens include Ebola virus, HPV, HIV, influenza, Hepatitis C Virus, Hepatitis B Virus. Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Respiratory syncytial virus (RSV), Norovirus, Sapovirus, and measles virus. Analytes that are indicative of the presence of viral pathogens include, but are not limited to, capsid protein or peptides, and other viral particles.
Non-limiting examples of protozoan pathogens include Trichomonas vaginalis, Cryptosporidium, Cyclospora cayetanensis, Giardia lamblia, and biomarkers for Amoebiasis derived from Entamoeba histolytica such as E. histolytica ADP-forming acetyl-CoA synthetase (EhACS) or related peptides [Huat (2014)], Leishmaniasis biomarkers such as the amastin signature peptide [Rafati (2006)].
Oncological disease agents include, but are not limited to, lung, breast, colorectum, prostate, stomach, liver, kidney or cervix cancer, leukemia, Kaposi sarcoma, Testis, Ovary, thyroid, and other cancer peptide biomarkers unique for certain cancer types, which can be identified by mass spectrometry.60-63
Neurodegenerative disease agents include, but are not limited to, peptide biomarkers indicated in Alzheimer's,64 [notably fungal biomarker for Alzheimer's were recently suggested in Pisa (2015)], the protein DJ-1 or peptides thereof as biomarkers for Parkinson disease,65 and biomarkers for prion disease such as proteins or peptides of the 14-3-3 family in cerebrospinal fluid for detection of Creutzfeldt-Jakob disease [Van Everbroeck (2005) and Huzarewich (2010)].
Clinical chemistry assay (for general health diagnostics) agents include, but are not limited to, peptide hormones. Peptide hormones include, but are not limited to, neurohypophysial hormones (e.g., oxytocin and vasopressin) and pancreatic hormones (e.g., glucagon, insulin and somatostatin).
Allergen and toxin agents include, but are not limited to, peptide derived from immunogenic wheat peptide (e.g., gluten), and carcinogen aflatoxin B1 derived from the fungi A. flavus.
Kidney disease agents include, but are not limited to, proteins and peptides identified as urinary biomarkers for kidney disease, such as β2-microglobulin, and differential patterns of peptides in type 2 diabetis66.
Cardiovascular disease agents include, but are not limited to, proteins and peptides indicative for atherothrombosis or risk markers for stroke. Markers for primary cardiovascular events include peptides derived from C-reactive protein, fibrinogen, cholesterol, apolipoprotein B, high density lipoprotein, and small molecules like vitamin D. Markers for secondary cardiovascular events include peptides derived from cardiac troponins I and T, C-reactive protein, serum creatinine, and cystatin C. Risk markers for primary stroke, include peptides derived from fibrinogen and serum uric acid [Van Holten et al. (2013)]
Agricultural agents include, but are not limited to, fungal pathogens of animals and plants, and fungal agents causing food spoilage. Fungal pathogens of animals and plants include, but are not limited, to animal fungal pathogens and plant fungal pathogens. Animal fungal pathogens include, but is not limited to, P. destructans. Non-limiting examples of plant fungal pathogens include F. graminearum, M. oryzea, B. cinerea, G. candidum, and C. purpurea. Non-limiting examples of fungal agents causing food spoilage include Z. bailii, Z. rouxii, and N. fischeri.
Industrial and model organism agents include, but are not limited to, fungal agents used for genetic studies and industrial applications such as food production, pharmaceutical production, fine chemical production, bioremediation, including, but not limited to, S. cerevisiae, K lactis, S. pombe, V. polyspora (receptor 1), V. polyspora (receptor 2), S. stipitis, S. japonicus, S. castellii, and S. octosporus.
Bioterrorism agents include, but are not limited to, peptide biomarkers for Bacillus anthracis (causative agent of anthrax—e.g., one of three polypeptides that comprise the anthrax toxin secreted by the pathogen: protective antigen (PA), lethal factor (LF) and edema factor (EF)),67 Clostridium botulinum (causative agent of botulism—e.g., Botulinum neurotoxin peptides such as the cyclic peptide C11-019),68 viral agents such as smallpox (Variola virus) and Viral encephalitis, Ebola virus.
Heavy metal contaminant include, but are not limited to, cadmium, mercury, lead or arsenic, as bound to biological receptors.
In certain embodiments, the agent is the same as the analyte, as disclosed herein. In certain embodiments, the agent is different from the analyte.
5.2. Sensor Cells
The sensor cell can be engineered to comprise one or more component of the assay system disclosed herein. As used herein, the term “engineered” means that one or more component is introduced into a sensor cell or its parent cell by a method selected from the group consisting of recombinant DNA techniques (e.g., Reiterative Recombination and CRISPR), natural genetic events, conjugation, and a combination thereof. Sensor cells can be prokaryotic cells or eukaryotic cells. In certain embodiments, a presently disclosed sensor cell is a microbe, including, but not limited to, bacteria, fungi, and slime molds. In certain embodiments, the sensor cell is a fungal cell. In certain embodiments, the fungal cell is a yeast cell. Non-limiting examples of yeast cells include Saccharomyces cerevisiae, Pichia pastoris and Schizosaccharomyces pombe. In one non-limiting embodiment, the sensor cell is Saccharomyces cerevisiae. Additional non-limiting examples of fungal cells include Candida albicans, Paracoccidioides brasiliensis, Fusarium graminearum, Magnaporthe oryzae, and Botrytis cinerea. In certain embodiments, the sensor cell is a bacterial cell. Non-limiting examples of bacterial cells include Escherichia coli, Bacillus subtilis, and Lactobacillus acidophilus.
5.3 Receptors and Coupling Systems
The present invention provides for receptors and coupling systems wherein a sensor cell comprises (e.g., bears) a receptor that binds to an analyte, where binding of the analyte triggers a detection event that is indicative of the presence of the agent (e.g., expression of a detectable reporter gene, including increased or decreased expression), release of a therapeutic molecule that directly remediates the agent, production of a redox active molecule, or a change in the membrane potential of the sensor cell). In certain embodiments, the sensor cell is engineered to bind to the analyte.
As used herein, the term “receptor” means a molecule (e.g., a ligand) that binds to a presently disclosed analyte that is indicative of the presence of an agent of interest. A presently disclosed receptor is positioned, either inherently or by association with a membrane protein, at the cell surface exposed to the extracellular environment. In certain embodiments, the receptor is a protein. In certain embodiment, the receptor is a naturally occurring (native) protein or a portion thereof. In certain embodiments, the receptor is a portion of a naturally occurring protein comprised in a fusion protein with one or more heterologous proteins. In certain embodiments, the receptor is a mutated version of a naturally occurring protein. In certain embodiments, the receptor is a synthetic protein. In certain embodiments, the receptor is a partly-synthetic protein. In certain embodiments, the receptor comprises one or more non-protein element.
In certain embodiments, the receptor is a non-protein molecule. In one non-limiting embodiment, the receptor is an aptamer or a riboswitch. The receptor may be comprised of a single element or may be comprised of a plurality of elements/subunits.
In certain non-limiting embodiments, the sensor cell comprises a receptor that binds to an analyte, wherein the receptor is coupled to a detectable reporter gene such that when the analyte binds to the receptor, expression of the reporter gene is increased or induced. In certain embodiments, the receptor is coupled to a detectable reporter gene such that when an analyte binds to the receptor, expression of the reporter gene is inhibited (for example, by binding of a transcriptional repressor). In certain embodiments, the analyte is a peptide, e.g., an agent-specific peptide.
As used herein, the term “coupled to” means that binding of an analyte to a receptor is causally linked, directly or indirectly, to and triggers a detection event that is indicative of the presence of the agent (e.g., expression of a detectable reporter gene (induced or inhibited expression), release of a therapeutic molecule that directly remediates the agent, production of a redox active molecule, or a change in the membrane potential of the sensor cell). In certain embodiments, the detection event is expression of a detectable reporter gene. In certain embodiments, the detection event is induced expression of a detectable reporter gene. The receptor may be linked to expression level of the reporter gene through, for example, a pathway of interacting molecules. This pathway may be host-endogenous or engineered.
In certain embodiments, the sensor cell is engineered to express the receptor, for example, by the introduction of a nucleic acid encoding the receptor. In certain embodiments, the nucleic acid is operably linked to a promoter element. In certain embodiments, the promoter element is constitutively active. In certain embodiments, the promoter element is inducibly active. In certain embodiments, the receptor is expressed on the surface of the sensor cell. In certain embodiments, the receptor is expressed on internal membranes of the sensor cell. In certain embodiments, the receptor is expressed in the cytoplasm of the sensor cell.
In certain embodiments, the analyte is a natural (cognate) ligand of the receptor; the coupled analyte-receptor system utilizes a receptor and its natural (cognate) ligand as the analyte. In certain embodiments, the coupled analyte-receptor system is a receptor engineered to bind a different non-cognate ligand as analyte, by way of directed evolution detailed below.
In certain non-limiting embodiments, the sensor cell expresses a single species of analyte receptor. In certain non-limiting embodiments, the sensor cell expresses a plurality of species of analyte receptor.
In certain non-limiting embodiments, the sensor cell comprises an analyte-specific receptor which is coupled to a detectable reporter gene by a G-protein signaling pathway. Hence, in certain embodiments, the receptor is a G-protein coupled receptor (GPCR) polypeptide or protein. In certain embodiments, the receptor is a non-native GPCR receptor.
In certain non-limiting embodiments, a yeast pheromone sensing system is used for analyte detection. The yeast pheromone signaling pathway is well studied structurally and is functionally similar to hormone and neurotransmitter signaling pathways in mammals.20 In certain non-limiting embodiments, the receptor is a variant of the yeast Ste2 receptor or Ste3 receptor, wherein the receptor is modified so that it binds to the analyte rather than yeast pheromone. In certain embodiments, the receptor or portion thereof is a polypeptide that is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% homologous, or at least about 99% homologous to the native yeast Ste2 or yeast Ste3 receptor. “Homologous” or “homology” can mean sequence (nucleotide sequence or amino acid sequence) homology or structural homology. In certain embodiments, “homology” or “homologous” refers to sequence (nucleotide sequence or amino acid sequence) homology. The sequence homology can be determined by standard software such as BLAST or FASTA. The receptor binds specifically to the analyte (e.g., agent-specific peptide) under assay conditions or under natural conditions (for example, but not limited to, at room temperature (e.g., 20-25° C., at or around body temperature (e.g., 30-40° C.), field temperature (e.g., 5-40° C.) or between about 20-40° C.). In certain non-limiting embodiments, the receptor is a chimeric protein comprising one or more fragment originating from other receptor proteins, or evolved from non-homologous receptor protein to bind to the analyte (e.g., agent-specific peptide) and interface with a signaling pathway. In certain non-limiting embodiments the receptor is a yeast GPCR polypeptide other than a pheromone binding receptor, such as Gpr1 putative sugar binding receptor and the cognate Gα protein Gpa2.
The present invention also provides a nucleic acid encoding the receptor and a host cell comprising said nucleic acid. The nucleic acid can be used to produce a presently disclosed sensor cell. The nucleic acid can be introduced into the host cell such that it is operably linked to an inducible or constitutively active promoter element. In certain embodiments, the sensor cell is a yeast cell, and a nucleic acid encoding a receptor is introduced into the yeast cell either as a construct or a plasmid in which it is operably linked to a promoter active in the yeast cell or such that it is inserted into the yeast cell genome at a location where it is operably linked to a suitable promoter. Non-limiting examples of suitable yeast promoters include, but are not limited to, constitutive promoters pTef1, pPgk1, pCyc1, pAdh1, pKex1, pTdh3, pTpi1, pPyk1, and pHxt7 and inducible promoters pGal1, pCup1, pMet15, and pFus1.
In certain non-limiting embodiments, receptor activation induces reporter gene expression under a FUS1 promoter, which allows for a convenient screen using reporter gene activation. In one non-limiting example, a GPCR polypeptide is expressed in a yeast cell and is coupled to the yeast pheromone mating system such that GPCR binding activates the yeast Fus1 promoter to express a downstream reporter gene.27 The GPCR DNA sequence can then be varied, and this library of altered receptors may be screened for binding of an analyte (e.g., an agent-specific peptide) using production of reporter gene as an indicator of binding.13,26
In certain non-limiting embodiments, where the pathway includes the yeast pheromone sensing pathway, a nucleic acid encoding the reporter is operably linked to at least a transcription controlling portion of the Fus1 promoter, for example, but not limited to, an activating sequence located in the region (−300) to (+400) of the Fus1 gene (Gene ID: 850330). In certain non-limiting embodiments, where the pathway includes the yeast pheromone sensing pathway, a nucleic acid encoding the reporter is operably linked to a Ste12-binding element [(A/T)GAAACA], such that binding of Ste12 acts as a transactivator of the expression of the reporter. In certain non-limiting embodiments, where the pathway includes the yeast pheromone sensing pathway, a nucleic acid encoding the reporter is alternatively linked to one or more inducible promoter other than pFus1, e.g., pFus2, pFig2, and/or pAga1. In certain embodiments, receptor-activation is linked to an engineered pheromone-responsive transcription factor, which binds a synthetic transcription controlling element distinct from the Ste12-binding element. The transcription factor Ste12 is composed of a DNA-binding domain, a pheromone responsive domain and an activation domain. The feasibility of engineering Ste12 to bind to non-natural control elements but remain to activate transcription in a pheromone-responsive manner has been shown [Pi et al (1997)].
In certain embodiments, a GPCR is engineered by directed evolution (DE) to alter its stability, specificity, and/or sensitivity. Hence, a receptor that is activated by a desired analyte can be generated by mutagenesis and selection in the laboratory. Several research groups have established DE in yeast as tool for changing mammalian GPCR ligand specificity.13,14,30-32 Non-limiting examples of such engineered GPCRs include mammalian tachykinin receptors, secretin receptors, opioid receptors, and calcitonin receptors. Non-limiting examples of DE to develop a stable reporter strain are provided in the Examples section.
In certain embodiments, the GPCR is a fungal GPCR. In certian embodiments, the GPCR is a fungal phermone GPCR. In certain non-limiting embodiments, a fungal Ste2-type or Ste3-type GPCR derived from one or more fungus is engineered into S. cerevisiae or other yeast cells to serve as a receptor for detecting an agent of interest. While any peptide-sensing GPCR can be repurposed as a detection element in a yeast cell, fungal pheromone GPCRs have several key advantages for biosensor engineering. First, this type of GPCRs (GPCRs homologous to the S cerevisiae Ste2) couple robustly to the host/native pheromone pathway (see
Based on these characteristics, fungal pheromone GPCRs offer a highly viable platform for DE towards binding of novel peptide ligands (e.g., non-cognate peptide ligands) through mutagenesis of specific portions of the receptor, the peptide or both.
In certain embodiments, the receptors are identified by searching protein and genomic databases (e.g., NCBI, UniProt) for proteins and/or genes with homology (structural or sequence homology) to S. cerevisiae Ste2 receptor. In certain embodiments, the receptor has an average amino acid sequence homology of 33% to S. cerevisiae Ste2, ranging from 66% to 15% as calculated with Clustal Omega [Sievers (2014)].
In certain embodiments, the receptors have seven transmembrane helices, an extracellular N-terminus, an intracellular C-terminus, three extracellular loops and three intracellular loops when analyzed by TMHMM v2.0 [Krogh et al. (2001)]. As shown in
In certain embodiments, when coupled to a lycopene reporter system, as described below, a fungal-derived GPCR, optionally further modified by directed evolution, generates lycopene in the sensor cell in response to the peptide pheromones produced by an agent of interest. Pheromone GPCRs from related fungi can naturally recognize different peptide pheromones based on the highly specific characteristics of this class of GPCRs, which mediate the species-specific mating reaction while preventing interspecies breeding. As described in the Example section, putative GPCRs can be cloned and screened against their putative cognate peptide pheromones using a detector gene, e.g., a fluorescent reporter gene.
The present invention provides a sensor cell (e.g., a yeast cell) comprising a receptor, which is a fungal receptor modified to bind to a bacterial pathogen-specific analyte, such as one from V. cholerae. In certain embodiments, this modification is achieved via directed evolution. The natural yeast pheromone mating receptors Ste2 or Ste3, evolved to bind to a peptide pheromone ligand, are not necessarily likely to adjust to bacterial pathogen-specific analyte and therefore can be deleted from the strain to prevent false activation of reporter gene. A mammalian or hybrid G-protein can be used to enhance GPCR signal transduction in a yeast cell. The remaining genes in the pathway may be endogenous to the yeast sensor cell, or may be engineered for improved performance.
One or more rounds of DE can be performed to generate a GPCR responsive to the natural cholera analytes and peptides. In certain embodiments, cholera-specific peptides can be generated by adding sequence-specific proteases (e.g., trypsin, chymotrypsin, LysN, or GluC) to a given sample. Also, using available computational methods, a peptide database of in-silico proteolized proteomes from bacterial pathogens (e.g., Vibrio cholerae, Staphylococcus aureus, Bacillus subtilis, Streptococcus pneumonia, Salmonella sp., Listeria monocytogenes), fungal pathogens (e.g., Aspergillus niger, Candida albicans, Cryptococcus neoformans, Cryptococcus gattii, Histoplasma capsulatum, Pneumocystis jirovecii and Stachybotrys) viral pathogens (e.g., Ebola virus, HPV, HIV, influenza viruses), or proteolysis pattern of any single protein of interst e.g. produced during an industrial process, can be generated. This peptide database can be searched using peptide motifs derived from analysis of the natural diversity of fungal pheromones.
A computational approach can also be used to discover target peptide analytes that are amenable to detection by an engineered fungal GPCR. This computational method generates a pool of high priority targets that can be highly amenable to a DE approach. Engineered receptors such as 15C11 and 31E4, that show increased ligand promiscuity as starting points to generate engineered GPCRs, can be used to detect these new target peptide ligands from a diverse set of bacterial pathogens. Additionally, some of the natural peptide pheromones produced by bacterial pathogens can be targeted.
DE can be implemented to optimize any engineered GPCR for improved signal levels, enhanced EC50 and/or signal transduction kinetics. Of the six GPCR families, the secretin and fungal pheromone receptor families naturally sense peptides. Moreover, the rhodopsin receptor family also contains members with peptide ligands. Representative members of each of these families have been heterologously expressed in yeast and functionally coupled to the pheromone response pathway: neurotensin NT1 (rhodopsin-like), growth-hormone-releasing-hormone receptor (secretin-like), Sordaria macrospora pheromone receptor (fungal pheromone-like). These GPCRs can be engineered into a yeast cell as a method for detecting their cognate peptide ligands, e.g., growth hormone or neurotensin, for monitoring or quantification.
Fungal Ste2-type or Ste3-type GPCRs as well as other peptide-specific GPCRs mentioned above can be used as a platform for developing engineered peptide-activated GPCRs to generically detect agent-specific analytes. In certain embodiments, the present disclosure provides a step-wise Directed Evolution (DE) strategy based on intermediate hybrid peptides to change the ligand specificity of the parent GPCRs to bind the target peptides.
In certain embodiments, the engineered GPCR is an engineered receptor for the detection of Vibrio cholerae. The receptor can detect a peptide derived from the Cholera toxin (CTx). Additionally, there is a reservoir of biochemical and mutational data of the yeast Ste2 and Ste3 receptor in the literature.35-37,39,40,43 The same strategy can be used for detection of other fungal, viral or bacterial analytes described below.
GPCRs constitute a large class of cell-surface receptors that can be activated by a variety of other ligands, e.g., full proteins, small molecules (e.g., nucleotides and lipids), or light. A variety of these non-peptide sensing receptors have been functionally expressed in yeast.44 These receptors can be employed and engineered into the biosensor to sense analytes other than peptides, e.g., small molecules, proteins or heavy metals.
Non-limiting examples of DNA encoding certain GPCRs of the invention are set forth in Tables 2 and 6 below; the invention further provides for proteins encoded by said DNA sequences.
5.4. Detection Events
Being of the analyte to the receptor triggers a detection event that is indicative of the presence of the agent. The detection events include, but are not limited to, appearance of a reporter (including expression (increased or decreased expression) of a detectable reporter gene), release of a therapeutic molecule that directly remediates the agent, production of a redox active molecule, and a change in the membrane potential of the sensor cell.
In certain embodiments, the detection event is appearance of a reporter. The reporter can be a result of expression of a reporter gene. A reporter can include an enzyme that can produce chromogenic product on a substrate. In certain embodiments, the detection event is increased expression of a reporter gene.
In certain embodiments, the reporter is a laboratory reporter. A “laboratory reporter” means a reporter that cannot be detected by the naked eye (e.g., the change or appearance of the color cannot be detected by the naked eye), and/or a reporter whose detection requires instrumentation. Suitable laboratory reporters include, but are not limited to, bioluminescent, fluorescent, and certain chromogenic reporters. Bioluminescent reporters include, but are not limited to, luciferase. Fluorescent reporters include, but are not limited to, various fluorescent proteins (e.g., a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, a blue fluorescent protein). Non-laboratory chromogenic reporters include, but are not limited to, beta-galactosidase, beta-glucoronidase, and horse-radish peroxidase. In certain embodiments, the reporter is a fluorescent protein.
In certain embodiments, the reporter does not comprise a laboratory reporter. In certain embodiments, the reporter is a non-laboratory reporter. A “non-laboratory reporter” means a reporter that can be detected by the naked eye (e.g., the change or appearance of the color can be detected by the naked eye), and/or whose detection does not require instrumentation (e.g., reporters that are not conventionally used as research tools). Non-laboratory reporters include, but are not limited to, enzymes in the biosynthetic pathways of pigments (biosynthesized pigments that absorb in the visible light spectrum, also referred to as “biosynthesized visible-light pigments”), electrochemical, and reporters which constitute release of one or more therapeutic molecule. Certain chromogenic reporters are non-laboratory reporters, e.g., lycopene.
Biosynthesized visible-light pigments include, but are not limited to, terpenoids, carotenoids, lycopene, violacein and its precursors, melanin, and indigo. In certain embodiments, the reporter is a terpenoid. In certain embodiments, the reporter is a carotenoid. In certain embodiments, the reporter is lycopene. In certain embodiments, the receptor does not comprise a fluorescent protein.
Binding of analyte can induce or alternatively repress reporter gene expression. In the absence of an analyte, there may be essentially no reporter gene expression, reporter gene expression may occur at an undetectable level (e.g., undetectable by the naked eye), or reporter gene expression may occur at a baseline level that detectably increases upon analyte binding.
Violacein and deoxyviolacein are blue pigments isolated from several bacteria. [Sánchez (2006)]. Heterologous expression of the involved genes vioABCDE and optimization of production yields has been shown in E. coli and S. cerevisiae. [Lee (2013)].
Melanin is a black diffusible macromolecule whose overproduction has been achieved from L-tyrosine as precursor by heterologous co-expression of a tyrosinase in E. coli [Santos (2008)].
Production of the blue pigment bio-indigo from tryptophan as a precursor using a bacterial flavin-containing monooxygenase from the methylotrophic bacteria Methylophaga aminisulfidivorans has been achieved and optimized in E. coli [Hwan Han (2008)].
Carotenoids are a class of terpenoids composed of 8 isoprene units totaling 40 carbon atoms. Lycopene is a specific naturally produced carotenoid pigment whose heterologous expression in E. coli using the genes CrtE, CrtB and CrtI has been extensively studied.45 If lycopene is used as a reporter, a presently disclosed sensor cell can be engineered to contain the genes required for synthesis and at least one of said genes can be the detectable reporter gene coupled to activation by peptide receptor binding (e.g., at least a portion of the Fus1 promoter). As a non-limiting example, the gene coupled may be CrtI, CrtE or CrtB.
Lycopene can be visualized by the naked eye, is widely validated in yeast metabolic engineering, and is non-toxic. Lycopene is the first intermediate in carotenoid biosynthesis that has a sufficiently conjugated π-system to absorb in the visible region.46 Thus, unlike standard laboratory reporters like lacZ that require exogenously added caged dyes (X-gal) or fluorescent proteins that require specialized equipment (fluorimeter), lycopene can be directly observed by a non-technical person. Additionally, the biosynthesis of lycopene from endogenous yeast farnesyl pyrophosphate is well established in yeast, requiring only three heterologous genes (
Use of a biosynthesized visible-light pigment as a simple visual readout has a number of advantages. Use of a biosynthesized visible-light pigment readout requires no complex equipment since it can be seen by the naked eye and requires no expensive externally added reagent, since it can be biosynthesized from endogenous substrates. In contrast, most whole-cell biosensors reported in the literature use laboratory readouts such as fluorescent proteins, lacZ, or luciferase, which require the use of expensive equipment, externally added chromogenic reagents or both.48-51
In certain embodiments, lycopene is modified to achieve better response times, signal-to-noise and robustness. For example, in certain embodiments, one or more alternate pheromone-responsive promoter is used.52 In certain embodiments, one or more synthetic Fus1-like promoter is used.53 In certain embodiments, one or more variant of the transcription factor Ste12 is used.54 In certain embodiments, one or more enhancement to the pheromone response pathway is made.55-58 In certain embodiments, one or more variant of the Crt genes including homologues is used.59 In certain embodiments, one or more codon optimized version and engineered version with enhanced activity or activation modality is used.
Additional biosynthesized visible-light pigments include mutants of CrtI disclosed in Schmidt-Dannert, C., Umeno, D. & Arnold, F. H. Molecular breeding of carotenoid biosynthetic pathways. Nat Biotech 18, 750-753 (2000), biosynthetic enzymes that generate alternate carotenoid pigments disclosed in Umeno, D. & Arnold, F. H. Evolution of a Pathway to Novel Long-Chain Carotenoids. J. Bacteriol. 186, 1531-1536 (2004), and lycopene enzymes from alternate organism disclosed in Verwaal, R. et al. High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous. Appl. Environ. Microbiol. 73, 4342-4350 (2007).
A presently disclosed sensor cell may also report in a non-measurable, non-visible way by releasing a therapeutic molecule that directly remediates the detected agent. In general, microbial cells have been used to produce therapeutic molecules such as peptides, proteins and other bioactive small-molecules. [Bourbonnais (1988); Miyajima (1985); Ro (2006)]. Similar to the generation of lycopene, a presently disclosed sensor cell can be coupled to the biosynthesis and secretion of such therapeutic molecule.
In certain embodiments, the detection event is release of a therapeutically relevant molecule, which can be reported through an electronic device. Interfacing to an electronic device can allow reporting to occur much more rapidly and produce a quantitative result. Additionally or alternatively, the release of a therapeutic molecule can be used to directly remediate the agent detected by a presently disclosed sensor cell.
In certain embodiments, the detection event is production of a redox active molecule. Others have in general coupled whole cells electrochemically to electrodes. This is usually done by mixing the cells with a redox-active molecule (a mediator) that couples a redox-active enzymatic process within the cell to a redox reaction on the electrode surface. [Su (2011); Eilam (1982); Garjonyte (2009)].
In certain embodiments, the production or release of a redox active molecule is detected by a redox reaction on an electrode. The redox active molecule can be biosynthesized in an analogous way as lycopene, e.g., by introducing the relevant biosynthetic enzymes into a presently disclosed sensor cell. Similarly, the production of this redox active molecule can be triggered by coupling one of the relevant biosynthetic enzymes to the pheromone signaling pathway. In certain embodiments, the redox active molecule is phenazine. The relevant biosynthetic enzymes are known [Mavrodi (2001)], and their secretion from a bacteria has been measured through the use of an electronic device [Bellin (2014)].
In certain embodiments, the detection event is a change in the membrane potential of the sensor cell. Electronic device that can measure changes in the membrane potential of cells are very common in neuroscience (e.g., multi electrode arrays). [Spira (2013)]. Such a device can be used to measure changes in membrane potential in our biosensor. In certain embodiments, the, a change in the membrane potential of the sensor cell is expression of a cAMP-activated ion channel in the sensor cell (e.g., a yeast cell). This type of channel has been shown to be functional in yeast. [Ali (2006)]
Signal Amplification:
In order to improve the robustness of the reporter signal, quorum sensing signal amplification strategy can be used. Specifically, binding of analyte not only induces expression of visible reporter gene but also induces the expression of enzymes responsible for synthesis of quorum sensing molecules in yeast, or alternative GPCR ligands such as a-factor or alpha-factor. Thus, enhanced sensitivity can be achieved by signal amplification using a positive feedback loop. Signal amplification in this form naturally exists in S. cerevisiae and other fungi using the same GPCRs described below such as Ste2
5.5. Analytes
Suitable analytes can be any ligand which is capable of binding to a receptor, where such binding triggers a detection event that is indicative of the presence of the agent, including triggering a cellular response by the sensor receptor. Suitable analytes include, but are not limited to, proteins, polypeptides (including amino acid polymers), and peptides. “Protein” generally refers to molecules having a particular defined 3-dimensional (3D) structure, whereas “polypeptide” refers to any polymers of amino acids, regardless of length, sequence, structure, and function. “Peptide” is generally reserved for a short oligomer that often but not necessarily lacks a stable conformation. [Creighton Proteins: Structures and Molecular Properties 2nd Edition, ISBN-10: 071677030X]. Proteins can be longer than 50 amino acid residues and peptides can be between 3 and 50 amino acid residues or longer.
In certain embodiments, an analyte is a peptide epitope. As used herein, the term a “peptide epitope” refers to a sub-region of amino acids within a larger polypeptide or protein. A peptide epitope can be composed of about 3-50 residues that are either continuous within the larger polypeptide or protein, or can also be a group of 3-50 residues that are discontinuous in the primary sequence of the larger polypeptide or protein but that are spatially near in three-dimensional space. The recognized peptide epitope can stretch over the complete length of the polypeptide or protein, the peptide epitope can be part of a peptide, the peptide epitope can be part of a full protein and can be released from that protein by proteolytic treatment or can remain part of the protein molecule.
Some sensor cells (e.g., yeast cells, e.g. S. cerevisiae or Candida albicans) are surrounded by a thick cell wall, which can cause a permeability barrier to large molecules. The permeability of the S. cerevisiae cell wall was shown to be strongly growth phase-dependent, being most porous and plastic during exponential phase. [Nobel et al. (1991)]. The cell wall was shown to be permeable to molecules of a hydrodynamic radius of 5.8 nm, corresponding to a globular protein of 400 kDa. [Nobel (1990)]. Similar sized proteins are functionally secreted from yeast cells like S. cerevisiae, C. albicans, C. glabrata by passaging the cell wall [Nobel (1991)]. Therefore, polypeptides or proteins of up to at least 400 kDa may be accessible to the cell surface receptor as analytes. However, proteins or polypeptides beyond this range can also be detected. In certain embodiments, proteolysis are used to fragment the polypeptide or protein to release smaller polypeptides that can serve as the analyte and be accessible to the cell surface receptors.
The analytes can be natural, engineered or synthetic analytes. Virtually any peptide and modified peptide can be assayed using the composition and methods of this invention, including secreted peptides or fragments of proteins which may be released from the protein by a protease. Proteolysis can be induced by one or more host-specific proteases and/or by addition to a given sample of sequence-specific proteases such as trypsin, chymotrypsin, Gluc, and LysN. Modifications of peptides include but are not limited to post-translational farnesylation, glycosylation, deamination, and proteolytic processing.
In certain embodiments, the peptide is a fungal mating pheromone, e.g., a peptide specific to a fungal pathogen. Non-limiting examples of fungal mating pheromones include human fungal mating pheromones (meaning mating pheromones of fungi that can colonize or infect humans), non-human fungal mating pheromones (meaning mating pheromones of fungi that colonize or infect a non-human animal), plant fungal mating pheromones (meaning mating pheromones of fungi that colonize or infect a plant), food fungal mating pheromones (e.g., food safety/spoilage) (meaning mating pheromones of fungi that colonize or infect human or non-human animal food items), and industrial/model fungal mating pheromones. In certain embodiments, the industrial/model fungal mating pheromones are fungi species that are used for making food (e.g., fermentation of alcohol). In certain embodiments, the industrial/model fungal mating pheromones are fungi species that are used for industrial microbiology, e.g., production of drugs, or pesticides in agriculture. In certain embodiments, the industrial/model fungal mating pheromones are fungi species that are used for academic research.
Non-limiting examples of human fungal mating pheromones include the mating pheromones of C. albicans, C. glabrata, P. brasiliensis, L. elongisporous, P. rubens, C. guillermondi, C. tropicalis, C. parapsilosis, C. lusitaniae, S. scheckii. and Candida krusei.
Non-limiting examples of non-human animal fungal mating pheromones include the mating pheromone of P. destructans.
Non-limiting examples of plant fungal mating pheromones include the mating pheromones of F. graminearum, M. oryzea, B. cinerea, G. candidum, and C. purpurea.
Non-limiting examples of food fungal mating pheromones include the mating pheromones of Zygosaccharomyces bailii, Zygosaccharomyces rouxii, and N. fischeri.
Non-limiting examples of industrial/model fungal mating pheromones include the mating pheromones of S. cerevisiae, K lactis, S. pombe, V. polyspora (receptor 1), V. polyspora (receptor 2), S. stipitis, S. japonicas, S. castellii, and S. octosporus, A. oryzae, T. melanosporum, D. haptotyla, C. tenuis, Y. lipolytica, T. delbrueckii, B. bassiana, K. pastoris, A. nidulans, N. crassa, and H. jecorina.
In certain embodiments, the peptide is a peptide disclosed in Table 5.
In certain embodiments, the physicochemical properties, e.g., peptide length, overall charge, charge distribution and hydrophobicity/hydrophilicity, of a peptide are determined by using the program ProtParam on the Expasy server [Walker (2005) ISBN 978-1-59259-890-8]. In certain embodiments, the peptide has a length of 3 residues or more, a length of 4 residues or more, a length of 5 residues or more, 6 residues or more, 7, residues or more, 8 residues or more, 9 residues or more, 10 residues or more, 11 residues or more, 12 residues or more, 13 residues or more, 14 residues or more, 15 residues or more, 16 residues or more, 17 residues or more, 18 residues or more, 19 residues or more, 20 residues or more, 21 residues or more, 22 residues or more, 23 residues or more, 24 residues or more, 25 residues or more, 26 residues or more, 27 residues or more, 28 residues or more, 29 residues or more, 30 residues or more, 31 residues or more, 32 residues or more, 33 residues or more, 34 residues or more, 35 residues or more, 36 residues or more, 37 residues or more, 38 residues or more, 39 residues or more, 40 residues or more, 41 residues or more, 42 residues or more, 43 residues or more, 44 residues or more, 45 residues or more, 46 residues or more, 47 residues or more, 48 residues or more, 49 residues or more, or 50 residues or more. In certain embodiments, the peptide has a length of 3-50 residues, 5-50 residues, 3-45 residues, 5-45 residues, 3-40 residues, 5-40 residues, 3-35 residues, 5-35 residues, 3-30 residues, 5-30 residues, 3-25 residues, 5-25 residues, 3-20 residues, 5-20 residues, 3-15 residues, 5-15 residues, 3-10 residues, 3-10 residues, 5-10 residues, 10-15 residues, 15-20 residues, 20-25 residues, 25-30 residues, 30-35 residues, 35-40 residues, 40-45 residues, or 45-50 residues. In certain embodiments, the peptide has a length of 9-25 residues. In certain embodiments, the peptide has a length of 9-23 residues. In one non-limiting embodiments, the peptide has a length of 9 residues. In one non-limiting embodiments, the peptide has a length of 10 residues. In one non-limiting embodiments, the peptide has a length of 11 residues. In one non-limiting embodiments, the peptide has a length of 12 residues. In one non-limiting embodiments, the peptide has a length of 13 residues. In one non-limiting embodiments, the peptide has a length of 14 residues. In one non-limiting embodiments, the peptide has a length of 15 residues. In one non-limiting embodiments, the peptide has a length of 16 residues. In one non-limiting embodiments, the peptide has a length of 17 residues. In one non-limiting embodiments, the peptide has a length of 18 residues. In one non-limiting embodiments, the peptide has a length of 19 residues. In one non-limiting embodiments, the peptide has a length of 20 residues. In one non-limiting embodiments, the peptide has a length of 21 residues. In one non-limiting embodiments, the peptide has a length of 22 residues. In one non-limiting embodiments, the peptide has a length of 23 residues.
In certain embodiments, the peptide is hydrophobic. In certain embodiments, the peptide is mildly hydrophilic.
In certain embodiments, the peptide is a S. cerevisiae pheromone alpha-factor. The C-terminus of the S. cerevisiae pheromone alpha-factor is involved in binding to the receptor. The N-terminus of the S. cerevisiae pheromone alpha-factor contributes to signaling due to receptor activation.
Non-limiting examples of classes of peptide analytes include the following.
5.5.1. Peptides as Analytes in Diseases
5.5.1.1. Peptides in Fungal Infections
Suitable analyte peptides associated with fungal infections include, but are not limited to, a peptide from Aspergillus (e.g., Aspergillus niger), Candida (e.g., C. albicans or C. glabrata), Cryptococcus (e.g., Cryptococcus neoformans or Cryptococcus gattii), Histoplasma (e.g., Histoplasma capsulatum), Pneumocystis (e.g., Pneumocystis jirovecii), or Stachybotrys (e.g., Stachybotrys chartarum).
In certain embodiments, the agent-specific peptide is a peptide pheromone produced by a pathogenic fungus or a proteolytic product from a pathogenic fungus.
5.5.1.2. Peptides in Bacterial Infections
Suitable analyte peptides associated with bacterial infections include, but are not limited to, a peptide from V. cholera (e.g., Cholera toxin), Staphylococcus aureus (e.g., staphylococcal auto-inducing peptide or portion of beta toxin), and Salmonella spec. (e.g., Salmonella Exotoxins). In certain embodiments, an agent-specific analyte is a peptide derived from the cholera toxin or a proteolytic product from cholera. The proteolytic product from cholera can be generated by a host-specific protease and/or by an exogenous protease. In certain embodiments, an agent-specific analyte is a small molecule secreted or derived from Vibrio cholera. In certain embodiments, an agent-specific peptide is Vibrio cholerae specific or at least specific to a small group of bacteria including Vibrio cholerae (for example a group of up to 10 known species or up to 5 known species).
In certain embodiments, the peptide derived from the cholera toxin is selected from the group consisting of the peptides disclosed in Table 7.
In certain embodiments, the peptide associated with V. cholera is selected from the group consisting of a peptide having an amino acid sequence set forth in VEVPGSQHIDSQKKA (SEQ ID NO: 26), a peptide having an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%) homologous to SEQ ID NO: 26, a peptide having an amino acid sequence set forth in VPGSQHIDS (SEQ ID NO: 27), and a peptide having an amino acid sequence that is at least 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%) homologous to SEQ ID NO: 27.
5.5.1.3. Peptides in Viral Infections
Suitable analyte peptides associated with viral infections include, but are not limited to, a peptide from Ebola virus (e.g., secreted glycoprotein), Influenza virus (e.g., Hemagglutinin), or HIV (e.g., HIV glycoprotein)
5.5.1.4. Peptides in Non-Infectious Disease
Patterns of peptide biomarkers unique for certain cancer types have been identified by mass spectrometry.60-63 Suitable analyte peptides associated with cancer include, but are not limited to, protein portions released from human endogenous proteins by tumor-specific exopeptidases or antibody-derived peptide biomarkers for well characterized disease states.
Peptide or protein biomarkers have been identified in other diseases, e.g., Alzheimers,64 Parkinson,65 or different kidney diseases.66 Such peptides and proteins may also function as analytes.
5.5.2. Peptides as Analytes in Food Safety
5.5.2.1. Toxins
Suitable analyte peptides associated with food toxins include, but are not limited to, a peptide from Clostridium botulinum (e.g., Botulinum toxin), Shiga toxin-producing Escherichia coli (STEC) (e.g., Shiga toxin), and Shigella dysenteriae (e.g., Verotoxin).
5.5.2.2. Immunogens and Allergens
Suitable analyte peptides associated with food immunogens and allergens include, but are not limited to, immunogenic wheat peptide (e.g., gluten).
5.5.3. Peptides in Plant & Crop Infections
Suitable analyte peptides associated with plant and crop infections include, but are not limited to, a peptide of Fusarium graminearum, Botrytis cinerea, Magnaporthe oryzae, and Geotrichum candidum.
5.5.4. Peptides in Bioterrorism
Suitable analyte peptides associated with bioterrorism include, but are not limited to, peptides of Bacillus anthracis (anthrax), e.g., one of three polypeptides that comprise the anthrax toxin secreted by the pathogen: protective antigen (PA), lethal factor (LF) and edema factor (EF),67 or Clostridium botulinum (botulism), e.g., Botulinum neurotoxin peptides such as the cyclic peptide C11-019.68
5.5.5. Other Analytes
Non-peptide analytes can include, but are not limited to, quorum sensing small molecules such as the Vibrio Cholera CAI-1,69 inter-species bacterial quorum sensing AL-2,70 aflatoxin B1 produced by Aspergillus flavus, components of the bacterial LPS, or heavy metals contaminants such as cadmium, mercury, lead or arsenic.
5.6. Methods of Use
The present invention provides for a method of detecting the presence of an agent of interest in a sample using the sensor cell disclosed herein. In certain embodiments, the method comprises contacting the sample with a sensor cell (e.g., a yeast sensor cell) comprising (e.g., bearing) a receptor (e.g., a non-native GPCR receptor) that binds to an analyte indicative of the presence of the agent, wherein binding of the analyte to the receptor triggers a detection event that is indicative of the presence of the agent (e.g., increased expression of a reporter gene).
In certain embodiments, the receptor is coupled to the reporter gene. The method further comprises culturing the sensor cell for an effective period of time; and determining expression of the reporter gene. In certain embodiments, determining whether expression of the reporter gene comprises detecting the expression of the reporter gene by the naked eye and does not require instrumentation. In certain non-limiting embodiments, the reporter is lycopene.
In certain embodiments, the detection event is release of a therapeutic molecule that directly remediates the agent.
In certain embodiments, the detection event is production of a redox active molecule. The method further comprises measuring the production of the redox active molecule. In certain embodiments, measuring the production of the redox active molecule comprises an electronic device. The redox active molecule can be phenazine.
In certain embodiments, the detection event is a change in the membrane potential of the sensor cell. The change in the membrane potential of the sensor cell comprises expression of a cAMP-activated ion channel in the sensor cell.
The particulars of the receptor, coupling, and reporter gene are described in the sections above.
The method for determining whether the reporter gene is or has been expressed depends upon the particular reporting gene used. If the reporter gene produces a visibly detectable product, such as lycopene, it can be detected with the naked eye or colorimetrically. Means of detection of reporter genes known in the art can be used.
In certain non-limiting embodiments, the receptor is a G-protein coupled receptor (GPCR) engineered to bind to the analyte.
By way of non-limiting example, a method of detecting the presence of Vibrio cholerae in a water sample can include detecting the presence of a peptide associated with Vibrio cholerae in the water sample by a method comprising:
contacting the water sample with a sensor yeast cell bearing a GPCR polypeptide that binds to the analyte coupled to a CrtI gene such that when the peptide binds to the receptor, expression of the CrtI gene is induced and lycopene is produced;
culturing the sensor yeast cell for an effective period of time; and
determining whether lycopene has been produced.
The analyte associated with Vibrio cholerae can be a peptide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% homologous to VEVPGSQHIDSQKKA (SEQ ID NO: 26) or VPGSQHIDS (SEQ ID NO: 27). The effective period of time can be hours (e.g., about 24 hours, about 18 hours, about 12 hours, about 8 hours, about 6 hours, about 4 hours, about 3 hours, or about 2 hours) or minutes (e.g., about 90 minutes, about 60 minutes, about 45 minutes, about 30 minutes, about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 3 minutes, about 2 minutes, or about 1 minute).
In certain non-limiting embodiments, the present invention provides for a method of detecting the presence of a fungus or a fungal pathogen, comprising detecting the presence of an analyte associated with said fungus or a fungal pathogen in a sample by a method comprising:
contacting the sample with a sensor cell comprising (e.g., bearing) a receptor that binds to the analyte coupled to a reporter gene such that when the analyte binds to the receptor, expression of a detectable reporter gene is induced;
culturing the sensor cell for an effective period of time; and
determining whether the reporter gene is expressed. In certain non-limiting embodiments, the receptor is a G-protein coupled yeast receptor engineered to bind to the analyte. In certain non-limiting embodiments, the reporter gene expression is detected by the naked eye and does not require instrumentation. In certain non-limiting embodiments, the reporter gene product is lycopene.
In certain embodiments, the sensor cell is a freeze-dried or other dried cell, e.g., a freeze-dried yeast cell. The cell can be activated for use by addition of a food source, e.g., sugar or agar.
Non-limiting examples of samples can include a water sample and a sample of body fluid. Non-limiting examples of water samples include fresh water, sea water, and sewage samples. Non-limiting examples of body fluid samples include intestinal fluids, diarrhea or other feces, mucus (e.g., sputum), blood, cerebrospinal fluid, lymph, pus, saliva, vomit, urine, bile, and sweat. In certain embodiments, the agent to be detected is a plant fungal pathogen. A plant can be shaken in water to provide a water sample containing the fungal pathogen, or a soil sample can be mixed with water and tested for the fungal pathogen, or a portion of plant material (e.g., a fluid obtained from the plant) can be used as a sample.
5.7. Kits
The present invention provides kits for detecting the presence of an agent of interest, for example but not limited to a chemical or a pathogen, as described above. Kits can include one or more sensor cells, as described above, and can be used to perform methods of detecting the presence of an agent, as described above. Kits can further include one or more controls. Kits can include both a positive and a negative control. Kits can include a substrate that comprises the sensor cells and on which or in which detection can occur, e.g., a dish, cup, bowl, plate, paper, chip, gel, bag, stick, syringe, jar, or bottle. Kits can include a food or nutrient source, e.g., sugar or agar. Kits can include components to improve cell viability, including one or more carbon sources, one or more nitrogen sources, one or more trace nutrient sources, and one or more additional nutrient sources to improve response speed. Kits can include additional assay components, including proteases to release target peptides, dyes, filters, and/or cryo-protectants. Kits can be produced by combining all required assay components (e.g., nutrients, sensor cells, and proteases) and freeze-drying, air-drying, or binding this component mix to a substrate. In certain embodiments, the kit comprises a protease (e.g., a protease from prokaryote sources or a protease from eukaryote sources) for digestion of the agent into smaller detectable peptides.
5.7.1 Dipstick Embodiments
In particular non-limiting embodiments, the invention provides for a kit comprising biosensor cells on a solid support comprised in a dipstick configuration. The solid support may be any natural and/or synthetic material, including but not limited to glass fiber, cellulose, quartz fiber, cellulose fiber, polytetrafluoroethylene, cotton, rayon, viscose, etc. In non-limiting examples, the support material may be configured such that the biosensor cells may be applied by filtration; for example, biosensor cells may be applied, by filtration, to a filter paper or disk, and then at least a portion of that paper or disk (e.g. a section of the filter paper or disk) may be incorporated into a dipstick configuration. Alternatively, the biosensor cells may be applied by direct application, for example, applying a volume of liquid culture. The solid support may be affixed, prior to or after (or concurrently with) application of biosensor cells, to a support strip to create a dipstick having a proximal end that may be directly or indirectly held by the user and a distal end bearing the solid support and biosensor cells, permitting dipping the biosensor into a sample to be tested. In certain embodiments, the support strip has liquid wicking activity (e.g., absorbent paper or other material). The proximal end of the dipstick may optionally fit into a holder (to form a dipstick device) that facilitates gripping the dipstick device. In certain non-limiting embodiments, the dipstick comprises a solid support having at least a portion of its surface bearing an amount of biosensor cells sufficient to generate detectable signal after contacting an analyte of interest, and optionally a portion bearing an amount of a negative control (e.g. cells that would not generate detectable signal after contact with the analyte of interest). In certain non-limiting embodiments, the dipstick comprises a solid support having at least several portions of its surface (e.g., an array) each bearing distinct biosensor cells with each type of biosensor cells present in an amount sufficient to generate detectable signal after contacting its corresponding analyte or analytes of interest. In certain non-limiting embodiments, the amount of biosensor is at least between about 1×106 and 5×108 cells, or between about 1×107 and 1×108 cells. Cells may be applied to the support, for example, by vacuum filtration. After application of biosensor to solid support, the composition may optionally be allowed to dry for at least about 20 minutes. The present invention provides for a kit comprising one or more dipstick, and optionally comprising one or more holder; in a particular embodiment, the kit comprises 1-3 holders, or one holder, and at least 3 or at least 5 or at least 10 dipsticks for testing for the same or different analytes. In certain non-limiting embodiments, a method is provided in which the dipstick described above may be used to detect an analyte of interest or an array of analytes of interest by dipping its distal end, bearing the biosensor cells and/or the negative control cells and/or the array of distinct biosensor cell types, into a sample such that the biosensor cells and/or the negative control cells and/or the array of distinct biosensor cell types contact the sample, and then incubating the dipstick at a temperature that is at least about 20° C., preferably greater than 20° C., and preferably greater than 25° C., for a period of time that allows signal to develop, for example, but not limited to, at least about 1 hour, at least about 3 hours, at least about 5 hours, at least about 7 hours, at least about 10 hours, at least about 12 hours or at least 15 hours. In certain situations, it may be desirable to add liquid (e.g. water, saline, or a medium that allows or promotes growth of biosensor cells) to a sample prior to testing; for example, where the biosensor is a yeast, a sample may be diluted with yeast growth medium. In certain exemplary non-limiting embodiments, urine or serum may be diluted 1:1 with yeast growth medium, and blood may be diluted about 2:98 with yeast growth medium. A solid sample, such as soil or stool, may be suspended in yeast growth medium prior to testing. In certain non-limiting embodiments, a kit is provided comprising at least one dipstick as described above, optionally a dipstick holder, and either liquid nutrient medium or powdered medium that can be reconstituted, by addition of water or other liquid, to form a liquid nutrient medium for growth of biosensor cells. In certain non-limiting embodiments, a kit is provided comprising at least one dipstick as described above, optionally a dipstick holder, and either liquid yeast nutrient medium or powdered medium that can be reconstituted, by addition of water or other liquid, to form a liquid yeast nutrient medium for growth of yeast biosensor cells, as described above.
A yeast strain producing lycopene in response to the activation of the endogenous GPCR, Ste2 was generated by the natural S. cerevisiae peptide pheromone, α-Factor (αF). A parental reporter strain was made by deleting the cyclin-dependent kinase inhibitor Far1 to prevent cell-cycle arrest and deleted the G-protein activating protein Sst2 to prevent signal attenuation. For general procedures, see Pausch, M. H. G-protein-coupled receptors in Saccharomyces cerevisiae: high-throughput screening assays for drug discovery. Trends Biotechnol. 15, 487-494 (1997). Then, the carotenoid genes derived from E. herbicola, CrtE, and CrtB were placed under the control of the constitutive promoters pTef1 and pPgk1, respectively. The final biosynthetic gene CrtI was placed under control of the Fus1 promoter, a downstream target of the pheromone response pathway. See Bardwell, L. A walk-through of the yeast mating pheromone response pathway. Peptides 26, 339-350 (2005). This lycopene reporter cassette was introduced into the parental reporter strain through Reiterative Recombination. See Wingler, L. M. & Cornish, V. W. Reiterative Recombination for the in vivo assembly of libraries of multigene pathways. Proc Natl Acad Sci USA 108, 15135-15140 (2011). This v1.0 reporter strain became visibly orange 36 hours after exposure to αF, as shown in
Through modification of the v1.0 strain, a lycopene response time of 2 hours under optimal culture conditions and less than 6 hours in a stringent product prototype assay was observed. To do so, the CrtI amount was increased with an additional chromosomal copy of the pFus1-CrtI construct. This led to a 9.8-fold improvement in response time. The catalytic activity of CrtI was improved by increasing FAD content in the cell through the overexpression of the FAD synthetase FAD1. See Schaub, P. et al. On the Structure and Function of the Phytoene Desaturase CRTI from Pantoea ananatis, a Membrane-Peripheral and FAD-Dependent Oxidase/Isomerase. PLoS ONE 7, e39550 (2012); Wu, M., Repetto, B., Glerum, D. M. & Tzagoloff, A. Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae. Mol. Cell. Biol. 15, 264-271 (1995). This modification independently led to a 10.3-fold improvement in the response time, and to a 21.1-fold improvement when combined with the increased CrtI copy number. These results are shown in
E.
herbicola
E.
herbicola
E.
herbicola
S.
cerevisiae
Several putative GPCRs were screened against their putative cognate peptide pheromones using a fluorescent reporter gene.33 Recognition of pheromones from the following pathogenic fungi was shown in S. cerevisiae:
Human Pathogens:
As shown in
Candida
albicans
Candida
glabrata
Paracoccidioides
brasiliensis
Fusarium
graminearum
Magnaporthe
oryzea
Botryhs
cinerea
6.3.1. Directed Evolution of Reporter Strain
A stable reporter strain to perform DE on plasmid-borne receptor variants based on previous methods for DE of GPCRs in yeast was established. This strain was analogous to the lycopene reporter with the lycopene biosynthetic genes replaced by the reporters: pFus1-mCherry (fluorescence), pFus1-His3 (growth advantage), pFus2-Ura3 (negative selection). The chromosomal copy of Ste2 was deleted.
6.3.2. Library Generation and Selection Scheme
The endogenous S. cerevisiae Ste2 pheromone receptor was mutated by error-prone PCR and selected for active mutants by fluorescence-activated cell sorting (FACS). The enriched libraries were screened in microtiter plates using a growth based assay using pFus1-His3 as previously reported.30
6.3.3. Peptide Ligand Design for Step-Wise DE
A stepwise selection framework that has been used to change substrate specificity of proteins and enzymes was used.72 Peptide targets that allow generation of a wide range of intermediate hybrid ligands that march from the native peptide ligand (e.g. native yeast α-Factor) to the desired target ligand (e.g. peptides derived from Cholera Toxin) were used for directed evolution.
6.3.4. Successful Demonstration of De Strategy
This DE strategy was applied to CTx and two intermediate peptides (as shown in
S. cereviseae
S. cereviseae
6.3.5. Demonstration of Proteases to Release Target Ligands
A simple proteolytic degradation of commercially purified CTx was performed. CTx was specifically degraded with either Trypsin or a combination of LysN and GluC. The expected target peptide was successfully detected by mass spectrometry showing it to be released from the full protein. The experiment resulted in a list of released peptides of different length and physicochemical properties which can be used as additional target analytes. Analogous degradation of CTx in the gut or the environment may make target peptides available in field samples. Additionally and alternatively, these extremely robust and cheap proteases may be incorporated into a product formulation.
The strain is engineered to respond to a cholera specific peptide by generating a color output.
To develop a cholera peptide binding receptor, the GPCR is subjected to mutagenesis and the resulting library is expressed in the same yeast host. All variants are screened against the peptide, which is synthetically synthesized or originates from bacterial cultures, and strains that show reporter gene expression are further investigated and optimized. Enhanced binding may be achieved by more stringent screening conditions such as lower concentration of target molecule or less copies of the receptor expressed on the cell surface. In certain embodiments, color change is rapid—for example 10 grams, 1 gram, 100 mg, 10 mg, or even 1 mg of freeze dried yeast may result in sufficient red color to be readily apparent to the naked eye, and the assay is desirably sensitive enough to detect low levels of peptide. Non-engineered yeast may be used as controls to test biosensor specificity and false-positive rate. Native alpha factor/Ste2 receptor activation can also be used as a control.
GPCRs were cloned into yeast using the Reiterative Recombination DNA assembly system. The desensitization of the receptor, where prolonged stimulation leads to an attenuated response, was eliminated by deletion of SST2, allowing cells to respond to doses of pheromone that are roughly two orders of magnitude lower than those detected by normal cells and prevent recovery from pheromone-induced growth arrest, even if the ligand was removed.20 Deletion of Far1 also prevented pheromone-induced cell cycle arrest. The endogenous pheromone receptor Ste2 was deleted to avoid cross talk with yeast mating signal.
Viability of S. cerevisiae was determined after different freeze-drying treatments.73 The results are shown in
The engineering of S. cerevisiae as a specific and sensitive biosensor for the presence of pathogenic fungi that may be easily used outside the laboratory. The sensor may be used by non-experts, and thus consists of non-technical mixing and color change output that is visible to the naked eye.
Baker's yeast, a safe organism broadly used in the food industry for centuries and easily grown in a robust manner was reprogrammed to express the tomato red pigment lycopene in response to binding of natural pathogen-specific peptides by expressing natural fungal binding receptors. This user-friendly and equipment-free signal is compatible with household use at local communities at-risk for fungi infections.
Fungal pathogens have recently been identified as increasing cause of human disease as well as a cause of population decline in animals and crops. The annual number of cases of sepsis caused by fungal organisms in the U.S. increased by 207% between 1979 and 2000 [Pfaller, Diekema, (2007)]. Several factors contribute to the increase in fungal infections, among which are the increasing number of immunocompromised HIV, cancer and transplantation patients, aging population, and increased global mobility which expands the habitats of endemic opportunistic fungal strains [Pfaller, Diekema, (2007)].
Candida fungal species are the major cause of opportunistic mycoses worldwide with 72.8 million annual candida species infections cases worldwide and a 33.9% case/fatality ratio [Pfaller, Diekema, (2007)]. Candida infections are associated with a high crude mortality of 46% to 75% and a long hospital stay which causes tremendous health care burden. Two fungal species, C. albicans and C. Glabrata, were shown to be the causative agents of 62% and 12% of Candidasis, respectively. [Ramirez-Zavaleta (2010)]. Candida albicans is a fungi naturally found in human gastrointestinal, genitourinary tracts and skin, but under compromised immunity it could result in kidney, heart or brain infection [Berman, Sudbery (2002)].
It is difficult to diagnose and distinguish fungal infections. While several anti-fungal therapeutics are available, mortality rates of invasive fungal diseases remain extremely high, often exceeding 50%. This is due to a major clinical bottleneck in early treatment, rooted in significant lack of rapid diagnosis [Brown et al. (2012)]. For example, although several methods are currently available for detection of pathogenic fungi in the laboratory, the current gold standard for confirming candida infection in patients remains slow methods such as cultures or cost prohibitive methods such as coagulation assays which are often unavailable in high risk areas for fungal infections. In this Example, a non-technical biosensor that could be used outside of the laboratory for detection of pathogenic fungi was developed.
In order to detect fungal pathogens, fungal receptors that are naturally binding the fungal peptide mating pheromone were generated. Candida albicans cells are diploid (a/alpha) and both homothallic and heterothallic mating have been observed in clinical samples, making mating peptide a relevant biomarker for fungal detection. C. albicans must switch its phenotype from white to opaque before secretion of pheromones can occur to induce mating, a transition triggered by different environmental signals. The opaque “mating” phenotype was found to be stabilized by the presence of CO2 and GlcNAc and observed during passage through mouse intestines, suggesting persistence of mating-compatible, pheromone producing C. albicans cells in the host [Ramirez-Zavala (2008); Huang (2010)]. Mating was also observed in systemic infections and colonization of the skin and intestines. [Hull et al. (2000), Lachke et al (2003), Dumitru (2007)]. C. glabrata population is mostly clonal, and while distinct mating types have been identified, pheromone genes are not expressed in most isolates and neither mating types responds to pheromone.
6.7.1. Fungal GPCRs as the Detection Element
Natural fungal GPCRs were cloned and tested for functionality with their respective natural ligands in S. cerevisiae biosensor strain. The results for GPCR activation experiments in biosensor strain are presented in
As shown in
Most receptor-pheromone pairs were found to be highly sensitive to their ligand peptide, with EC50 values of 4 nM, 51 nM and 34 nM for C. albicans, L. elongisporous, and P. brasiliensis, respectively, notably higher than the natural activation of the S. cerevisiae GPCR-pheromone pair (EC50=190 nM). C. glabrata was less active EC50=3.6 μM) in biosensor settings (see
6.7.2. Lycopene as a Simple, Low-Cost Readout
Having established fungal GPCRs as the detection element, the inventors then implemented and optimized a lycopene biosynthetic pathway as a direct, low-cost readout for the biosensor (see
6.7.3. An Integrated Biosensor
A product profile that satisfies the unique requirements of a live yeast cell sensor as diagnostic device was developed. Specifically, a core product component, the “Yeast Reporter Tab”, maintaining viable, functional yeast cells while enhancing color contrast and ease of use (see
The integrated biosensor properly responded to a synthetic peptide derived from the human pathogen C. albicans. Importantly, the biosensor retained a high level of sensitivity and speed while producing a signal visible to the naked eye (see
Furthermore,
EXAMPLE 8 is an updated study of EXAMPLE 2. Whole-cell diagnostic device enables the use of integral membrane receptors to mediate highly specific and sensitive detection of biologically relevant ligands. Notably, membrane proteins such as GPCRs have not been amenable for in vitro diagnostics as they are notoriously difficult to express outside of their natural membrane environment. A whole-cell provides access to the untapped repertoire of molecular recognition of GPCRs in much the same way ELISAs allowed access to antibody recognition [Lequin (2005)]. The inventors focused on implementing the highly specific fungal peptide-activated GPCRs, such as Ste2 from S. cerevisiea, for detection of fungal peptides.
Fungal GPCRs have several key advantages for biosensor engineering. First, GPCRs homologous to the S. cerevisiae Ste2 robustly coupled to the host pheromone pathway. (see
aThe GRAVY value is the average hydropathy of the given sequence. Positive values indicate overall hydrophilicity of the sequence and negative values relative hydrophobicity. Index range is −4.5 to 4.5
6.8.1. Key Characteristics of Fungal GPCRs
Candidate receptors for biosenosor engineering were identified by searching protein and genomic databases (NCBI, UniProt) for proteins and/or genes with homology to S. cerevisiae Ste2 receptor. Functionally characterized receptors (described below) had an average amino acid sequence homology of 33% to S. cerevisiae Ste2, ranging from 66% to 15% as calculated with Clustal Omega [Sievers (2014)].
Additionally, all receptors were predicted to have seven transmembrane helices, an extracellular N-terminus, an intracellular C-terminus, three extracellular loops and three intracellular loops when analyzed by TMHMM v2.0 [Krogh et al. (2001)]. Notably, while large portions of the extracellular loops and transmembrane helices had low conservation across receptors, three key regions with increased homology (see
6.8.2. List of Functionally Characterized Receptors
Twenty three receptor-peptide pairs were cloned and functionally characterized in sensor strain, as shown in
A. nidulans, A. oryzae, B. bassiana, C. lusitaniae, C. tenuis, N. fischeri, N. crassa, P. destructans, H. jecorina, T. melanosporum, D. haptotyla, S. scheckii, Y. lipolytica, T. delbrueckii, K. pastoris
Saccharomyces
cerevisiae
Candida
albicans
Candida
glabrata
Paracoccidioides
brasiliensis
Fusarium
graminearum
Magnaporthe
oryzea
Botrytis
cinerea
Lodderomyces
elongisporous
Penicillium
rubens
Candida
guilliermondii
Candida
tropicalis
Candida
parapsilosis
Geotrichum
candidum
Zygosaccharomyces
bailii
Zygosaccharomyces
rouxii
Kluyveromyces
lactis
Schizosaccharomyces
pombe
Vanderwaltozyma
polyspora
Vanderwaltozyma
polyspora
Scheffersomyces
stipitis
Schizosaccharomyces
japonicus
Saccharomyces
castellii
Schizosaccharomyces
octosporus
Aspergillus
nidulans
Aspergillus
oryzae
Beauvaria
bassiana
Candida
lustianiae
Candida
tenuis
Neosartorya
fischeri
Neurospora
crassa
Pseudogymnoascus
destructans
Hypocrea
jecorina
Tubermelanosporum
Dactylellina
haptotyla
Sporothrix
scheckii
Yarrowia
lipolytica
Torulaspora
delbrueckii
Komagataella
pastoris
6.8.4. Key Characteristics of Peptide Ligands
Twenty three natural fungal peptides were synthesized and tested for activation of their corresponding receptor in the biosensor strain. Physico-chemical properties, e.g., peptide length, overall charge, charge distribution and hydrophobicity/hydrophilicity were determined for all 23 functionally verified peptide ligands using the program ProtParam on the Expasy server [Walker (2005) ISBN 978-1-59259-890-8]. Sequence variability and conserved sequence motifs within the set of peptide ligands were determined using an alignment and clustering method described in [Andreatta et al. (2013)].
A. Physicochemical Characteristics of Peptide Ligands
Natural mating peptide ligands featured diversity in length (9-23 residues), overall charge and number of charged residues as well as hydrophobicity (GRAVY, Grand average of hydropathy [Kyte and Doolittle (1982)] ranging from hydrophobic to mildly hydrophilic (see Table 9).
B. Sequence-Function Relationship and Sequence Diversity
Functional Domains within Alpha-Factor:
previously reported Alanine scanning mutagenesis revealed defined functional domains within the S. cerevisiae mating pheromone alpha-factor [Naider et al. (2004)]. Residues at the C-terminus were found to be mainly involved in binding to the receptor, while residues at the N-terminus were shown to contribute to signaling due to receptor activation. NMR studies also showed that alpha factor adopts a bended secondary structure due to the tendency of the internal residue stretch to form a loop [Higashijima et al. (1983)].
Sequence Motifs of Peptide Ligands:
A motif search for the peptides listed below was performed using a 13-residue motif length as an input parameter, because this is the length of the well characterized alpha factor. The peptides were clustered into 3 groups by conservation of residues (see
Correlation Between Sequence Motifs and Physicochemical Properties:
The peptide alignments within the clusters showed that sequences within the same cluster varied in length, overall charge, distribution of charged residues and hydrophobicity/hydrophilicity (see
The design of S. cerevisiae biosensor allowed for simple plug-and-play engineering of new receptor-ligand pairs into the existing biosensor strain. The first step in developing yeast biosensors for additional targets using this platform was the identification of specific peptide biomarkers, for which specific receptors can be adapted via receptor engineering and directed evolution. As shown in
First, mass spectrometric analysis is used to identify the peptidome of a given sample. A sample can be anything from a blood sample to a nasal swab or water sample. The peptidome of a sample includes peptides a priori present in the sample or otherwise released after proteolytic treatment (e.g. treatment with trypsin or chemotrypsin).
The resulting peptides are then compared against our existing fungal ligand library to identify the highest homology match. The inventors' fungal ligand library is a list of fungal peptide pheromones—unmodified peptides between 9-15 residues in length—which are predicted or have been validated to activate their cognate fungal mating GPCR. The GPCR corresponding to homologous library peptide is then used as parent for biosensor engineering and provides an advantageous starting point for directed evolution experiments towards the peptide target.
Cholera toxin (CTx) is a heteromeric protein complex secreted by the bacterium Vibrio cholerae. It is responsible for the massive, watery diarrhea characteristic of cholera infection and it was shown to be an abundant protein in stool samples of cholera-infected patients. [LaRocque et al. (2008)]. CTx is composed of 2 subunits, CtxA (27 kDa) and CtxB (11.6 kDa), where CtxB assembles in a pentameric ring around a single CtxA subunit.
Trypsin digestion of un-denatured, completely folded Ctx (the protein form expected in an untreated stool sample) was performed and the resulting peptidome was determined by mass spectrometry (see peptide list in Table 7). Then, a similarity search of the resulting Ctx peptidome was performed with the inventors' existing library of functional peptides tested in their sensor strain. A peptide HFGVLDEQLHR (SEQ ID NO: 132) with 36% identity to a functional member of the inventors' fungal peptide library, the fungi Zygosaccharomyces rouxii (see
The conservation of N-termini of these peptides is encouraging since the N-terminal end of mating pheromones was shown to be significant for receptor activation. [Naider et al. (2004)]. In addition, while tryptic release of some peptides may be less efficient than others because several predicted trypsin cleavage sites might not be solvent exposed and accessible, the high peptide count of the identified peptide (Table 7) indicates its high abundance in the analyzed sample. Importantly, the same peptide identified in this work was previously reported in tryptic digests of clinical stool samples from cholera infected patients. [LaRocque et al. (2008)]. Directed evolution experiments towards GPCR binding of the identified Ctx peptide is performed.
Materials and Methods.
To assemble the dipstick, the biosensor strains were pre-cultured in 50 mL of yeast extract peptone dextrose media (YPD) at 30° C. at 300 RPM for 72 hours. The culture was diluted with water to an OD600 of 2.5 and vacuum filtered onto a glass fiber filter paper (Thermo Scientific, DS0281-7500) using a plastic stencil to generate spots with a diameter of 5 mm. An appropriate culture volume was used to give about 5×107 cells per spot. The filter paper with biosensor spots was cut into small squares (8×8 mm, 1 biosensor spot) and placed onto a strip of wicking paper made of a standard brown paper towel (FIG. S8B, C). Each paper-based dipstick assay contained two different spots—an indicator (biosensor) spot and a control spot composed of S. cerevisiae carrying off-target receptor as a negative control.
To characterize its functionality, the dipstick was dipped into 1 mL of liquid sample and incubated at 30° C. The lycopene readout was inspected visually and quantitatively measured using time-lapse photography analyzed with ImageJ. A 24-well plate was used to easily array several dipsticks in the field of view of the camera. For all assays, a 10× stock of media was used and diluted to reach the appropriate 1× concentration. All measurements were performed in three or more replicates. For YPD assays (
Additionally, we designed a small plastic holder to facilitate the ease of use of this dipstick assay. This plastic holder was 3D printed out of acrylonitrile butadiene styrene (ABS). We validated the holder it did not negatively impact the assay functionality.
To assay the long-term stability of the paper-dipstick, the biosensor spots were prepared on filter paper as described above and allowed to air-dry for 20 minutes at room temperature. The filter papers were then placed in plastic pouches, flushed with argon, sealed and stored in the dark at room temperature. After 38 weeks of storage the filter papers were removed from the storage pouches, and assembled with the paper towel wicking paper as described above. To characterize the functionality, the assembled paper dipsticks were rehydrated by dipping directly into 1 mL of liquid sample made of 1×YPD media supplemented either with 1 μM of the indicated fungal pathogen peptide or water as a control and incubated at 30° C. The lycopene readout was inspected visually and quantitatively measured using time-lapse photography. All measurements were performed in three or more replicates.
We also determined a visibility threshold for paper-based dipstick assay when measured by time-lapse photography and pixel color analysis. This was done by visually inspecting time-lapse clips. The visible threshold for the dipstick assay was determined to be 4 Δ Red Color units and is shown by a grey line in
To enable quantitative characterization of the paper-based dipstick assay we developed a method to measure lycopene production based on time-lapse photography and pixel color value analysis. Specifically, dipsticks dipped in samples and a tripod-mounted digital single-lens reflex camera (DSLR, Nikon D7000) were placed in a dark box kept at 30° C. Flash photographs were taken automatically every 5 minutes. The resulting sequence of photographs was analyzed using ImageJ139. For each time point, the average pixel color values were measured for each of the two dipstick spots using constant measurement areas. The apparent level of red color of each spot was first calculated by the following:
where R, G, B are the measured red, green and blue color values, respectively. Since the color of the biosensor spots ranges from off-white to red-orange the color values are such that R>G>B is always true. Therefore, Rapparent is a value that scores the level of red from 0 to 1. We then calculated the total level of positive lycopene readout produced by the dipstick by the following:
ΔRed Color=Rapp,indicator−Rapp,negative (E2)
where Rapp, indicator and Rapp, negative are the apparent red color values of the indicator biosensor spot and the negative control yeast spot, respectively given by Eq. E1. Importantly, since the two yeast spots of the dipstick assay are always in close proximity to each other, the A Red Color value is not sensitive to variations in light levels and can be used to compare dipsticks placed anywhere in the field of view of the camera. Using these sequences of photographs we also generated time-lapse clips showing that the lycopene color change can be visualized by the naked eye. These clips are motion and exposure equalized to remove flicker between frames.
Results and Discussion.
Biosensor and control cells were spotted onto filter paper, and detection was performed by simply dipping the paper into liquid samples containing synthetic mating peptides (
Using a P. brasiliensis dipstick assay, we observed a robust and highly reproducible signal that surpassed the visible lycopene threshold to give a clear Yes/No readout (
Materials and Methods.
Preparation of Culture Supernatant from Clinically Isolated Fungal Pathogens.
H. capsulatum—Strains Hc01 and Hc06 are clinical isolates representing North America class 2 (NAm2) and North America class 1 (NAm1), respectively.127H. capsulatum strains were added to liquid SDA medium (40 g/L glucose, 10 g/L peptone) at 105 cells/mL and incubated for 10 days at 26° C. without agitation to induce conversion to mycelia. Conversion to mycelia was confirmed by phase-contrast microscopy. Mycelia were then transferred to HMM media.128 and the cultures incubated at 26° C. After 3 weeks of growth, mycelia were separated from the supernatant by filtration through a cellulose filter (Whatman qualitative filter paper #2, 8 μm-diameter pores) and the filtrate subsequently filtered through a polyethersulfone membrane (0.45 μm diameter pores) to obtain the final culture filtrate. The supernatants were lyophilized, resuspended in 0.1 volume of H2O (10× concentration) and kept at −20° C.
Paracoccidioides—Strains P. brasiliensis Pb18 and P. lutzii Pb01 are clinical isolates containing mating loci MAT1-2 and MAT1-1, respectively.129 The mycelium form was grown at 24° C. at 150 rpm in synthetic McVeigh Morton (MMvM) liquid medium.130 Supernatants were collected by filtration 10 days after the yeast-mycelium transition. The supernatants were lyophilized, resuspended in 0.1 volume of H2O (10× concentration) and kept at −20° C.
C. albicans—Human isolates GC75 with MTLα/MTLα131 and ySB36132 were utilized, the latter being found to be heterozygous for its mating loci, MTLa/MTLα. Homozygous MTLα/MTLα derivatives of ySB36 were obtained by selection on sorbose as previously described.133 In brief: ySB36 was cultured for 16 hours in YPD liquid media at 30° C., washed once with water and ˜105 cells were plated on 2% sorbose media (0.67% yeast nitrogen base without amino acids, 2% sorbose). Colonies were visible after 4 days incubation at 30° C. Several colonies were re-streaked on 2% sorbose media, followed by re-streaking on YPD media and genotyping by colony PCR (see primers Listed in Table 8 below). One homozygous MTLα/MTLα isolate (ySB45) was used for supernatant preparation. Phenotypically switched opaque colonies of GC75 and ySB45 were isolated by Phloxine B staining as previously described.134 In brief: A single colony of GC75 or ySB45 was incubated for 24 h at 25° C. in liquid YPD media without agitation. In total ˜2×103 cells were plated on YPD agar supplemented with 5 μg/ml Phloxine B (Sigma Aldrich) and incubated at 25° C. for 4 days. Opaque colonies stained pink on Phloxine B containing media. For supernatant preparation, a single opaque colony of C. albicans GC75 or ySB45 was cultured overnight in YPD media at 25° C., and used to inoculate 50 ml of YPD liquid media. Cells were cultured for ˜24 h at 25° C. to a final OD600 of 9.5 (˜2.8×108 cells/ml) and 7.9 (˜2.3×108 cells/ml), respectively. Cells were pelleted by centrifugation, the supernatant was reduced to dryness by vacuum concentration and resuspended in 0.1 volume H2O (10× concentration) and kept at −20° C.
C. albicans
S. cerevisiae—samples were obtained from S. cerevisiae strain FY250 with MTLα135 and W303-1B with MTLα (ATCC 201238). Cells were cultured in 50 ml YPD media for 20 h at 30° C. to a final OD600 of 9.8 (˜2.9×108 cells/ml) and 8.5 (˜2.5×108 cells/ml), respectively. Cells were pelleted by centrifugation, the supernatant of FY250 was reduced to dryness by vacuum concentration and resuspended in 0.1 volume H2O (10× concentration) and kept at −20° C. The supernatant of W303-1B was kept at 1× concentration at −20° C.
Detection of Mating Peptides in Supernatants of Clinically Isolated Fungal Strains.
P. brasiliensis or C. albicans biosensor strains (yMJ258 and yMJ260, respectively) and a control S. cerevisiae strain (yMJ251) were used to test for the presence of the respective mating peptides in supernatants derived from clinically isolated pathogenic fungi or S. cerevisiae (supernatants preparation described above). Cells were seeded at an OD600 of 2 in the indicated supernatant mixed with standard complete synthetic media (2% dextrose) supplemented with 5% YPD in 96-well microtiter plates, cultured at 30° C. and 800 RPM, and lycopene production was measured by absorbance as described above. A 2× stock of media and a 10× stock of the supernatant were used and diluted to reach the appropriate 1× concentration. The control supernatant for W303-1B was diluted to 50% in the final assay. Statistical significance of signal (i.e. biosensor strain treated with its cognate-supernatant) over noise (same biosensor strain treated with non-cognate supernatants) was determined by performing a paired parametric t-test in Prism (GraphPad). The highest P-value resulting from sample comparisons is given as ** P≤0.01, *** P≤0.001 (
Determination of Lycopene Content in Microtiter Plate Format.
To determine the relative lycopene content directly in a cell suspension, we adapted the method proposed by Myers et al.140 to characterize pigmented cells through optical density measurements at multiple wavelengths. This method greatly reduces the noise due to variations in cell growth phase, cell density and other sample irregularities. This enabled the precise evaluation of lycopene content in a high throughput microtiter plate format.
As described by Myers et al.140, the optical density of the cell suspension measured at a sensitive wavelength (i.e. corresponding to an absorption maxima of the pigment) is approximately composed of two additive components: scatter due to cells and absorbance due to the pigment. Therefore the pigment content in a cell suspension is proportional to the measured optical density corrected for the scattering component as follows:
[pigment]∝AbsS,P=ODS−ODS,scat (E3)
where AbsS,P is the absorbance due to the pigment at the sensitive wavelength S, ODS is the measured optical density at the sensitive wavelength S, and ODS,scat is a calculated scattering component at the sensitive wavelength S. Since there was noticeable Raleigh-like wavelength dependence in the scatter of lycopene null strains we chose the following functional form to approximate scatter at a particular wavelength λ:
where A and B are constants that reflect changes in cell density and other sample irregularities. At each time point and for each sample, we can calculate the corresponding values of A and B by using the optical density values measured at two robust wavelengths (i.e. corresponding to wavelengths where scatter is the only or dominant component). Substituting these additional scatter-only optical density measurements into Eq. E4 and solving for A and B we get:
where ODR1 and ODR2 are the measured optical densities at the robust wavelengths R1 and R2. Therefore, by setting λ=S and substituting Eq. E4 into Eq. E3, the relative content of lycopene in a cell suspension is given by:
To apply this method to lycopene in yeast, we determined the appropriate sensitive and robust wavelengths by obtaining the absorbance spectrum of lycopene directly in yeast cells. The spectrum was determined by subtracting the optical density spectrum of a lycopene null strain yMJ105 from that of a constitutive lycopene producing strain LW2671 (
Three additional considerations were crucial to yield reproducible lycopene measurements in a microtiter plate format. First, all three optical density measurements (at 395 nm, 520 nm and 600 nm) were taken at the same time for each well to reduce errors due to the settling of cells during the measurement of a whole microtiter plate. Second, assay wells were blanked using a reference well on the same microtiter plate containing identical media conditions as the assay wells but with no cells. This was particularly important when colored media was used. Finally, high cell densities (OD600≥2) were used to yield larger bulk lycopene signals even with the short path length of micro titer plates (˜3 mm). Since these high optical density values were outside the linear range of the photodetector, all optical density values were first corrected using the following formula to give true optical density values:
where ODmeas is the measured optical density, ODsat is the saturation value of the photodetector and k is the true optical density at which the detector reaches half saturation of the measured optical density. Appropriate values for ODsat and k were determined by plotting direct optical density measurements of a range of cultures of several strains, against the true optical densities determined by dilution to the linear range. Optical densities were taken at 395 nm, 520 nm and 600 nm. All points were fit once with Eq. E8 using Prism (GraphPad) to give ODsat=3.57 and k=3.16. These values were used to correct all optical density measurements in this study.
Results and Discussion.
Next, we challenged our biosensor for detection of naturally secreted mating peptides using clinically-isolated Paracoccidioides strains. Paracoccidioidomycosis (PCM), an invasive fungal infection endemic to Latin America, is one of many neglected tropical diseases that primarily affect poor populations and lack systematic surveillance.141 PCM is caused by inhalation of airborne conidia produced by mycelium of the soil ascomycete P. brasiliensis.136 Recent identification of the genetic components underlying its mating system137 enabled us to pursue specific yeast-based detection of P. brasiliensis, which could facilitate detection of its environmental reservoir.
Specifically, we challenged our yeast biosensor to detect cultured mycelial P. brasiliensis isolated from human patients. Biosensor cells expressing P. brasiliensis mating receptor, which exhibited specific and sensitive detection of its synthetic mating peptide (
Saccharo-
myces
cerevisiae
Candida
glabrata
Candida
albicans
Lodderomyces
elongisporus
Para-
coccidioides
brasiliensis
(lutzii)
Botrytis
cinerea
(Botryotinia
fuckeliana)
Fusarium
graminearum
(Gibberella
zeae)
Magnaporthe
oryzae
Zygo-
saccharo-
myces
bailii
Zygo-
saccharo-
myces
rouxii
Histoplasma
capsulatum
S. cerevisiae biosensor
C. albicans biosensor
B. cinerea biosensor
C. glabrata biosensor
F. graminearum biosensor
L. elongisporous biosensor
M. oryzea biosensor
P. brasiliensis biosensor
Z. bailii biosensor
Z. rouxii biosensor
H. capsulatum biosensor
C. glabrata biosensor
Candida albicans, MTLα/MTLα
Candida albicans, MTLa/MTLα
Candida albicans, MTLα/MTLα
Paracoccidioides lutzii, MAT1-1
Paracoccidioides brasiliensis, MAT1-2
Histoplasma capsulatum, NAm2
Histoplasma capsulatum, NAm1
Various references are cited herein, the contents of which are hereby incorporated by reference in their entireties.
This application is a divisional of U.S. patent application Ser. No. 15/596,837, filed May 16, 2017, which is a continuation-in-part of International Patent Application No. PCT/US2015/061373, filed Nov. 18, 2015, which claims priority to U.S. Provisional Patent Application Ser. No. 62/081,441, filed Nov. 18, 2014, priority to all of which are claimed, and the contents of all of which are incorporated by reference in their entireties herein. International Patent Application No. PCT/US2015/061373 includes a Sequence Listing which is incorporated by reference herein.
This invention was made with government support under grant AI110794 awarded by the National Institutes of Health, grant HR0011-15-2-0032 awarded by the Department of Defense/Defense Advanced Research Projects Agency, and grant 1144155 awarded by the National Science Foundation. The government has certain rights in the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20200319180 A1 | Oct 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62081441 | Nov 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15596837 | May 2017 | US |
| Child | 16906669 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2015/061373 | Nov 2015 | US |
| Child | 15596837 | US |
