US20180258435A1 - Biosynthetic amyloid-based materials displaying functional protein sequences - Google Patents

Biosynthetic amyloid-based materials displaying functional protein sequences Download PDF

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US20180258435A1
US20180258435A1 US15/564,619 US201615564619A US2018258435A1 US 20180258435 A1 US20180258435 A1 US 20180258435A1 US 201615564619 A US201615564619 A US 201615564619A US 2018258435 A1 US2018258435 A1 US 2018258435A1
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linker
polypeptide
csga
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Neel S. Joshi
Peter Q. NGUYEN
Anna Duraj-Thatte
Pichet Praveschotinunt
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Harvard University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Definitions

  • the technology described herein relates to engineered polypeptides, bacteria comprising such polypeptides, engineered bacteria, biofilms comprising said bacterial cells, biofilms comprising the engineered polypeptides produced by the engineered bacterial cells, and amyloid-based extracellular matrix components produced by engineered bacterial cells.
  • Biofilms have been investigated for beneficial purposes such as wastewater treatment and biotransformations, but these efforts focus on the use of naturally occurring organisms. Efforts to engineer the structure of biofilms exist. See WO/2012/166906 and PCT/US2014/035095.
  • Embodiments of the present disclosure are directed to methods of genetically modifying bacteria to create amyloid-based materials, such as biofilms created by amyloid fibers, having non-native functional polypeptides expressed thereon and connected thereto by a linker domain optimized for functioning of the non-native functional polypeptides.
  • the linker domain is optimized for functioning of a CsgA protein as is it assembled into an amyloid state and for functioning of the functional polypeptide.
  • Exemplary biofilms may include living bacterial cells, non-living bacterial cells or combinations of living bacterial cells and non-living bacterial cells.
  • Exemplary bacteria as described herein include E. coli .
  • Exemplary bacteria as described herein include non-pathogenic bacteria.
  • Exemplary bacteria as described herein include Nissle strain 1917 (EcN), MG1655, K12-derived strains, such as LSR10 and PHL628.
  • Exemplary bacteria as described herein include bacteria that have been genetically modified to remove the nucleic acid sequence or nucleic acid sequences encoding the CsgA protein, i.e. the CsgA gene.
  • Exemplary bacteria as described herein include bacteria that have been genetically modified to include a genomic deletion of the nucleic acid sequence or nucleic acid sequences encoding the CsgA protein, i.e., the CsgA gene.
  • Exemplary bacteria as described herein are useful in the methods also described herein, such as the therapeutic or diagnostics methods described herein.
  • a functional polypeptide is linked to the CsgA protein by a linker to form a CsgA-linker-functional polypeptide structure.
  • one or more nucleic acid sequences which encode for the CsgA-linker-functional polypeptide structure are included in a bacteria cell or the bacteria cell is genetically modified to include one or more nucleic acid sequences which encode for the CsgA-linker-functional polypeptide structure.
  • the one or more nucleic acid sequences which may be foreign nucleic acid sequences, are inserted into the bacteria using methods known to those of skill in the art and the bacteria expresses the CsgA-linker-functional polypeptide structure.
  • a naturally occurring bacteria is modified to include one or more foreign nucleic acid sequences thereby resulting in a non-naturally occurring bacteria.
  • the non-naturally occurring CsgA-linker-functional polypeptide structure is secreted and assembled to produce an amyloid nanofiber network which includes the functional polypeptide.
  • the functional polypeptide is on the surface of the amyloid nanofiber network and provides the amyloid nanofiber network with the property or characteristic of the functional polypeptide.
  • Functional polypeptides of various lengths, secondary structures, properties, characteristics, functions, and the like are envisioned as being attached to the CsgA protein while still allowing amyloid formation and while imparting the properties, characteristics, functions of the functional polypeptide to the biofilm.
  • an engineered bacteria is provided as is an engineered CsgA protein insofar as the CsgA protein includes the linker and the functional polypeptide and the engineered bacteria includes one or more nucleic acid sequences encoding the CsgA-linker-functional polypeptide structure.
  • the engineered bacteria may or may not include a genomic deletion of the natural CsgA gene as described above. According to this aspect, both the engineered bacteria and the engineered CsgA protein are non-naturally occurring.
  • the CsgA may have attached thereto two or more linkers to each of which is attached a functional polypeptide or functional group.
  • the amyloid network may include two or more CsgA species, each attached to its own linker.
  • a CsgA protein may have attached thereto two or more or a plurality of linkers each of which may or may not have a functional polypeptide or functional group attached thereto to provide a CsgA protein with a two or more or a plurality of functional polypeptides or functional groups attached thereto.
  • Each functional polypeptide or functional group may be the same or different.
  • one functional polypeptide or group attached to the CsgA protein via a first linker may be a cell specific binding functional polypeptide or functional group and a different functional polypeptide or functional group attached to a different CsgA via a second linker may be a therapeutic or diagnostic functional polypeptide or functional group.
  • the assembled hybrid amyloid fiber, composed of the two CsgA variants can target a particular tissue or cell type and also deliver a therapeutic or diagnostic agent to the tissue or cell type.
  • Linkers may be attached at any location of the CsgA protein as is feasible including the N-terminus, the C-terminus or at locations in between the N-terminus and the C-terminus.
  • two or more or a plurality of linker domains may be attached in series with a functional polypeptide or group attached to each linker, thereby providing two or more or a plurality of functional polypeptides or groups to the CsgA protein.
  • methods described herein include the proliferation of the bacteria cell or bacterial cells to create a population of bacteria cells that expression the foreign nucleic acid to create curli fibers and a biofilm including the functional polypeptide.
  • aspects according to the present disclosure include administering the bacteria as described herein to an organism for therapeutic or diagnostic purposes.
  • An organism includes a mammal, such as a human or a non-human mammal.
  • FIG. 1 depicts results of a Congo-Red plate assay where the presence of extracellular amyloid fibers is shown by red staining of the culture spots. The data was obtained from a LSR10 strain.
  • FIG. 2 depicts results of a whole-cell immunodotblot analysis for FLAG tag functionality.
  • Accessible FLAG epitopes on CsgA-FLAG chimeras with different linker domains were probed using anti-FLAG antibodies conjugated to fluorescent DyLight 680.
  • the data was obtained from a LSR10 strain.
  • FIG. 3 depicts in schematic a strategy for optimized linker design showing the effect of optimized linker on functional polypeptide (“material”) performance.
  • Unoptimized linker domains may hinder the performance of the biofilm-based material by occluding binding sites for the functional domains, preventing proper folding of either domain, hindering assembly of the amyloid fibers, or hindering secretion.
  • Optimized linkers minimize these issues, thereby improving the desired function of the material overall.
  • FIG. 4 is a graph depicting expression of curli nanofibers fused with trefoil factor proteins determined by a CR absorbance assay for various CsgA-TFF constructs with various length flexible linker domains. The data was obtained from a PHL628 strain.
  • FIG. 5 is a graph depicting expression of curli nanofibers fused with gut binding short peptide domains using a Congo red binding assay of wild type CsgA compared to CsgA fused to four different peptides that have bene shown to bind to specific gut tissues. The data was obtained from a PHL628 strain.
  • FIG. 6 is a graph depicting adhesion of LSR10 cells harboring gut-binding domains to Caco-2 cells where constructs with the F48 linker and containing trefoil domains TTF1, TTF2, and TTF3, or small peptides T18, A1, CP15 and P8 were tested for in vitro binding to Caco-2 monolayers. Bound cells were recovered and quantified by CFU analysis.
  • FIG. 7 is a graph depicting adhesion of PHL628 cells producing engineered curli fibers to Caco-2 cell monolayers. When no curli is produced, adhesion decreases significantly, suggesting that the wild type curli fibers may play a role in adhesion to epithelial surfaces. TTF1 and TTF3 increase adhesion to the epithelial surface.
  • FIG. 8A-8D depict construction of a construction of a csgA deletion mutant of Nissle 1917.
  • FIG. 8A depicts a Lambda red recombination strategy for the deletion of the csgA gene in Nissle.
  • FIG. 8B depicts PCR validation of the chloramphenicol cassette insertion at the csgA locus identifies two positive clones (black arrows).
  • FIG. 8C depicts these two clones were verified for the presence of the 265, 158, and 1113 bp amplicons.
  • FIG. 8D depicts sequencing verification of the regions flanking the csgA gene indicates successful CAT cassette integration (highlighted in yellow) (SEQ ID NO:2).
  • FIG. 9 is a graph depicting adhesion of Nissle PBP17 harboring gut-binding TFFs to Caco-2 cells. Constructs with the F48 linker and containing trefoil domains (TFF1, TFF2, and TFF3) were expressed in a Nissle ⁇ csgA mutant (PBP17) and tested for in vitro binding to Caco-2 monolayers. Bound cells were recovered and quantified by CFU analysis and the data is presented as the percent bound compared to the initial inoculum.
  • FIG. 10 depicts a diagram of CsgA-linker-AMP constructs to be synthesized and screened.
  • Sec is a periplasmic secretion tag that is cleaved after transport.
  • N22 is the outer membrane secretion tag.
  • CsgA is the amyloidogenic region of the protein.
  • the total panel size will be 9 members, with fused domains ranging from 22-28 amino acids.
  • aspects of the present disclosure are directed to structures having a self-assembling domain and a variable functional domain interconnected by a linker domain.
  • the self-assembling domain is a bacterial matrix protein.
  • the self-assembling domain is an amyloidogenic protein.
  • the self-assembling domain is the amyloidogenic protein, CsgA.
  • CsgA amyloidogenic protein
  • CsgA as distinguished from an engineered CsgA polypeptide refers to the major structural subunit of curli.
  • the sequences of CsgA and its homologs are known in a number of species, e.g. the sequence of E. coli CsgA is known (NCBI Gene ID NO: 949055; SEQ ID NO: 1 (polypeptide)).
  • CsgA refers to E. coli CsgA.
  • CsgA refers to a polypeptide having at least 80% homology to SEQ ID NO: 1 (e.g. 80% or greater homology, 90% or greater homology, or 95% or greater homology), e.g. naturally occurring mutations or variants of CsgA, homologs of CsgA, or engineered mutations or variants of CsgA.
  • an “engineered CsgA polypeptide” refers to a CsgA polypeptide comprising a linker and a functional polypeptide attached to the CsgA at either the C-terminus or the N-terminus or both, but without interrupting the sequence of the CsgA polypeptide.
  • structures according to the present disclosure include a CsgA protein, a linker and a functional polypeptide.
  • a CsgA protein is attached to a functional polypeptide by a linker.
  • the functional polypeptide is a heterologous peptide or protein domain.
  • the functional polypeptide is a heterologous peptide or protein domain that is foreign to the bacterial cell which will express the heterologous peptide or protein domain.
  • the CsgA protein, linker and functional polypeptide are attached in series, for example, having the structure CsgA-linker-functional polypeptide.
  • the CsgA protein and the functional polypeptide each exhibit functionality while bound together through the linker.
  • the combined length of the linker and functional polypeptide can be within the range of 10-500 amino acids, 10-450 amino acids, 10-400 amino acids, 10-350 amino acids or 10-300 amino acids.
  • bacteria are modified to include a nucleic acid encoding the CsgA-linker-functional polypeptide structure.
  • Methods of introducing a nucleic acid to a bacteria cell are known to those of skill in the art.
  • the modified bacteria secrete the CsgA-linker-functional polypeptide structure which results in curli fiber production followed by biofilm formation.
  • the CsgA, linker and functional protein structure are produced by engineered or non-naturally occurring bacteria and the CsgA and functional protein exhibit proper folding and exhibit functionality.
  • methods are provided for engineering a bacteria to produce a CsgA-linker-functional polypeptide structure which is exported from the bacteria and assembled into extracellular amyloid fibers.
  • the CsgA is nucleated to form an amyloid at the cell surface, and then continues to polymerize into long fibers that eventually encapsulate the cells and provide the biofilm with structural support. Attached to each CsgA is the linker and the functional polypeptide as a fusion. The structure is secreted and the functional polypeptide is displayed on the surface of the extracellular amyloid network.
  • the domains are chosen such that they may have functions that can alter or enhance the properties of the biofilm as a whole and the linkers are chosen to allow the domains to have their particular functions.
  • Functional polypeptides within the scope of the present disclosure include peptides or proteins having a desired function. Such functions include catalytic function, recognition function or structural function. Exemplary functional polypeptides include targeting domains. Exemplary functional polypeptides include therapeutic polypeptides. Exemplary functional polypeptides include diagnostic polypeptides. Exemplary functional polypeptides include anticancer polypeptides. Exemplary functional polypeptides include antimicrobial polypeptides. Exemplary functional polypeptides include anti-inflammatory polypeptides. Exemplary functional polypeptides include polymer binding polypeptides. Exemplary functional polypeptides include metabolite binding polypeptides. Exemplary functional polypeptides include targeting polypeptides.
  • Exemplary functional polypeptides include functional polypeptides that bind to tissues or cells or substrates. For example, by appending a domain with known steel binding capabilities to CsgA, a biofilm is produced with the ability to adhere to steel surfaces, whereas the wild-type biofilm does not have this capability.
  • Exemplary functional polypeptides include a first member of a known binding pair. When expressed, the first member of the binding pair is available for binding to a second member of the binding pair which may have attached to it a functional polypeptide, such as for therapeutic or diagnostic purposes. In this manner, the functional polypeptide with the second member of the binding pair may be contacted to the biofilm to add the functional polypeptide to the biofilm, such as to provide the biofilm with the characteristic of the functional polypeptide.
  • Exemplary functional polypeptides may be those to which a functional group may be covalently attached either directly or through a linker.
  • a functional group may be covalently attached either directly or through a linker.
  • CsgA a peptide capable of undergoing spontaneous covalent modification
  • a biofilm whose surface can be modified with any protein or compound of interest can be created by subsequent addition of the protein or compound of interest.
  • Exemplary therapeutic polypeptides include engineered polypeptides with therapeutic function, polypeptides with anti-inflammatory bioactivity (trefoil factors—e.g. TFF1-3, interleukins—e.g. IL-10, other anti-inflammatory cytokines, anti-TNF ⁇ factors), polypeptides with anti-microbial bioactivity (e.g. coprisin, cathelicidin, LL-37, thuricin CD, lantibiotics), polypeptides with anti-cancer bioactivity (growth inhibiting biologics).
  • trefoil factors e.g. TFF1-3, interleukins—e.g. IL-10, other anti-inflammatory cytokines, anti-TNF ⁇ factors
  • polypeptides with anti-microbial bioactivity e.g. coprisin, cathelicidin, LL-37, thuricin CD, lantibiotics
  • polypeptides with anti-cancer bioactivity growth inhibiting biologics.
  • Exemplary diagnostic polypeptides include those known to those of skill in the art and identified by literature search.
  • anticancer polypeptides include polypeptides with anti-cancer bioactivity (growth inhibiting biologics) and anticancer polypeptides include those known to those of skill in the art and identified by literature search.
  • antimicrobial polypeptides include coprisin, cathelicidin, LL-37, thuricin CD, lantibiotics and antimicrobial polypeptides known to those of skill in the art and identified by literature search.
  • anti-inflammatory polypeptides include trefoil factors—e.g. TFF1-3, interleukins—e.g. IL-10, other anti-inflammatory cytokines, anti-TNF ⁇ factors and anti-inflammatory polypeptides known to those of skill in the art and identified by literature search.
  • trefoil factors e.g. TFF1-3
  • interleukins e.g. IL-10
  • other anti-inflammatory cytokines e.g. IL-10
  • anti-TNF ⁇ factors anti-inflammatory polypeptides known to those of skill in the art and identified by literature search.
  • Exemplary polymer binding polypeptides include those known to those of skill in the art and identified by literature search.
  • Exemplary metabolite binding polypeptides include those known to those of skill in the art and identified by literature search.
  • Exemplary targeting polypeptides include those known to those of skill in the art and identified by literature search.
  • tissue-binding polypeptides include T18, CP15 and those known to those of skill in the art and identified by literature search.
  • Exemplary cell-binding polypeptides include T18, CP15 and those known to those of skill in the art and identified by literature search.
  • Exemplary polypeptides that are a first pair of a binding pair of molecules include coiled-coil domains such as SynZips, Trp-Zip domains, affinity tags such as FLAG, and the like and those known to those of skill in the art and identified by literature search
  • Linkers within the scope of the present disclosure are characterized in terms of amino acid content, length, rigidity and secondary structure. Linkers within the scope of the present disclosure separate the amyloid domain and the functional polypeptide domain and allow proper folding and functioning of each domain. In this manner, a linker can be tailored to the particular amyloid domain and the particular functional polypeptide domain. According to one aspect, functional independence of the structural (i.e., CsgA) and fused (heterologous) domains is maximized by a suitable linker to limit steric interference between domains during the export and assembly processes of the bacterial cell. According to one aspect, longer and more flexible linkers of the type (GGGS) n (SEQ ID NO:4) are exemplary. According to an additional aspect, cell stress is minimized by limiting the overall length of the fusion protein. Longer linker sequences and higher induction levels stress the biosynthetic machinery of the cells, inhibiting cell growth and leading to cell lysis in extreme cases.
  • Linkers within the scope of the present disclosure facilitate functioning of the CsgA domain and the functional peptide domain.
  • Linkers within the scope of the present disclosure allow efficient protein processing and export through the bacterial curli secretion machinery as well as provide the proper spatial and physicochemical separation of the amyloid and functional domains to retain their respective functions.
  • Linkers within the scope of the present disclosure include amino acid residues.
  • the amino acid residues may be any of the naturally occurring amino acid residues.
  • Amino acid residues may also be synthetic amino acids known to those of skill in the art.
  • Representative amino acids which may be used in linkers include Glycine, Alanine, Valine, Leucine, Isoleucine, Serine, Cysteine, Selenocysteine, Threonine, Methionine, Proline, Phenylalanine, Tyrosine, Tryptophan, Histidine, Lysine, Arginine, Aspartate, Glutamate, Asparagine, and Glutamine.
  • Linkers within the scope of the present disclosure include a cleavage site.
  • Cleavage sites and enzymes for cleavage are known to those of skill in the art.
  • the functional polypeptide may be cleaved from the linker and released into the surrounding environment, for example for therapeutic or diagnostic purposes.
  • Exemplary enzymes include those from the family of matrix metalloproteinases (MMPs), which have their own recognition sequences, proteases secreted by pathogens such as CD2830 from C. difficile and the like.
  • MMPs matrix metalloproteinases
  • CsgA within the scope of the present disclosure includes an amyloid domain which self-assembles into an amyloid structure.
  • a linker may be attached to either the C terminus or the N-terminus or separate linkers may be attached to both the C terminus and the N terminus.
  • the linker length can be any length which may be expressed from a cell, such as a bacterial cell when linking a CsgA protein and a functional polypeptide.
  • the functional polypeptide length can be any length which may be expressed from a cell, such as a bacterial cell when linked to a CsgA protein by a linker.
  • the combination of a linker and functional polypeptide includes no more than 500 amino acids. In some embodiments, the combination of a linker and functional polypeptide includes no more than 400 amino acids. In some embodiments, the combination of a linker and functional polypeptide includes no more than 300 amino acids.
  • the combination of a linker and functional polypeptide includes no more than 200 amino acids. In some embodiments, the combination of a linker and functional polypeptide includes no more than 100 amino acids. In some embodiments, the combination of a linker and functional polypeptide includes no more than 50 amino acids. In some embodiments, the combination of a linker and functional polypeptide includes no more than 40 amino acids. In some embodiments, the combination of a linker and functional polypeptide includes no more than 30 amino acids.
  • a linker sequence is a polypeptide sequence of at least 7, 8, 9, 10, 11, 12, 24, 48 or more amino acids. In some embodiments, the linker sequence comprises from about 7 amino acids to about 250 amino acids. In some embodiments, the linker sequence comprises from about 7 amino acids to about 200 amino acids. In some embodiments, the linker sequence comprises from about 7 amino acids to about 150 amino acids. In some embodiments, the linker sequence comprises from about 7 amino acids to about 100 amino acids. In some embodiments, the linker sequence comprises from about 12 amino acids to about 250 amino acids. In some embodiments, the linker sequence comprises from about 12 amino acids to about 200 amino acids. In some embodiments, the linker sequence comprises from about 12 amino acids to about 150 amino acids.
  • the linker sequence comprises from about 12 amino acids to about 100 amino acids. In some embodiments, the linker sequence comprises from about 24 amino acids to about 100 amino acids. In some embodiments, the linker sequence comprises from about 48 amino acids to about 250 amino acids. In some embodiments, the linker sequence comprises from about 48 amino acids to about 200 amino acids. In some embodiments, the linker sequence comprises from about 48 amino acids to about 150 amino acids. In some embodiments, the linker sequence comprises from about 48 amino acids to about 100 amino acids. In some embodiments, the linker sequence comprises from about 7 amino acids to about 30 amino acids. In some embodiments, the linker sequence comprises from about 20 amino acids to about 50 amino acids. In some embodiments, the linker sequence comprises from about 30 amino acids to about 50 amino acids.
  • the linker sequence comprises from about 40 amino acids to about 50 amino acids. In some embodiments, the linker sequence comprises from about 6 amino acids to about 20 amino acids. In some embodiments, the linker sequence comprises from about 7 to about 10 amino acids. In some embodiments, the linker sequence comprises a flexible polypeptide, e.g a polypeptide not having a rigid secondary and/or tertiary structure. In some embodiments, the linker sequence comprises glycine and serine residues. In some embodiments at least 50% of the amino acids comprised by the linker sequence are glycine or serine residues, e.g. at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more are glycine or serine residues. In some embodiments, the linker sequence consists of glycine and serine residues.
  • a functional polypeptide includes a polypeptide having an activity or function, such that when it is present in a biofilm, it confers upon the biofilm a property, function, or activity which it did not have in the absence of the activity of the polypeptide.
  • an activity polypeptide can be, e.g. an enzyme, a polypeptide that binds another molecule, a binding domain, a peptide that is bound by another molecule (e.g. a ligand or epitope), or the like.
  • polypeptides for use as activity polypeptides include, but are not limited to Metal binding domain (MBD); SpyTag; graphene binding (GBP); carbon nanotube binding (CBP); gold binding (A3); CT43; FLAG; Z8; E14; QBP1; CLP12; and AFP8.
  • MBD Metal binding domain
  • GBP graphene binding
  • CBP carbon nanotube binding
  • Au3 gold binding
  • the functional polypeptide when present as part of an engineered CsgA polypeptide is functional.
  • a polypeptide is said to be “functional” or expressed as a “functional” polypeptide if the polypeptide retains at least about 50% of the activity (e.g. enzymatic activity or binding activity) that it has as an isolated polypeptide.
  • One of skill in the art can readily detect increases in reaction products and/or detect decreases in reaction substrates, e.g. by mass spectroscopy (MS, including, e.g., MADLI/TOF, SELDI/TOF, LC-MS, GC-MS, HPLC-MS, etc., among others) or detect increases or decrease in binding to a binding partner, e.g.
  • MS mass spectroscopy
  • a functional activity polypeptide can retain at least 50% of the activity of the isolated polypeptide, e.g. 50% or more of the activity, 60% or more of the activity, 75% or more of the activity, or 90% or more of the activity of the isolated polypeptide.
  • the functional polypeptide can be a conjugation domain.
  • Such embodiments can permit immobilization of target proteins in the biofilm, e.g., when the target protein is too large to be expressed as a fusion with CsgA.
  • the conjugation domain present on the engineered CsgA polypeptide can specifically bind to a partner conjugation domain present as part of the target protein, thereby incorporating the target protein into the biofilm.
  • Such conjugation domains are also referred to herein as a first member of a binding pair of molecules and a second member of a pair of binding pair of molecules.
  • conjugation domain includes a polypeptide that can specifically bind to and/or be specifically bound by a partner conjugation domain, e.g.
  • a conjugation domain can be, e.g., about 100 amino acids or less in size, about 75 amino acids or less in size, about 50 amino acids or less in size, about 40 amino acids or less in size or smaller.
  • a partner conjugation domain can be about the same size as the conjugation domain or larger, e.g., a partner conjugation domain can be about 4000 amino acids or less in size, about 3000 amino acids or less in size, about 2000 amino acids or less in size, about 1000 amino acids or less in size, about 500 amino acids or less in size, about 200 amino acids or less in size, about 100 amino acids or less in size, about 75 amino acids or less in size, about 50 amino acids or less in size, about 40 amino acids or less in size, or smaller.
  • the binding of the conjugation domain and partner conjugation domain is covalent.
  • conjugation domains are known in the art and include, but are not limited to, SpyTag; biotin acceptor peptide (BAP); biotin carboxyl carrier protein (BCCP); and a peptide comprising a LPXTG (SEQ ID NO:30) motif.
  • partner conjugation domains are known in the art and include but are not limited to, respectively, SpyCatcher, streptavidin; streptavidin; and peptides comprising aminoglycine. Further discussion of conjugation systems comprising a conjugation domain and a partner conjugation domain can be found, e.g., in Mao et al.
  • the target polypeptide comprising the partner conjugation domain can further comprise a functional agent.
  • the functional agent has an activity or function, such that when it is present in a biofilm, it confers upon the biofilm a property, function, or activity which it did not have in the absence of the polypeptide.
  • a functional agent can be of any size and is not part of the engineered CsgA polypeptide.
  • Exemplary functional agents include, e.g. an enzyme, a polypeptide that binds another molecule, an antibody, a therapeutic agent, a diagnostic agent, a metal, an antimicrobial agent, an anti-inflammatory agent, an anticancer agent or the like.
  • a polypeptide comprising a functionalizing polypeptide and a conjugation domain can further comprise an extracellular localization tag, e.g. a sequence which will cause a cell expressing the polypeptide to secrete the polypeptide.
  • a functionalized engineered CsgA polypeptide or functionalized biofilm can be provided by contacting an engineered CsgA polypeptide comprising a conjugation domain (or a cell and/or biofilm comprising that polypeptide) with a polypeptide comprising the partner conjugation domain.
  • the engineered CsgA polypeptide and the polypeptide comprising the partner conjugation domain are maintained in contact for a period of time, i.e. the “binding step.”
  • the binding step is followed by a washing step, e.g. to remove excess unbound polypeptide.
  • an engineered CsgA polypeptide comprising a conjugation domain is bound to (or binds) the partner conjugation domain in the presence of albumin (i.e. the “binding step”).
  • the albumin is BSA.
  • the albumin is present at about 0.1% to about 10%.
  • the albumin is present at about 0.5% to about 5%.
  • the albumin is present at about 1% to about 2%.
  • the binding step is allowed to proceed for at least about 2 hours, e.g. about 2 hours or more, about 6 hours or more, about 12 hours or more, or about 24 hours or more. In some embodiments, the binding step is allowed to proceed in the presence of albumin.
  • the washing step proceeds for about 10 minutes to about 6 hours. In some embodiments, the washing step proceeds for about 30 minutes to about 3 hours. In some embodiments, the washing step proceeds for about 90 minutes.
  • the polypeptides are agitated (e.g. shaken) during the washing step.
  • the washing step comprises washing the polypeptides in a solution of albumin.
  • the albumin is BSA. In some embodiments, the albumin is present at about 0.01% to about 3%. In some embodiments, the albumin is present at about 0.1% to about 1%. In some embodiments, the albumin is present at about 0.3%. In some embodiments, the washing step comprises 2 or more successive washes. In some embodiments, the washing step comprises 3 successive washes.
  • Specific binding includes a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target.
  • specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity.
  • a reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.
  • a vector comprising a nucleic acid sequence encoding an engineered CsgA polypeptide as described herein.
  • a “vector” includes a nucleic acid construct designed for delivery to a host cell or transfer between different host cells.
  • a vector can be viral or non-viral. Many vectors useful for transferring genes into target cells are available, e.g. the vectors may be episomal, e.g., plasmids, virus derived vectors or may be integrated into the target cell genome, through homologous recombination or random integration.
  • a vector can be an expression vector.
  • An “expression vector” can be a vector that has the ability to incorporate and express heterologous nucleic acid fragments in a cell.
  • An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms.
  • the nucleic acid incorporated into the vector can be operatively linked to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that polynucleotide sequence.
  • a nucleic acid encoding an engineered CsgA polypeptide can be present within a portion of a plasmid.
  • Plasmid vectors can include, but are not limited to, pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/ ⁇ or KS +/ ⁇ (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see Studier et. al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technology, vol. 185 (1990), which is hereby incorporated by reference in its entirety).
  • a “viral vector” may be a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle.
  • the viral vector can contain a transgenic gene in place of non-essential viral genes.
  • the vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo.
  • Numerous viral vectors are known in the art and can be used as carriers of a nucleic acid into a cell, e.g. lambda vector system gill, gt WES.tB, Charon 4.
  • the nucleic acid encoding an engineered CsgA polypeptide can be constitutively expressed. In some embodiments, the nucleic acid encoding an engineered CsgA polypeptide can be operably linked to a constitutive promoter. In some embodiments, the nucleic acid encoding an engineered CsgA polypeptide can be inducibly expressed. In some embodiments, the nucleic acid encoding an engineered CsgA polypeptide can be operably linked to an inducible promoter. In some embodiments, the nucleic acid encoding an engineered CsgA polypeptide can be operably linked to a native CsgA promoter.
  • an “inducible promoter” may be one that is characterized by initiating or enhancing transcriptional activity when in the presence of, influenced by, or contacted by an inducer or inducing agent than when not in the presence of, under the influence of, or in contact with the inducer or inducing agent.
  • An “inducer” or “inducing agent” may be endogenous, or a normally exogenous compound or protein that is administered in such a way as to be active in inducing transcriptional activity from the inducible promoter.
  • the inducer or inducing agent e.g., a chemical, a compound or a protein
  • can itself be the result of transcription or expression of a nucleic acid sequence e.g., an inducer can be a transcriptional repressor protein
  • an inducer can be a transcriptional repressor protein
  • inducible promoters include but are not limited to, the lac operon promoter, a nitrogen-sensitive promoter, an IPTG-inducible promoter, a salt-inducible promoter, and tetracycline, steroid-responsive promoters, rapamycin responsive promoters and the like.
  • Inducible promoters for use in prokaryotic systems are well known in the art, see, e.g. the beta.-lactamase and lactose promoter systems (Chang et al., Nature, 275: 615 (1978, which is incorporated herein by reference); Goeddel et al., Nature, 281: 544 (1979), which is incorporated herein by reference), the arabinose promoter system, including the araBAD promoter (Guzman et al., J. Bacteriol., 174: 7716-7728 (1992), which is incorporated herein by reference; Guzman et al., J.
  • An inducible promoter useful in the methods and systems as disclosed herein can be induced by one or more physiological conditions, such as changes in pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, and the concentration of one or more extrinsic or intrinsic inducing agents.
  • the extrinsic inducer or inducing agent may comprise amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs, hormones, and combinations thereof.
  • the inducible promoter is activated or repressed in response to a change of an environmental condition, such as the change in concentration of a chemical, metal, temperature, radiation, nutrient or change in pH.
  • an inducible promoter useful in the methods and systems as disclosed herein can be a phage inducible promoter, nutrient inducible promoter, temperature inducible promoter, radiation inducible promoter, metal inducible promoter, hormone inducible promoter, steroid inducible promoter, and/or hybrids and combinations thereof.
  • Appropriate environmental inducers can include, but are not limited to, exposure to heat (i.e., thermal pulses or constant heat exposure), various steroidal compounds, divalent cations (including Cu2+ and Zn2+), galactose, tetracycline, IPTG (isopropyl- ⁇ -D thiogalactoside), as well as other naturally occurring and synthetic inducing agents and gratuitous inducers.
  • Inducible promoters useful in the methods and systems as disclosed herein also include those that are repressed by “transcriptional repressors” that are subject to inactivation by the action of environmental, external agents, or the product of another gene. Such inducible promoters may also be termed “repressible promoters” where it is required to distinguish between other types of promoters in a given module or component of the biological switch converters described herein. Preferred repressors for use in the present invention are sensitive to inactivation by physiologically benign agent.
  • a lac repressor protein is used to control the expression of a promoter sequence that has been engineered to contain a lacO operator sequence
  • treatment of the host cell with IPTG will cause the dissociation of the lac repressor from the engineered promoter containing a lacO operator sequence and allow transcription to occur.
  • a tet repressor is used to control the expression of a promoter sequence that has been engineered to contain a tetO operator sequence
  • treatment of the host cell with tetracycline will cause the dissociation of the tet repressor from the engineered promoter and allow transcription of the sequence downstream of the engineered promoter to occur.
  • an engineered microbial cell comprising an engineered CsgA polypeptide and/or comprising a vector or nucleic acid encoding such a polypeptide.
  • the engineered CsgA polypeptide can comprise a functional polypeptide comprising a conjugation domain.
  • a cell encoding and/or comprising an engineered CsgA polypeptide can comprise an activity polypeptide comprising a conjugation domain can further encode and/or comprise a second engineered polypeptide comprising a partner conjugation domain and a functionalizing polypeptide.
  • described herein is a population of cells comprising two cell types, the first cell type encoding and/or comprising an engineered CsgA polypeptide comprising an activity polypeptide comprising a conjugation domain and the second cell type encoding and/or comprising a second engineered polypeptide comprising a partner conjugation domain and a functionalizing polypeptide.
  • a single cell can comprise a CsgA polypeptide with a conjugation domain and also comprise the polypeptide which will bind to and/or be bound by that CsgA polypeptide or that a first cell can comprise a CsgA polypeptide with a conjugation domain and a second cell can comprise the polypeptide which will bind to and/or be bound by that CsgA polypeptide.
  • an engineered CsgA polypeptide with a conjugation domain can be contacted with a second polypeptide comprising a partner conjugation domain and a functionalizing polypeptide, e.g. the second polypeptide can be produced (e.g.
  • a bacterial cell of the methods and compositions described herein can be any of any species.
  • the bacterial cells are of a species and/or strain which is amenable to culture and genetic manipulation.
  • the bacterial cell can be a gram-positive bacterial cell.
  • the bacterial cell can be a gram-negative bacterial cell.
  • the parental strain of the bacterial cell of the technology described herein can be a strain optimized for protein expression.
  • Non-limiting examples of bacterial species and strains suitable for use in the present technologies include Escherichia coli, E. coli BL21, E. coli Tuner, E. coli Rosetta, E. coli JM101, and derivatives of any of the foregoing.
  • Bacterial strains for protein expression are commercially available, e.g. EXPRESSTM Competent E. coli (Cat. No. C2523; New England Biosciences; Ipswich, Mass.).
  • the cell is an E. coli cell.
  • the nucleic acid encoding an engineered CsgA polypeptide is comprised by a cell expressing wild-type CsgA. In some embodiments, the nucleic acid encoding an engineered CsgA polypeptide is comprised by a cell with a mutation and/or deletion of the wild-type CsgA gene, e.g. such that the cell does not express wild-type CsgA. In some embodiments, the nucleic acid encoding an engineered CsgA polypeptide is introduced into a cell by homologous recombination, e.g. such that the nucleic acid encoding an engineered CsgA polypeptide replaces the wild-type CsgA gene in the cell.
  • a biofilm comprising an engineered microbial cell comprising one or more engineered CsgA polypeptide and/or comprising a vector or nucleic acid encoding such a polypeptides.
  • a “biofilm” refers to a mass of microorganisms which can adhere or is adhering to a surface.
  • a biofilm comprises a matrix of extracellular polymeric substances, including, but not limited to extracellular DNA, proteins, glyopeptides, and polysaccharides.
  • the nature of a biofilm such as its structure and composition, can depend on the particular species of bacteria present in the biofilm. Bacteria present in a biofilm are commonly genetically or phenotypically different than corresponding bacteria not in a biofilm, such as isolated bacteria or bacteria in a colony.
  • the technology described herein relates to a biofilm that is produced by culturing an engineered microbial cell comprising an engineered CsgA polypeptide (and/or comprising a vector or nucleic acid encoding such a polypeptide) under conditions suitable for the production of a biofilm.
  • Conditions suitable for the production of a biofilm can include, but are not limited to, conditions under which the microbial cell is capable of logarithmic growth and/or polypeptide synthesis. Conditions may vary depending upon the species and strain of microbial cell selected. Conditions for the culture of microbial cells are well known in the art. Biofilm production can also be induced and/or enhanced by methods well known in the art, e.g.
  • conditions suitable for the production of a biofilm can also include conditions which increase the expression and secretion of CsgA, e.g. by exogenously expressing CsgD.
  • the biofilm can comprise the cell which produced the biofilm.
  • described herein is a composition comprising an engineered CsgA polypeptide as described herein.
  • CsgA units When expressed by a cell capable of forming curli, e.g. a cell expressing CsgA, CsgB, CsgC, CsgD, CsgE, CsgF, and CsgG or some subset thereof, CsgA units will be assembled to form curli filaments, e.g. polymeric chains of CsgA.
  • filaments of the polypeptide can be present in the composition.
  • the filaments can be part of a proteinaceous network, e.g. multiple filaments which can be, e.g. interwoven, overlapping, and/or in contact with each other.
  • the proteinaceous network can comprise additional biofilm components, e.g. materials typically found in an E.
  • biofilm components can include biofilm proteins (e.g. FimA, FimH, Ag43, AidA, and/or TibA) and/or non-proteinaceous biofilm components (e.g. cellulose, PGA and/or colonic acid).
  • the composition can further comprise an engineered microbial cell comprising an engineered CsgA polypeptide and/or comprising a vector or nucleic acid encoding such a polypeptide.
  • a cell, composition, or biofilm comprising an engineered CsgA polypeptide (and/or comprising a vector or nucleic acid encoding such a polypeptide) to display a polypeptide, e.g. within the biofilm, within the composition, and/or on the cell surface.
  • display refers to expressing the polypeptide (e.g. as an activity polypeptide) in such a manner that it can come in contact with the extracellular environment.
  • a displayed polypeptide can be capable of binding with a binding partner, catalyzing an enzymatic reaction, and/or performing any other activity which it would perform as an isolated polypeptide.
  • a polypeptide displayed within a biofilm will retain more activity than a soluble version of that polypeptide. It is contemplated herein that a polypeptide displayed within a biofilm (e.g. an activity polypeptide and/or functionalizing polypeptide) will retain more activity than a soluble version of that polypeptide when exposed to activity degrading conditions such as, e.g., high or low pH, organic solvents, desiccation, high or low temperature, radiation, etc.
  • a cell, composition, or biofilm comprising an engineered CsgA polypeptide (and/or comprising a vector or nucleic acid encoding such a polypeptide), in an application selected from the group consisting of biocatalysis; industrial biocatalysis; immobilized biocatalysis; chemical production; filtration; isolation of molecules from an aqueous solution; water filtration; bioremediation; nanoparticle synthesis; nanowire synthesis; display of optically active materials; biosensors; surface coating; therapeutic biomaterial; biological scaffold; structural reinforcement of an object; and as a delivery system for therapeutic agents.
  • an application selected from the group consisting of biocatalysis; industrial biocatalysis; immobilized biocatalysis; chemical production; filtration; isolation of molecules from an aqueous solution; water filtration; bioremediation; nanoparticle synthesis; nanowire synthesis; display of optically active materials; biosensors; surface coating; therapeutic biomaterial; biological scaffold; structural reinforcement of an object; and as a delivery system for therapeutic agents.
  • a cell, composition and/or biofilm can comprise multiple different engineered CsgA polypeptides, each of which comprises a different activity polypeptide, e.g. an engineered CsgA polypeptide comprising an enzymatic activity polypeptide and an engineered CsgA polypeptide comprising a binding domain activity polypeptide.
  • a cell, composition, and/or biofilm can comprise 1 or more engineered CsgA polypeptides, e.g. 1, 2, 3, 4, 5, 6, or more engineered CsgA polypeptides.
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • protein and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • protein and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
  • Protein and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • polypeptide proteins and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
  • nucleic acid or “nucleic acid sequence” may be any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof.
  • the nucleic acid can be either single-stranded or double-stranded.
  • a single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA.
  • the nucleic acid can be DNA.
  • nucleic acid can be RNA.
  • Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.
  • a test amyloid chimeric protein library was constructed to test various linker designs between an amyloid domain (CsgA) and a functional peptide domain (FLAG).
  • CsgA amyloid domain
  • FLAG functional peptide domain
  • the CsgA protein is secreted by the bacterium Escherichia coli and the protein then self-assembles into highly robust functional amyloid nanofibers with a diameter of ⁇ 4-7 nm. These amyloid fibers are known as ‘curli’ and exist as extended tangled networks encapsulating the cells.
  • the FLAG domain is an octapeptide polypeptide tag used for affinity chromatography and epitope-tagged protein detection.
  • a linker may include from between 7 and 50 amino acids. According to one aspect, a linker may include from between 8 and 50 amino acids. According to one aspect, a linker may include from between 9 and 50 amino acids. According to one aspect, a linker may include from between 10 and 50 amino acids. According to one aspect, a linker may include from between 11 and 50 amino acids. According to one aspect, a linker may include from between 12 and 50 amino acids. According to one aspect, a linker may include from between 15 and 50 amino acids. According to one aspect, a linker may include from between 20 and 50 amino acids. According to one aspect, a linker may include from between 24 and 50 amino acids. According to one aspect, a linker may include from between 45 and 50 amino acids.
  • a linker may be flexible or rigid.
  • a linker may include one or more or a plurality of repeating amino acid subunits.
  • a linker may be hydrophobic or hydrophilic.
  • a linker may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or greater amino acid subunits.
  • a linker may include 2 repeating subunits or more, 3 repeating subunits or more, 4 repeating subunits or more, 5 repeating subunits or more, 6 repeating subunits or more, 7 repeating subunits or more, 8 repeating subunits or more, 9 repeating subunits or more, 10 repeating subunits or more, 11 repeating subunits or more, 12 repeating subunits or more, 13 repeating subunits or more, 14 repeating subunits or more, or 15 repeating subunits or more.
  • a plurality of amino acid subunits results in a flexible linker.
  • a flexible linker may include a linker with a sequence that lacks inherent secondary or tertiary structure in solution.
  • a plurality of amino acid subunits results in a rigid linker.
  • a rigid linker may include a linker with a sequence that has a secondary or tertiary structure that allows it to maintain a defined conformation in solution.
  • GGGS An exemplary amino acid subunit is GGGS (SEQ ID NO:4).
  • a linker has the structure [GGGS] n where n is an integer from 1 to 20 (SEQ ID NO:12). Exemplary values for n include 3, 6, and 12.
  • a linker having a plurality of GGGS (SEQ ID NO:4) subunits is a linker that is flexible in whole or in part.
  • a linker has the structure [P] n where n is an integer from 1 to 30 (SEQ ID NO:13).
  • linkers with repetitive Ps include extended type II trans helices.
  • Exemplary values for n include 12 and 24.
  • a linker having a plurality of P subunits is a linker that is rigid in whole or in part.
  • An exemplary amino acid subunit is an alpha-helix motif such as EAAAK (SEQ ID NO:14).
  • a linker has the structure [EAAAK] n where n is an integer from 1 to 15 (SEQ ID NO:15). Exemplary values for n include 3 and 9.
  • a linker having a plurality of EAAAK (SEQ ID NO:14) subunits is a linker that is rigid in whole or in part and includes a hydrophilic portion and a hydrophobic portion.
  • Exemplary amino acid residues include 12 or more amino acids, 24 or more amino acids or 48 or more amino acids.
  • the amino acids are flexible amino acids.
  • the amino acids are one or more of glycine, serine, alanine or leucine.
  • a corresponding increase in extracellular curli fibers results from increased linker length.
  • a corresponding increase in functional domain accessibility results from increased linker length.
  • Exemplary linkers include:
  • CsgA-linker-FLAG chimeras with different intervening linkers were tested for optimal self-assembly into curli nanofibers.
  • the self-assembly of the protein library with different linkers into curli nanofibers was detected using Congo Red (CR), a standard stain used for the colorimetric detection of amyloids.
  • CR Congo Red
  • E. coli cells expressing fusion proteins that are successfully secreted and are competent for self-assembly into amyloid nanofibers will stain red.
  • all the flexible linkers show some intermediate levels of CR staining, with the longest linker showing the greatest amount.
  • the longer polyproline linker showed no CR staining and the highest amount of CR staining was obtained for the EK3 linker.
  • the CsgA-linker-FLAG structure with a flexible linker demonstrated a monotonic increase in fluorescence as a function of the linker length.
  • the CsgA-linker-FLAG structure with a polyproline rigid linker lacked fluorescence. Without wishing to be bound by scientific theory, these hydrophobic linkers may impede proper functioning of the peptide tag.
  • the CsgA-linker-FLAG structure with an alpha-helical EAAAK (SEQ ID NO:14) linker showed that the EK3 linker construct, which had more CR staining than the EK9 construct (see FIG. 1 ), had lower fluorescence. In contrast, the lower CR-binding EK9 linker construct had a high fluorescent signal, indicating greater accessibility of the FLAG epitope tag. See FIG. 2 .
  • aspects of the present disclosure are directed to methods of optimizing linker design for linking CsgA and a functional polypeptide.
  • a functional polypeptide is selected for linkage with CsgA.
  • a linker design is then selected to form the CsgA-linker-functional polypeptide structure.
  • a bacteria is then modified to include a nucleic acid sequence encoding the CsgA-linker-functional polypeptide structure. The modified bacteria may then be caused to proliferate into a population of bacteria and is assayed for the expression of the CsgA-linker-functional polypeptide structure.
  • the modified bacteria may then be caused to proliferate into a population of bacteria and is assayed for the formation of a biofilm of the CsgA-linker-functional polypeptide structure.
  • the biofilm may be assayed for the functional characteristics of the functional polypeptide.
  • exemplary linkers described herein allow the self-assembly and functional domains of the CsgA-linker-functional polypeptide structure to operate or function without interfering with each other.
  • An exemplary method of determining the effect of a linker on biofilm formation and functional characteristics of the functional polypeptide is depicted in FIG. 3 .
  • Unoptimized linker domains may hinder the performance of the biofilm-based material by occluding binding sites for the functional domains, preventing proper folding of the CsgA or functional polypeptide domains, hindering assembly of amyloid fibers or hindering secretion.
  • Optimized linkers minimize these issues and improve the desired function of the biofilm.
  • Optimized linker domains facilitate the fabrication of biofilm based materials with superior mechanical and biophysical stability under a wide range of conditions (extreme temperatures, extreme pH, in the presence of unfavorable solvents, under exposure to the elements in outdoor applications, etc.).
  • Optimized linker domains enable each functional peptide domain to operate independently, effectively increasing the number of functional peptide domains in the material that are contributing to a desired behavior on the micro- or macro-scale. For example, if the desired function of the biofilm-based material is metal binding, the material may have inhibited stability or metal binding affinity because of molecular crowding or undesirable molecular interactions that result from inappropriate linker domains Optimized linker domains reduce undesirable molecular interactions from occurring, ultimately enhancing the performance of the material.
  • the bacteria is a non-pathogenic bacteria.
  • Exemplary non-pathogenic bacteria include Nissle strain 1917 (EcN), MG1655, K12-derived strains, PHL628, LSR10, LSR6 and the like.
  • Wild-type EcN which is marketed as a probiotic under the trade name Mutaflor, can be delivered orally, survive transit through the upper GI tract, then transiently colonize the ileum and colon for several days after initial administration. During this colonization process, EcN produces curli fibers.
  • Exemplary binding domains include those identified in Table 2 below.
  • Optimal linker Optimal CsgA-peptide domain [IPTG] Temperature Duration construct (SEQ ID NO: 40) (mM) (C.) (h) CsgA-TFF1 48 AA (GGGS) n 0.3 37 24 CsgA-TFF2 24 AA (GGGS) n 0.3 37 24 CsgA-TFF3 24 AA (GGGS) n 0.3 37 24 CsgA-lunasin 36 AA (GGGS) n 0.3 37 24 CsgA-MAM 36 AA (GGGS) n 0.3 37 24 CsgA-T18 48 AA (GGGS) n 0.3 25 48 CsgA-CP15 48 AA (GGGS) n 0.3 25 48 CsgA-P8 48 AA (GGGS) n 0.3 25 48
  • Functional polypeptides include a polypeptide or protein sequence that displays binding affinity with the epithelial surfaces of the gastrointestinal (GI) tract.
  • GI gastrointestinal
  • Exemplary polypeptide or protein sequences are known to those of skill in the art or can be determined from phage display on biological tissues, from sequences that exist in naturally occurring organisms or from sequences that have been engineered in some other way to bind to specific surfaces.
  • exemplary polypeptide or protein sequences having affinity with tissues or cells associate with the GI tract can be determined from a microfluidic system that mimics the structure of a gut epithelium. Such systems may be referred to as a “Gut on a CHIP.” Such systems incorporate flow and cyclic strain motions to mimic peristalsis.
  • Engineered bacteria strains as described herein having gut binding functional groups may be introduced into such a system and the micro-scale localization and residence time in the system may be monitored to determine binding of the cells through expression of the CsgA-linker-functional polypeptide to the system.
  • Such a system is described in Lab Chip, 2012, 12, 2165-2174, hereby incorporated by reference in its entirety.
  • Exemplary polypeptide or protein sequences having affinity with tissues or cells associate with the GI tract can be determined from monitoring or determining spatiotemporal distribution in mouse models.
  • engineered bacteria strains such as engineered Nissle strains
  • Residence time of the engineered strains will be measured by CFU counting from fecal samples collected daily. Spatial localization within the gut will be monitored by harvesting the gut tissue, sectioning, and tracking the presence of engineered strains by CFU counts from homogenized tissues and immune staining of histological tissue slices.
  • TFFs trefoil factors
  • TFF1 contains a single trefoil domain of 60 amino acids.
  • TFF2 contains two homologous trefoil domains, resulting in a total of 6 disulfide bonds, and is over 100 amino acids in length.
  • Various constructs were made containing CsgA fused to the TFF2 via the flexible linkers identified in the preceding experiments.
  • TFF1 single trefoil domain construct
  • longer flexible linker domains resulted in a marked improvement in expression levels as measured by a quantitative Congo Red binding assay ( FIG. 4 ).
  • TFF1 showed nearly a 5-fold increase when the flexible linker length was increased from 24 to 48 residues.
  • TFF2 showed no improvement as a function of linker length, which may be due to the complexity of the protein (6 disulfides) or larger length (>100 amino acids).
  • FIG. 6 shows adhesion of LSR10 cells expressing CsgA constructs with gut-binding domains to Caco-2 cells.
  • FIG. 7 shows adhesion of PHL628 cells expressing CsgA constructs with gut-binding domains to Caco-2 cells.
  • Nissle 1917 which expresses curli nanofibers.
  • the Nissle strain was isolated during WWI from the fecal samples of a soldier who was resistant to infectious enteropathogenic diarrhea. Further studies have shown the Nissle strain to be a profound probiotic, with the ability to protect against gastroinvasive bacteria and to ameliorate other gastrointestinal disorders such as ulcerative colitis, irritable bowel syndrome, and Crohn's disease. Accordingly, aspects of the present disclosure are directed to attaching Nissle E. coli to gut tissue.
  • FIG. 8A-8D shows construction of a CsgA deletion mutant of Nissle 1917.
  • FIG. 8A shows a lambda red recombination strategy for the deletion of the csgA gene in Nissle.
  • FIG. 8B shows PCR validation of the chloramphenicol cassette insertion at the csgA locus identifies two positive clones (black arrows).
  • FIG. 8C shows two clones verified for the presence of the 265, 158, and 113 bp amplicons.
  • FIG. 8D shows sequencing verification of the regions flanking the csgA gene indicates successful CAT cassette integration (highlighted in yellow).
  • an in vitro binding study to Caco-2 cells was performed as described above. Constructs with the F48 linker and containing trefoil domains (TFF1, TFF2, and TFF3) were expressed in a Nissle ⁇ csgA mutant (PBP17) and tested for in vitro binding to Caco-2 monolayers. Bound cells were recovered and quantified by CFU analysis and the data is presented in FIG. 9 as the % of total inoculated bacterial cells that remained adhered to the Caco-2 monolayer.
  • Exemplary structures include
  • aspects of the present disclosure are directed to methods of treating chronic inflammatory diseases of the gut, such as inflammatory bowel disease and Crohn's disease by administering to an individual in need thereof a certain engineered bacterial strain or strains as described herein.
  • the bacterial strain expresses a CsgA-linker-anti-inflammatory polypeptide construct.
  • the bacterial strain expresses the CsgA-linker-anti-inflammatory polypeptide construct and the anti-inflammatory polypeptide treats the chronic inflammatory disease of the gut.
  • the bacterial strain expresses a CsgA-linker-first member of a binding pair polypeptide construct.
  • the second member of the binding pair attached to an anti-inflammatory compound is then administered.
  • the first member of the binding pair and the second member of the binding pair bind thereby localizing the anti-inflammatory compound, for example, to the gut, for the treatment of chronic inflammatory diseases of the gastrointestinal tract.
  • the bacterial strain is a non-pathogenic bacterial strain, such as the Nissle strain or any other non-pathogenic strain known to those of skill in the art such as MG1655, K12-derived strains, and the like.
  • the anti-inflammatory compound can be a protein sequence capable of abrogating inflammatory processes in the gut.
  • Exemplary protein sequences include the trefoil factor family of peptides (TFFs) because they are endogenous signaling molecules in the mammalian gut, have demonstrated efficacy in treating IBD and Crohn's in the clinic, and are under development in their soluble form as biologics for these indications.
  • TNFs trefoil factor family of peptides
  • the anti-inflammatory compound, protein or polypeptide may exert its effects while still attached to the engineered curli fibers.
  • the anti-inflammatory compound, protein or polypeptide may exert its effects as a soluble protein or compound after cleavage from the curli fibers by a protease.
  • proteases in the MMP family MMP2, MMP9 and the like are upregulated during inflammation and may be used to cleave the anti-inflammatory compound, protein or polypeptide from the linker domain.
  • methods are provided to deliver bioactive proteins and peptides locally to the inflamed gut. According to one aspect, methods are provided to deliver bioactive proteins and peptides locally to the inflamed gut in response to specific inflammatory cues.
  • Anti-inflammatory compound, protein or polypeptide may be identified by the exemplary methods described below for TTF domains.
  • Caco-2 intestinal epithelial cells grown in 2D culture are treated with purified curli fibers displaying TFF domains.
  • Biomarkers indicative of TFF bioactivity are monitored such as cell migration speed as TFFs are known to promote cell migration.
  • the migration speeds for assembled curli fibers composed of either wt-CsgA (neg. ctrl), CsgA-TFF, or soluble TFF (positive ctrl) are compared. Migration speeds are measured using a wound healing assay on confluent monolayers. If TFF is active, it will increase migration speeds, comparable to the soluble TFFs.
  • levels of COX-2 expression in the three cell populations are compared by Western blot. Expression above the negative control confirms the bioactivity of the curli-bound TFFs.
  • a Gut-on-a-Chip system is used to measure the bioactivity of the curli-bound TFFs using a more physiologically-relevant model system.
  • Chronic inflammation is simulated by adding inflammatory agents (e.g. LPS) to a chip containing an epithelial layer, endothelial layer, and circulating immune cells.
  • inflammatory agents e.g. LPS
  • LPS inflammatory agents
  • the expression of key inflammatory markers NF- ⁇ B, IL-1 ⁇ , IL-8, COX-2, EGFR activation, etc.
  • NF- ⁇ B, IL-1 ⁇ , IL-8, COX-2, EGFR activation, etc. is then monitored in response to treatment with curli-bound TFFs.
  • DFS established mouse models of intestinal inflammation
  • IL-10 knockout etc.
  • Abrogated inflammatory responses are measured qualitatively by histology and quantitatively with qPCR of key inflammatory cytokines.
  • aspects of the present disclosure are directed to methods of treating cancer such as cancer of the GI tract by administering to an individual in need thereof a certain engineered bacterial strain or strains as described herein.
  • the bacterial strain expresses a CsgA-linker-cancer treating polypeptide construct.
  • Certain cancer treating polypeptides include those having known activity against cancers including growth inhibiting factors such as bevacizumab, cetuximab, panitumumab and the like.
  • the bacterial strain expresses the CsgA-linker-cancer treating polypeptide construct and the cancer treating polypeptide treats the cancer tissue or cancer cells.
  • the bacterial strain expresses a CsgA-linker-first member of a binding pair polypeptide construct.
  • the bacterial strain expresses the CsgA-linker-first member of a binding pair polypeptide construct.
  • the second member of the binding pair attached to a cancer treating compound is then administered.
  • the first member of the binding pair and the second member of the binding pair bind thereby localizing the cancer treating compound, for example, to the gut, for the treatment of cancer, such as cancers of the gastrointestinal tract.
  • Useful bacterial strains include non-pathogenic E. coli , such as the E. coli Nissle 1917 (EcN), MG1655, K12-derived strains and the like.
  • the cancer treating protein or polypeptide may exert its effects while still attached to the engineered curli fibers.
  • the cancer treating protein or polypeptide domain may exert its effects as a soluble protein after cleavage from the curli fibers by a protease.
  • proteases in the MMP family are upregulated during inflammation and may be used to cleave the cancer treating polypeptide from the linker domain.
  • aspects of the present disclosure are directed to methods of delivering a diagnostic agent, such as a marker, to a site within a mammal by administering to the mammal a certain engineered bacterial strain or strains as described herein.
  • the bacterial strain expresses a CsgA-linker-diagnostic polypeptide construct.
  • the bacterial strain expresses the CsgA-linker-diagnostic polypeptide construct and the diagnostic polypeptide is detected.
  • the bacterial strain expresses a CsgA-linker-first member of a binding pair polypeptide construct.
  • the bacterial strains express the CsgA-linker-first member of a binding pair polypeptide construct.
  • the second member of the binding pair attached to a diagnostic compound is then administered.
  • the diagnostic compound may include an imaging agent or dye. The first member of the binding pair and the second member of the binding pair bind thereby localizing the diagnostic compound to the location of interest, such as the gut.
  • Useful bacterial strains include non-pathogenic E. coli , such as the Nissle strain, MG1655, K12-derived strains and the like.
  • the diagnostic polypeptide or diagnostic compound may be still attached to the engineered curli fibers.
  • the diagnostic polypeptide or the diagnostic compound may be cleaved from the curli fibers by a protease.
  • proteases in the MMP family are upregulated during inflammation and may be used to cleave the diagnostic polypeptide or diagnostic compound from the linker domain.
  • the diagnostic polypeptide or diagnostic compound may be released from the curli fibers using a protease-cleavable linker.
  • a recombinant construct of CsgA-linker-diagnostic polypeptide is made where the linker domain includes an amino acid sequence that is recognized and cleaved by a protease enzyme.
  • Such amino acid sequences and associated protease enzymes are known to those of skill in the art and include MMPs, CD2830 and the like.
  • the linker may be selected such that it is susceptible to cleavage by enzymes that are produced locally at sites of inflammation (MMP2, MMP9, etc.). Methods described above can be used to confirm both in vitro and in vivo activity of this embodiment of the disclosure.
  • aspects of the present disclosure are directed to methods of treating gut borne pathogens by administering to an individual in need thereof a certain engineered bacterial strain or strains as described herein.
  • the bacterial strain expresses a CsgA-linker-antimicrobial polypeptide construct.
  • Certain antimicrobial polypeptides or proteins include any polypeptide or protein sequence having antimicrobial activity.
  • Antimicrobial peptides for use in a therapeutic context are known to those of skill in the art. See Cotter, P. D., Ross, R. P. & Hill, C. Bacteriocins—a viable alternative to antibiotics? Nat Rev Micro 11, 95-105 (2012); Hing, T. C. et al.
  • the antimicrobial peptide cathelicidin modulates Clostridium difficile -associated colitis and toxin A-mediated enteritis in mice.
  • gut borne pathogens include Clostridium difficile, Salmonella typhimurium , Enteropathogenic E. coli, Helicobacter pylori and the like.
  • the bacterial strain expresses the CsgA-linker-antimicrobial polypeptide construct and the antimicrobial polypeptide treats the gut borne pathogens in a manner to reduce or eliminate the gut borne pathogens.
  • the bacterial strain expresses a CsgA-linker-tissue or cell binding polypeptide construct.
  • the bacterial strain expresses the CsgA-linker-tissue or cell binding polypeptide construct and the bacterial strain treats the gut borne pathogens in a manner to reduce or eliminate the gut borne pathogens.
  • the bacterial strain expresses a CsgA-linker-first member of a binding pair polypeptide construct.
  • the bacterial strains express the CsgA-linker-first member of a binding pair polypeptide construct.
  • the second member of the binding pair attached to an antimicrobial compound is then administered.
  • the first member of the binding pair and the second member of the binding pair bind thereby localizing the antimicrobial compound to the gut for the treatment of the gut borne pathogens in a manner to reduce or eliminate the gut borne pathogens.
  • Useful bacterial strains include non-pathogenic E. coli , such as the E. coli Nissle 1917 (EcN), MG1655, K12-derived strains and the like.
  • the antimicrobial protein or polypeptide may exert its effects while still attached to the engineered curli fibers.
  • the antimicrobial protein or polypeptide may exert its effects as a soluble protein after cleavage from the curli fibers by a protease.
  • proteases in the MMP family are upregulated during inflammation and may be used to cleave the bioactive domain from the linker domain.
  • the antimicrobial proteins or peptides may be released from the curli fibers using a protease-cleavable linker.
  • a recombinant construct of CsgA-linker-antimicrobial is made where the linker domain includes an amino acid sequence that is recognized and cleaved by a protease enzyme.
  • the linker may be selected such that it is susceptible to cleavage by enzymes that are produced locally at sites of inflammation (MMP2, MMP9, etc.).
  • antimicrobial proteins are fused to the curli fibers via a linker that is susceptible to cleavage by the proteolytic virulence factor CD2830.
  • antimicrobial proteins are released inside the gut only in the presence of C. difficile virulence factors. The antimicrobial protein is delivered locally to kill the invading pathogen.
  • antimicrobial proteins with demonstrated activity against C. difficile , including thurcin CD, lantibiotics like nisin and actagardine, cathelicidins and LL-37.
  • One exemplary antimicrobial protein is coprisin, a peptide that was originally isolated from Copris tripartitus (a Korean dung beetle) that has recently shown promise as a treatment for C. difficile infections.
  • Coprisin has a high potency against C. difficile (MIC of 1.5 ⁇ g/mL compared to 3.0 ⁇ g/mL for vancomycin) and a lack of activity against common gut commensals like Lactobacillus and Bifidobacterium .
  • the full coprisin peptide is 43 amino acids, but a 9 amino acid truncated analog (LLCIALRKK)(SEQ ID NO:29) exhibits higher antibiotic activity.
  • the coprisin-derived sequence is fused to CsgA through a linker that is susceptible to cleavage in the presence of a protease virulence factor secreted by C. difficile during infection. See Hensbergen, P. J. et al. A novel secreted metalloprotease (CD2830) from Clostridium difficile cleaves specific proline sequences in LPXTG (SEQ ID NO:30) cell surface proteins. Molecular & Cellular Proteomics 13, 1231-1244 (2014).
  • CD2830 is a metalloprotease that is actively secreted by C. difficile into the extracellular space and is thought to play a role in pathogen motility by cleaving adhesions that bind to the epithelial cell surface. Importantly, CD2830 is known to exhibit specificity for cleavage of proline-rich sequences, especially Pro-Pro and sortase-like LPXTG (SEQ ID NO:30) sequences.
  • CsgA is the amyloidogenic region of the protein.
  • the total panel size is 9 members, with fused domains ranging from 22-28 amino acids.
  • the plasmids harbor ampicillin resistance genes for antibiotic selection and the genes encoding for the CsgA fusion proteins are placed under the control of an IPTG inducible promoter.
  • the bioactive peptide is cleaved and released from the curli fibers in response to a protease.
  • Suitable proteases can be identified using the following method which is exemplified by protease CD2830.
  • the CD2830 protease is actively secreted by C. difficile , and is purported to act as a virulence factor by cleaving host protein-binding adhesions produced by the pathogen, thereby promoting motile phenotypes.
  • CD2830 cleaves proline-rich sequences. Accordingly, linker domains include one or more prolines.
  • CD2830 is expressed recombinantly in E. coli and purified.
  • a 96-well filter plate assay is used to subject biofilms to various conditions and to monitor the capture or release of soluble entities from the biofilm.
  • a panel of engineered EcN variants are grown and induced in suspension culture, then immobilized with their associated curli fibers on the filter plate.
  • the biofilms are washed to remove all soluble or weakly bound biomolecules.
  • the biofilms are treated with the recombinant CD2830 at various concentrations.
  • Release of the AMPs is monitored at a range of time points to determine the kinetic parameters of the cleavage from assembled curl fibers.
  • AMP release is monitored by LC-MS analysis of the collected fractions after protease treatment. Similar release studies is performed with a live C. difficile strain (ATCC 43255) by exposure of the filtered biofilms to the pathogen inside an anaerobic chamber.
  • Antimicrobial activity of antimicrobial proteins, such as coprisin by two different assays of the modified curli fibers—one that mimics a conventional minimum inhibitory concentration (MIC) assay, and another that monitors the cytotoxicity of C. difficile cells after culture in the presence of modified curli fibers.
  • MIC minimum inhibitory concentration
  • EcN variants that are selected for their ability to release active AMP in response to recombinant CD2830 are induced in YESCA media to form modified curli fibers.
  • C. difficile cultures are prepared by growing them overnight under anaerobic conditions to stationary phase.
  • the altered CsgA genes are incorporated into the genome of EcN.
  • a mutant of EcN is generated wherein the csgA gene has been replaced with an antibiotic selection marker by using a lambda Red recombineering technique wherein a double stranded DNA insert containing a desired selection marker flanked by homology domains specific for the csgA locus is introduced into the bacterial cells along with the lambda Red recombination factors.
  • a lambda Red recombineering technique wherein a double stranded DNA insert containing a desired selection marker flanked by homology domains specific for the csgA locus is introduced into the bacterial cells along with the lambda Red recombination factors.
  • the genome edited clones are selected by plating on antibiotic selective plates and the presence of the correct insertion is confirmed by PCR and sequencing.
  • the resulting strain referred to as EcN- ⁇ csgA
  • EcN- ⁇ csgA does not produce curli fibers.
  • a similar lambda Red recombineering strategy is used to replace the csgA gene in wild-type EcN with genes encoding for CsgA-linker-AMP variants.
  • the genetic insertion cassettes contain both the chimeric sequences and an antibiotic resistance marker for the selection of successfully edited clones.
  • the resistance gene is flanked by flippase recognition target (FRT) domains that enable self-resection if treated by mild heat.
  • FRT flippase recognition target
  • Successfully edited clones are subjected to a range of characterization protocols to ensure that they produce curli fibers.
  • Whole cell ELISA is used to confirm the presence of assembled CsgA in the fibers.
  • SEM is used to confirm that the gross morphology of the engineered fibers resembles that of wild-type curli fibers.
  • MALDI-MS analysis of purified curli fibers is used to confirm that the linker and AMP domains are not degraded during the secretion and assembly process.
  • growth rates in suspension culture are monitored for wild-type EcN, EcN- ⁇ csgA, and each of the CsgA-linker-AMP constructs.
  • the viability of the EcN genomic mutants in the healthy mouse gut is tested.
  • the engineered EcN mutants survive and transiently colonize the mouse gut without harming the host.
  • Genomically altered EcN mutants are fed to healthy mice at CFU values of ⁇ 108.
  • the inoculation occurs once, and the mice are fed a normal diet for 10 days.
  • the residence time of the engineered EcN variants in the mouse gut is monitored by collecting fecal samples daily and counting viable colonies on Macconkey agar plates with the appropriate antibiotic selection. Mice are observed and their body weight measured daily in order to confirm that there are no symptoms of bacterial infection.
  • the mice are sacrificed and their organs harvested in order to determine the spatial distribution of EcN cells.
  • the GI tract is sectioned into upper, lower, and middle sections and homogenized before counting CFUs by serial dilution on selective plates. A similar protocol is applied to the liver and spleen to check for invasive phenotypes.
  • the therapeutic effects of the engineered EcN variants on gut inflammation following C. difficile infection are monitored using the mouse model of antibiotic-induced C difficile -associated disease (CDAD) developed by Kang, et al., which more closely resembles human disease responses compared to other available models.
  • CDAD antibiotic-induced C difficile -associated disease
  • Mice in similar test groups are housed together and pre-treated with water containing a cocktail of antibiotics (gentamicin, metronidaxole vancmycin, and colistin at appropriate dosages).
  • Control mice receive a single dose of clindamycin (10 mg/kg) intraperitoneally, whereas experimental mice receive clindamycin intraperitoneally plus 10 8 CFU of the engineered EcN variant via oral gavage.
  • mice are infected by oral gavage with 0.5 ml of a suspension of C. difficile strain VPI 10463 (5 ⁇ 10 8 CFU/ml). Control mice are further given drinking water alone, and experimental mice are also further administered the EcN variants orally for 6 days and monitored for weight loss and survival. Fecal samples are collected daily and tissues are collected on day 10, and the localization of the EcN is measure by CFU counting and immunohistochemistry.
  • aspects of the present disclosure are directed to methods of capturing capture targets such as harmful agents, toxins or metabolites by administering to an individual in need thereof a certain engineered bacterial strain or strains as described herein.
  • the bacterial strain expresses a CsgA-linker-capture agent polypeptide construct.
  • Certain capture agents and associated capture targets include cholesterol and cholesterol-binding-peptides, phosphate and phosphate-binding-peptides, gliadin and gliadin-binding peptides and the like and those readily identified through literature search. Representative examples are shown in Table 5 below.
  • the bacterial strain expresses the CsgA-linker-capture agent polypeptide construct and the capture agent binds to the capture target.
  • the bacterial strain expresses a CsgA-linker-first member of a binding pair polypeptide construct.
  • the second member of the binding pair attached to a capture agent is then administered.
  • the first member of the binding pair and the second member of the binding pair bind thereby localizing the capture to the bacteria cell.
  • Useful bacterial strains include non-pathogenic E. coli , such as the Nissle strain, MG1655, K12-derived strains and the like.
  • the capture agent protein or polypeptide may exert its effects while still attached to the engineered curli fibers.
  • the capture agent protein or polypeptide may exert its effects as a soluble protein after cleavage from the curli fibers by a protease.
  • proteases in the MMP family are upregulated during inflammation or may be otherwise available in the gastrointestinal tract and may be used to cleave the bioactive domain from the linker domain.
  • the capture agent proteins or peptides may be released from the curli fibers using a protease-cleavable linker.
  • a recombinant construct of CsgA-linker-capture agent is made where the linker domain includes an amino acid sequence that is recognized and cleaved by a protease enzyme.
  • the linker may be selected such that it is susceptible to cleavage by enzymes that are produced locally at sites of inflammation (MMP2, MMP9, etc.). Methods described above can be used to confirm both in vitro and in vivo activity of this embodiment of the disclosure.
  • aspects of the present disclosure are directed to methods of delivering a diagnostic agent, such as a marker, to a site within a mammal by administering to the mammal a certain engineered bacterial strain or strains as described herein.
  • the bacterial strain expresses a CsgA-linker-diagnostic polypeptide construct.
  • the bacterial strain expresses the CsgA-linker-diagnostic polypeptide construct and the diagnostic polypeptide is detected.
  • the bacterial strain expresses a CsgA-linker-first member of a binding pair polypeptide construct.
  • the bacterial strains express the CsgA-linker-first member of a binding pair polypeptide construct.
  • the second member of the binding pair attached to a diagnostic compound is then administered.
  • the first member of the binding pair and the second member of the binding pair bind thereby localizing the diagnostic compound to the location of interest, such as the gut.
  • Useful bacterial strains include non-pathogenic E. coli , such as the Nissle strain, MG1655, K12-derived strains and the like.
  • the diagnostic polypeptide or diagnostic compound may be still attached to the engineered curli fibers.
  • the diagnostic polypeptide or the diagnostic compound may be cleaved from the curli fibers by a protease.
  • proteases in the MMP family are upregulated during inflammation and may be used to cleave the diagnostic polypeptide or diagnostic compound from the linker domain.
  • the diagnostic polypeptide or diagnostic compound may be released from the curli fibers using a protease-cleavable linker.
  • a recombinant construct of CsgA-linker-diagnostic polypeptide is made where the linker domain includes an amino acid sequence that is recognized and cleaved by a protease enzyme.
  • the linker may be selected such that it is susceptible to cleavage by enzymes that are produced locally at sites of inflammation (MMP2, MMP9, etc.). Methods described above can be used to confirm both in vitro and in vivo activity of this embodiment of the disclosure.
  • aspects of the present disclosure utilize the modified bacterial cells which express a fusion of a CsgA protein linked to a non-native functional polypeptide by a linker for biocatalysis.
  • methods are provided for the use of curli derived materials and biofilms as described herein as functionalizable surfaces for the immobilization of enzymes, such as enzymes used to perform chemical transformations in industrial applications (water purification, biofuel generation, etc.) and pharmaceutical applications (synthesis of drug intermediates).
  • the CsgA-linker-immobilized enzyme includes the linkers described herein.
  • methods are provided for metal removal or recovery. According to this aspect, methods are provided for the use of curli derived materials and biofilms as described herein as functionalizable surfaces for the immobilization of metals.
  • the CsgA-linker-metal binding polypeptide or agent includes the linkers described herein.
  • the metal binding polypeptide or agent may bind specific metals in ionic and metallic form.
  • methods are provided for bioremediation and for purification, such as by providing affinity purification matrices.
  • the curli system described herein is a biologically produced peptide-functionalized surface coating capable of being programmed to specifically immobilize another chemical or biological entity or to exhibit specific binding properties.
  • the displayed peptide may possess intrinsic properties such as binding to other exogenously added functional components, such as inorganic nanoparticles (especially those with interesting opto-electronic properties or magneto-responsiveness), carbon-based nanostructures (i.e., graphene or nanotubes, which may confer conductivity), or environmental toxins (i.e., hormones or toxic metals).
  • the engineered biofilms can also be used to display peptides that template the formation of inorganic or organic materials.
  • biofilm Functionalizing the biofilm with peptides that specifically bind to different materials allows the surface coating of these materials in a genetically programmable manner.
  • applications whereby the living biofilm is used to immobilize and present any arbitrary protein, as might be useful for applications in biocatalysis, biotemplating, or biosensing are specifically contemplated.
  • the synthesis and assembly of the material described herein is accomplished entirely by the bacterial cell, which acts as a factory for the production of programmed nanomaterials.
  • Additional specific applications include biologically-produced nanomaterials that have programmable optical, magnetoresistive or semiconductor properties from either the peptide/immobilized protein itself or by the induction of templated materials.
  • a system for high-efficiency immobilized biocatalysis in which various immobilization substrates can be used for the adhesion of the biofilm and which can be used in any bioreactor design is provided.
  • the peptide/immobilized proteins can also encode for biologically active biomolecules that will allow the biofilm to act as a tissue scaffold or vaccine delivery material.
  • peptide/immobilized proteins that bind to or enzymatically neutralize environmental toxins such as synthetic hormones, small molecules, or toxic metals is used as a biofilm-based technology for bioremediation.
  • environmental toxins such as synthetic hormones, small molecules, or toxic metals
  • peptides that are able to specifically bind to precious metals such as gold, silver, platinum, and rhodium on the biofilms described herein, an active surface area for the profitable recovery of such precious materials is provided.
  • the curli nanofibers can be engineered as conductive nanowires for numerous advanced materials applications by the display of peptides/proteins that are inherently conductive, or by the templating/anchoring of materials that are conductive.
  • bacteria to generate nanowires for energy storage based upon the expression on the curli biofilm of peptides capable of templating conductive or semiconductive materials, such as FePO4, is provided.
  • Bacteria can be specifically engineered via the displayed peptide to bind strongly to specific substrates, such as steel, glass, or gold. Such material-specific binding can provides a biofilm-based biosensing apparatus.
  • the curli nanofiber matrix can also be engineered to display peptides/proteins that interact with other molecules in order to enhance or alter the mechanical properties of another material.
  • the biofilm can act as a living coating capable of providing adaptive and regenerative benefits, such as biocatalysis on a wide variety of immobilization substrates, corrosion resistance to the material, enhanced biofilm coverage for microbial fuel cell applications, or act as an environmentally responsive organic (biofilm)-inorganic (substrate) material.

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