# Patent

## Title
Methods for detecting live pathogens

## Abstract

Disclosed are methods and kits pertaining to detecting live bacterial pathogens using sortase enzymes and reflective spectroscopy such as reflective interferometry.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Stage of PCT/US2012/035993, filed on May 1, 2012, the entire contents of which is incorporated herein by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 21, 2014, is named 091619-0791_SL.txt and is 7,900 bytes in size.
TECHNICAL FIELD
This disclosure relates generally to methods pertaining detection of live pathogens. In certain embodiments, the disclosure relates to detecting enzymatic by-products of live pathogens using reflective interferometry.
BACKGROUND
The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art.
Sensitive and accurate detection of bacterial contamination in food products and liquids is important for ensuring the protection of human and animal safety. Presently, many methods of detecting bacterial contamination in food and liquid utilize monoclonal antibody binding to detect specific bacterial pathogens. However, the sensitivity of assays using antibodies is limited since a typical bacterial cell includes few binding sites for a typical selective antibody. Moreover, existing antibody detection schemes react to their binding target regardless of whether the target is part of a live cell. Thus, these techniques are prone to false positives and increased background from dead cells and/or cell detritus. Consequently, even with sophisticated detectors, live food pathogen detection limits are still typically high, approximately 104 to 105 colony forming units per milliliter.
SUMMARY
The methods described herein relate to detecting live bacterial pathogens in a sample and involve cleavage of peptides by enzymes in the pathogen, and detecting products of the cleaved enzymes using light reflectance.
In one aspect, the present technology provides methods for detecting one or more live bacterial pathogens in a sample. The methods may include: contacting the sample with one or more bacterial sortase substrates and a biosensor having a porous membrane, wherein the porous membrane excludes intact sortase substrates and admits the product of a cleaved sortase substrate; measuring the reflectivity spectrum of the biosensor; and comparing the reflectivity spectrum to that of a biosensor not contacted with the sample, wherein an altered reflectivity spectrum indicates the presence of one or more live bacterial pathogens in the sample.
In another aspect, the present technology provides methods for detecting one or more live foodborne bacterial pathogens in a food matrix. The methods may include: contacting the food matrix with a biosensor having a porous membrane and one or more bacterial sortase substrates, wherein the porous membrane excludes intact sortase substrates and admits the product of a cleaved sortase substrate; measuring the reflectivity spectrum of the biosensor using Reflective Interferometric Fourier Transform Spectroscopy (RIFTS); and comparing the reflectivity spectrum to that of a biosensor not contacted with the food matrix, wherein an altered reflectivity spectrum indicates the presence of one or more live bacterial pathogens in the food matrix.
In yet another aspect, the present technology provides a kit for detecting one or more live bacterial pathogens in a sample. The kit includes one or more bacterial sortase substrates; and a biosensor comprising a porous membrane, wherein the porous membrane excludes intact sortase substrates and admits the product of a cleaved sortase substrate.
In some embodiments of the methods described herein, measuring the reflectivity spectrum of the biosensor is accomplished using one or more of Reflective Interferometric Fourier Transform Spectroscopy (RIFTS), grazing angle x-ray reflectivity, gonioreflectometry, or spectral reflectivity.
In some embodiments of the methods and kits described herein, the biosensor comprises a self-compensating interferometric biosensor. In some embodiments the porous membrane of the biosensor is made of at least silicon dioxide or aluminum oxide. In some embodiments, the porous membrane has at least a base layer and a surface layer, each layer comprising interconnected pores, wherein the base layer has pores of a smaller diameter than the surface layer and excludes the product of a cleaved sortase substrate, and wherein the base layer contacts a solid support.
In some embodiments of the methods and kits described herein, the one or more sortase substrates are attached to the porous membrane. In some embodiments, the one or more sortase substrates are selected from one more peptides comprising the amino acid sequence Leu-Pro-X-Thr-Gly (SEQ ID NO. 1), wherein X may be any proteinogenic amino acid. In some embodiments, the one or more sortase substrates are selected from one more peptides comprising the amino acid sequence Asn-X-Thr-Asn (SEQ ID NO: 2), wherein X may be any proteinogenic amino acid. In some embodiments, the one or more sortase substrates are selected from one more peptides comprising the amino acid sequence Leu-Pro-Asn-Thr-Ala (SEQ ID NO: 3). In some embodiments, the one or more sortase substrates are one or more peptides of the amino acid sequence Leu-Pro-X-Thr-Gly (SEQ ID NO. 1), wherein X may be any proteinogenic amino acid. In some embodiments, the one or more sortase substrates are one or more peptides comprising the amino acid sequence Asn-X-Thr-Asn (SEQ ID NO: 2), wherein X may be any proteinogenic amino acid. In some embodiments, the one or more sortase substrates are one or more peptides comprising the amino acid sequence Leu-Pro-Asn-Thr-Ala (SEQ ID NO: 3). In some embodiments, the proteinogenic amino acid is selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, and tyrosine.
In some embodiments of the methods and kits described herein, the presence of one or more live bacterial pathogens in the sample results in proteolytic cleavage of the one or more peptides, thereby releasing the product of a cleaved sortase substrate. In some embodiments, the sortase substrate is a sortase A substrate. In some embodiments, the product of a cleaved sortase substrate is Gly, Asn, or Ala.
In some embodiments of the methods and kits described herein, the product of a cleaved sortase A substrate enters the surface layer of the porous membrane, thereby causing an alteration in the reflectivity spectrum of the surface layer.
In some embodiments of the methods and kits described herein, the one or more bacterial pathogens have at least genera selected from the group consisting of Listeria, Clostridium, Enterococcus, Staphylococcus, Streptococcus, Actinobacter, Bacillus, and Corynebacterium. 
In some embodiments of the methods described herein, the food matrix comprises fluids associated with dairy, meat, fish, fruit, and vegetables.
In some embodiments of the kits described herein, the kit has at least one or more positive controls, that have one or more bacterial sortase enzymes specific for the one or more bacterial sortase substrates. In some embodiments, the one or more positive controls have live bacteria. In some embodiments, the one or more positive controls have recombinant enzymes. In some embodiments of the kits described herein, the kit has at least one or more negative controls. In some embodiments the one or more negative controls include a solution that does not contain protein. In some embodiments, the one or more negative controls include a protein or enzyme that does not cleave sortase substrates. In some embodiments, the one or more negative controls include a bacterial protease that does not cleave sortase substrates. In some embodiments, the one or more negative controls include non-pathogenic live bacteria that do not express sortase enzymes.
The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic showing the steps of sortase cleavage of a peptide.
FIG. 2 is a schematic representation of a cross-section of a two-layer biosensor and light beams being applied and reflected.



DETAILED DESCRIPTION
Unless otherwise stated, the singular forms “a,” “an,” and “the” as used herein include plural reference.
As used herein, the term “sortase” or “sortase enzyme” refers to a prokaryotic enzyme having a catalytic domain with activity capable of selectively cleaving a backbone peptide bond of a polypeptide at a sortase recognition sequence and catalyzing a transpeptidation reaction which results in the formation of an amide bond between the terminal carboxyl group created by the cleavage and a surface protein of the cell wall of a cell. Sortase is present in almost all Gram-positive bacteria, as well as a few Gram-negative bacteria and Archaea.
The term “polypeptide” or “peptide” as used herein refers to two or more amino acids linked by a peptide (i.e., amide) bond between the carboxyl terminus of one amino acid and the amino terminus of another. The term “polypeptide” or “peptide” includes a protein. Where the polypeptide includes a sortase recognition sequence, the polypeptide may be cleave by a sortase enzyme in the manner described above. The term “peptide” may be combined with a prefix indicating the number of amino acids in the peptide, e.g., a “pentapeptide” is a peptide of five amino acids.
Sortase Enzymes and Specificity
Sortases are a family of bacterial enzymes (transpeptidases) that covalently anchor proteins made by the bacteria to the cell wall envelope of gram-positive, some gram-negative bacteria. Many gram-positive bacteria are pathogenic to humans. Table 1 below lists exemplary gram-positive bacteria and the diseases they cause in humans.







TABLE 1







Gram-Positive Bacterial Pathogens and Corresponding Diseases










Pathogen
Disease






Listeria
meningitis



Clostridium
botulism, tetanus, gas gangrene, and




pseudomembranous colitis



Enterococcus
Urinary tract infections, bacteremia, bacterial




endocarditis, meningitis



Staphylococcus 
Sialadenitis (food poisoning)



Streptococcus
Strep throat, meningitis, bacterial pneumonia



Bacillus
anthrax and gastroenteritis



Corynebacterium
Diphtheria









The transpeptidation reaction catalyzed by sortases is a cleavage of a surface protein substrate at a peptide motif near the cell wall sorting signal, leaving an acyl protein intermediate and a short peptide that includes the cell wall sorting signal. The acyl protein intermediate then undergoes a transpeptidation reaction with a peptidoglycan precursor, and binds to the peptidoglycan in the cell wall.
Sortases are characterized in four different classes (A, B, C, D). Each of the sortase classes cleaves a different peptide motif, and some class members cleave multiple peptide motifs. Class A sortases are used to anchor cell wall proteins involved with cell adhesion, immune evasion, internalization, and can act as phage receptors. Class B sortases anchor proteins to the cell wall that are used to acquire iron. Class C sortases assemble pili on the bacterial surface, and class D sortases anchor proteins to the cell wall during sporulation of bacilli and streptomyces. 
Class A Sortase typically cleaves the consensus peptide motif LPXTG (Leu-Pro-X-Thr-Gly; SEQ ID NO. 1), where X represents any proteinogenic amino acid. (As will be understood by those of skill in the art, the one and three letter codes for L-alpha amino acids used throughout this disclosure are well known in the art and have their art-accepted meanings.) Class A Sortase cleaves between the threonine and the glycine. The cleaved cell sorting peptide maintains the glycine residue at its amino terminus, and the cleaved protein binds to the peptidoglycan at the threonine residue. Class B Sortase cleaves a consensus motif of NXTN (Asn-X-Thr-Asn; SEQ ID NO: 2) between threonine and asparagine where X represents any proteinogenic amino acid. Class C Sortase cleaves the consensus motif LPXTG (SEQ ID NO: 1) where X represents any proteinogenic amino acid and accepts the lysine within the sequence YPKN (Tyr-Pro-Lys-Asn; SEQ ID NO: 4) as a residue for covalently linking the threonine on the cell wall peptide. Class D Sortase cleaves the consensus motif LPNTA (SEQ ID NO: 3) between the threonine and the alanine.
Organism-specific cleavage motifs also exist for substrates cleaved by sortase A. For example, Lactobacillus johnsonii has a conserved substrate cleavage motif of LPQTG (Leu-Pro-Gln-Thr-Gly; SEQ ID NO: 5), and its sortase A cleaves between the threonine and glycine, while Listeria monocytogenes has a conserved substrate cleavage motif of LPXTGD (Leu-Pro-X-Thr-Gly-Asp; SEQ ID NO: 7), in which peptide cleavage by sortase A occurs between the threonine and the glycine, resulting in a two-residue peptide of GD. Variation in cleavage motifs among bacterial genera is described in Boekhurst et al., J Bacteriol. 2005 July; 187(14):4928-34. Table 2 lists exemplary cleavage motifs for sortase A that are specific to certain gram-positive bacteria.







TABLE 2







Examples of Class A Sortase cleavage motifs










Sortase



Bacteria
Class
Cleavage Motif





Lactobacillus johnsonii
A
LPQTG




(SEQ ID NO: 5)


 


Lactobacillus plantarum
A
LPQTXE




(SEQ ID NO: 6)


 


Listeria monocytogenes
A
LPXTGD




(SEQ ID NO: 7)


 


Streptomyces coelicolor
A
LAXTG




(SEQ ID NO: 8)









Boekhurst et al used in silico analysis of 199 bacterial genomes to predict sortase cleavage sequences. In particular, species-specific pentapeptides were predicted for: Bacillus anthracis A2012, Bacillus anthracis Ames, Bacillus anthracis Ames 0581, Bacillus anthracis str Sterne, Bacillus cereus ATCC14579, Bacillus cereus ATCC 10987, Bacillus cereus ZK, Bacillus halodurans, Bacillus licheniformis ATCC 14580, Bacillus licheniformis DSM 13, Bacillus subtilis, Bacillus thuringiensis konkukian, Bifidobacterium longum, Bradyrhizobium japonicum, Clostridium acetobutylicum, Clostridium perfringens, Clostridium tetani E88, Corynebacterium diphtherias, Enterococcus faecalis V583, Lactobacillus johnsonii NCC 533, Lactobacillus plantarum, Lactococcus lactis, Listeria innocua, Listeria monocytogenes, Listeria monocytogenes 4b F2365, Methanopyrus kandleri, Oceanobacillus iheyensis, Shewanella oneidensis, Staphylococcus aureus MW2, Staphylococcus aureus Mu50, Staphylococcus aureus N315, Staphylococcus aureus aureus MRSA252, Staphylococcus aureus aureus MSSA476, Staphylococcus epidermidis ATCC 12228, Streptococcus agalactiae 2603, Streptococcus agalactiae NEM316, Streptococcus mutans, Streptococcus pneumoniae R6, Streptococcus pneumoniae TIGR4, Streptococcus pyogenes, Streptococcus pyogenes MGAS10394, Streptococcus pyogenes MGAS315, Streptococcus pyogenes MGAS8232, Streptococcus pyogenes SSI-1, Streptomyces avermitilis, Streptomyces coelicolor, Tropheryma whipplei TW08 27, and Tropheryma whipplei Twist. The amino acid sequences associated with the species-specific pentapeptides disclosed by =Boekhorst et al., for the above microorganisms are summarized in Table 3, below.







TABLE 3







Amino acid residues known or predicted


to be present in pentapeptide


sortase recognition sequences.


(N-terminus) Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 (C-terminus)











Xaa1
Xaa2
Xaa3
Xaa4
Xaa5





L, N,
P, S, E,
K, A, N, Q,
T, A,
G, A, S,


A, I,
G, K,
E, T, P, S, H,
K, G,
D, N,


G, V,
A, D,
D, I, R, V,
S, Y,
V, Q, E,


P, F,
V, or L
M, F, L, Y,
E, M, V,
K, or P


or Y

or G
or L









The sortase substrates of the present technology may include, but are not limited to, any of the sortase recognition sequences embraced by Table 3. Thus, in some embodiments, the sortase substrate of the present technology includes the peptide sequence of Formula I.








Xaa1-Xaa2-Xaa3-Xaa4-Xaa5
I






wherein each of Xaa1, Xaa2, Xaa3, Xaa4, and Xaa5 is selected from the indicated amino acids in Table 3. For example, the polypeptide may be a pentapeptide with any of the following sequences: LPKTG (SEQ ID NO: 9), LPNTG (SEQ ID NO:10), LPETG (SEQ ID NO:11), LPQTG (SEQ ID NO:12), LPATG (SEQ ID NO:13), LPNTA (SEQ ID NO:14), LAETG (SEQ ID NO:15), or NPQTN (SEQ ID NO:16). It will be understood by those of skill in the art that the peptide sequences represented by Formula I are read from left to right, with the N-terminus on the left of each residue and the C-terminus on the right, and that each pair of adjacent residues are linked via a peptide bond between the C-terminus of one residue and the N-terminus of the adjacent residue.

In some embodiments of the present technology, the sortase substrate of the present technology is selected from the group consisting of the formulae in Table 4, wherein each of Xaa1, Xaa2, Xaa3, Xaa4, and Xaa5 are selected from the indicated amino acids in Table 3.









TABLE 4





Formula
Sequence
SEQ ID NO:







IVA
Leu-Pro-Xaa3-Thr-Xaa5
17


 


IVB
Leu-Pro-Xaa3-Thr-Gly
18


 


IVC
Leu-Pro-Xaa3-Xaa4-Gly
19


 


IVD
Leu-Pro-Xaa3-Ala-Gly
20


 


IVE
Leu-Pro-Xaa3-Thr-Ser
21


 


IVF
Leu-Pro-Xaa3-Thr-Asn
22


 


IVG
Leu-Ala-Xaa3-Thr-Gly
23


 


IVH
Leu-Ser-Xaa3-Thr-Gly
24


 


IVI
Ile-Pro-Xaa3-Thr-Gly
25


 


IVJ
Phe-Pro-Xaa3-Thr-Gly
26


 


IVK
Leu-Pro-Xaa3-Thr-Ala
27









In some embodiments of the present technology, the sortase substrate includes Formula IVD and Xaa3 is selected from any of A, E, H, K, L, N, Q, S, or T. In other embodiments, the sortase substrate has Formula IVE and Xaa3 is selected from any of D, K, N, Q, S, or T. In other embodiments, the sortase substrate has Formula IVF and Xaa3 is selected from any of K, M, N, Q, or T. In others, the sortase substrate has Formula IVG and Xaa3 is selected from any of A, D, E, F, H, K, L, N, R, S, or Y. In other embodiments, the sortase substrate has Formula IVH and Xaa3 is selected from any of F, N, or S. In other embodiments, the sortase substrate has Formula IVI and Xaa3 is selected from any of D, E, K, M, N, Q, or R. In other embodiments, the sortase substrate has Formula IVJ and Xaa3 is selected from any of K, Q, or S. In other sortase substrate, the polypeptide has Formula IVK and Xaa3 is selected from any of D, E, K, or N.
In certain embodiments, the sortase substrate of Formula I includes a sequence selected from AKKEK (SEQ ID NO: 28), FPKTG (SEQ ID NO: 29), FPQTG (SEQ ID NO: 30), FPSTG (SEQ ID NO: 31), GPDTA (SEQ ID NO: 32), IPALG (SEQ ID NO: 33), IPDTG (SEQ ID NO: 34), IPETG (SEQ ID NO: 35), IPKTG (SEQ ID NO: 36), IPMTG (SEQ ID NO: 37), IPNTG (SEQ ID NO: 38), IPQTG (SEQ ID NO: 39), IPRTG (SEQ ID NO: 40), IVKTG (SEQ ID NO: 41), LAATG (SEQ ID NO: 42), LADTG (SEQ ID NO: 43), LAETG (SEQ ID NO: 44), LAHTG (SEQ ID NO: 45), LAFTG (SEQ ID NO: 46), LAKTG (SEQ ID NO: 47), LALTG (SEQ ID NO: 48), LANTG (SEQ ID NO: 49), LARTG (SEQ ID NO: 50), LASTG (SEQ ID NO: 51), LAYTG (SEQ ID NO: 52), LAETP (SEQ ID NO: 53), LEKTN (SEQ ID NO: 54), LGATG (SEQ ID NO: 55), LGNTG (SEQ ID NO: 56), LLKTG (SEQ ID NO: 57), LPAAG (SEQ ID NO: 58), LPEAG (SEQ ID NO: 59), LPHAG (SEQ ID NO: 60), LPKAG (SEQ ID NO: 61), LPLAG (SEQ ID NO: 62), LPNAG (SEQ ID NO: 63), LPQAG (SEQ ID NO: 64), LPSAG (SEQ ID NO: 65), LPTAG (SEQ ID NO: 66), LPKAN (SEQ ID NO: 67), LPEKG (SEQ ID NO: 68), LPALG (SEQ ID NO: 69), LPQMN (SEQ ID NO: 70), LPDTA (SEQ ID NO: 71), LPETA (SEQ ID NO: 72), LPKTA (SEQ ID NO: 73), LPNTA (SEQ ID NO: 74), LPFSG (SEQ ID NO: 75), LPSSG (SEQ ID NO: 76), LPQTD (SEQ ID NO: 77), LPATG (SEQ ID NO: 78), LPDTG (SEQ ID NO: 79), LPETG (SEQ ID NO: 80), LPFTG (SEQ ID NO: 81), LPGTG (SEQ ID NO: 82), LPHTG (SEQ ID NO: 83), LPITG (SEQ ID NO:129), LPKTG (SEQ ID NO: 84), LPLTG (SEQ ID NO: 85), LPMTG (SEQ ID NO: 86), LPNTG (SEQ ID NO: 87), LPQTG (SEQ ID NO: 88), LPRTG (SEQ ID NO: 89), LPSTG (SEQ ID NO: 90), LPTTG (SEQ ID NO: 91), LPVTG (SEQ ID NO: 92), LPYTG (SEQ ID NO: 93), LPKTN (SEQ ID NO: 94), LPMTN (SEQ ID NO: 95), LPNTN (SEQ ID NO: 130), LPQTN (SEQ ID NO: 96), LPTTN (SEQ ID NO: 97), LPDTS (SEQ ID NO: 98), LPKTS (SEQ ID NO: 99), LPNTS (SEQ ID NO: 100), LPQTS (SEQ ID NO: 0101), LPSTS (SEQ ID NO: 102), LPETV (SEQ ID NO: 103), LPIVG (SEQ ID NO: 104), LPIYS (SEQ ID NO: 105), LSNTG (SEQ ID NO: 106), LSSTG (SEQ ID NO: 107), LSFTG (SEQ ID NO: 108), NAKTN (SEQ ID NO: 109), NAKTS (SEQ ID NO: 110), NKKSA (SEQ ID NO: 111), NPKTG (SEQ ID NO: 112), NPQTG (SEQ ID NO: 113), NPQTN (SEQ ID NO: 114), NPTKQ (SEQ ID NO: 115), NDTAV (SEQ ID NO: 131), NPKSS (SEQ ID NO: 116), NSKTA (SEQ ID NO: 117), PETGE (SEQ ID NO: 118), PKTGE (SEQ ID NO: 119), VPTGV (SEQ ID NO: 120), VANTG (SEQ ID NO: 121), VPDTG (SEQ ID NO: 122), VPPTG (SEQ ID NO: 123), YPKTG (SEQ ID NO: 124), or YPRTG (SEQ ID NO: 125).
Engineered Peptides as Sortase Substrates
Because of the variety of sortases and species-specific cleavage motifs, peptides containing a sortase cleavage motif can be used as a cleavable marker attached to the biosensor contemplated herein. FIG. 1 is a schematic of a bacterium contacting peptides containing a cleavage motif and cleaving the peptide to release one portion of the peptide and covalently binding the other portion.
Peptides containing a sequence susceptible to sortase cleavage may be modified or synthesized in a variety of configurations. In one peptide configuration, the sortase cleavage sequence is at the carboxy terminus of the peptide. In that case, cleavage by a sortase results in a peptide shortened by one or two amino acids. For example, a peptide having a carboxy terminus with the sequence LPXTG (SEQ ID NO: 1), will give two cleavage products: a peptide terminating in LPXT and a free glycine (G). Similarly, a peptide with a carboxy terminus of LPKTGD (SEQ ID NO: 126) can be cleaved by sortase A from Listeria monocytogenes, leaving a two-residue peptide of glycine and aspartic acid (GD).
In another peptide configuration, the sortase cleavage sequence is at least two amino acid residues away from the carboxy terminus (two residues towards the N-terminus), so that sortase cleavage results in two peptides. For example, the sortase cleavage sequence has 5 amino acid residues, such as 5 lysines, that follow the cleavage sequence and make up the peptide's carboxy terminus. In another example, the sortase cleavage sequence is in the middle of a longer peptide.
Peptides can also be modified with small molecules other than amino acids. Peptides having sortase recognition sequences have been engineered to contain heterologous molecular labels such as fluorescein and biotin (Nelson et al, ACS Chem Biol. 2010 Dec. 17; 5(12):1147-55). These engineered peptides were used to incorporate the molecular labels into the cell wall of Staphylococcus aureus up to 6500 polypeptides per cell.
The sortase substrates of the present technology are readily synthesized using standard peptide coupling techniques and reagents well known in the art, such as those described in Peptide Synthesis Protocols, Pennington, M. W., Dunn, B. M. (Eds.) Humana Press, Inc., New Jersey, 1994 and in Bodansky, M. and A. Bodansky, A. The Practice of Peptide Synthesis, Springer-Verlag, New York 1984. The polypeptides may be synthesized in solution phase, or may be synthesized using solid phase resins and/or supports (e.g., rink resins, amide resins, polystyrene resins, and the like). The synthesis may be automated.
Fabrication of Two-Layer Biosensor
The biosensor contemplated herein may be fabricated in two layers, stacked one on top of the other. Each layer is porous, and has different diameter pores. The top layer of the biosensor that will be exposed to sample has larger diameter pores than the bottom layer. The pores in each layer are connected, such that a single pore is formed, with a wider section above and a narrower section below. FIG. 2 shows a schematic representation of a cross-section of a two-layer biosensor and its pores. The diameter of the larger diameter pore in layer 1 is configured such that an amino acid tag or small peptide tag cleaved by sortase A can easily enter the length of the pore. The diameter of the smaller pore in layer 2 is configured to exclude an amino acid tag or small peptide tag from the length of the pore.
The biosensor can be fabricated from mesoporous silicon or from anodic aluminum oxide. Methods of etching and electrochemistry techniques can be used to form the porous layers of the biosensor, for example as described in Pacholski et al 2005, Pacholski et al 2006, and Losic et al (Pacholski et al, J. Am. Chem. Soc. 2005, 127, 11636-11645; Pacholski et al, J. Am. Chem. Soc. 2006, 128, 4250-4252; Losic et al, Australian Journal of Chemistry, 64(3) 294-301, 2011). Example 1 describes an etching technique for forming the two-layer biosensor.
Attachment of Peptides to Surface of Biosensor
Peptides used to detect the presence of a live bacteria can be attached to a two-layer biosensor either covalently or non-covalently, both of which are known in the art. See Jonkheijm et al, Angew. Chem. Int. Ed. 2008, 47, 9618-9647 for descriptions of potential methods of peptide attachment.
Peptide adsorption to silicon can be used as a method of attachment. Nanoporous silicon has been shown to be capable of adsorbing peptides without covalent modification by Pacholski et al, 2005 and Pacholski et al 2006 (J. Am. Chem. Soc. 2005, 127, 11636-11645 and J. Am. Chem. Soc. 2006, 128, 4250-4252).
Covalent attachment of peptides can be accomplished by adding the peptides to functionalized silicon, for example, silicon having N-hydroxysuccinimide (NHS)-activated carboxylic acids. Following exposure of the peptide to the functionalized silicon, the amino terminus of the peptides reacts with the functional group and become covalently bound. Functionalization of silicon can be accomplished, for example, by removing the oxide layer of the silicon with hydrofluoric acid (HF) and then reacting the hydrogen terminated surface with functionalized alkenes. In another method, the silicon can be oxidized with ionized gas and functionalized using organo silanes. Microcontact printing and microstamping are also potential methods of precisely applying and covalently attaching peptides to silicon (see Jonkheijm et al, Angew. Chem. Int. Ed. 2008, 47, 9618-9647; Blinka et al Nanotechnology. 2010 Oct. 15; 21(41):415302).
Detection of Light Reflectance from Two-Layer Biosensor
Light reflectance from a two-layer biosensor can be measured in a variety of ways. Reflective Interferometric Fourier Transform Spectroscopy (RIFTS) is a method of detecting the reflectivity spectrum of the two-layer biosensor and resolving the individual components by fast Fourier transform (FFT). Light beams applied to the two-layer biosensor create three different interfering light beams that correspond to three FFT peaks. FIG. 2 shows a schematic of a two-layer biosensor, with the applied light beam (labeled as lambda), and the reflected beams labeled a, b, and c. Following detecting of the interfering light beams and fast Fourier transformation, FFT peaks corresponding to layer 1, layer 2, and layer 3 (the sum of layers 1 and 2 of the biosensor) are seen (see FIG. 2). The FFT peak for layer 2 can be calculated by subtracting the FFT peak intensity for layer 1 from layer 3.
A shift in the FFT peaks that correlate with a given layer indicates changes in amino acid or peptide concentration of the solution in the pores of that layer. Thus, changes in layer 1 following exposure of the biosensor to a sample, indicate entry of peptides and/or peptide fragments into the solution. Layer 2 of the biosensor is the portion of the biosensor that allows the biosensor to be self-compensating. Because peptides and/or amino acids are excluded from layer 2, the FFT spectrum for that layer will only change relative to reflectivity changes brought about by exposure to a sample liquid (as well as instrument variability) and allow the data to be corrected accordingly. Detection of amino acid and/or peptide fragments is accomplished by either calculating the ratio of intensities of the FFT spectrum peaks for layer 1 and layer 2, or by calculating the weighted difference in the frequencies of the FFT spectrum peaks from layer 1 and layer 2. Specific methods and algorithms disclosing RIFTS and FFT may be found, for example, in Pacholski et al, J. Am. Chem. Soc. 2005, 127, 11636-11645 and Pacholski et al, J. Am. Chem. Soc. 2006, 128, 4250-4252.
Other methods of measuring and detecting changes in spectral reflectance include: grazing angle x-ray reflectivity, also known as grazing-incidence small-angle X-ray scattering (GISAXS); gonioreflectometry, and spectral reflectivity.
Controls
The methods and kits described herein may contain one or more positive controls, one or more negative controls, or both positive and negative controls. Examples of positive controls may include lyophilized or soluble sortase enzymes that can cleave a sortase substrate (e.g. sortase A, sortase B, etc.), as well as non-pathogenic live bacteria that have extracellular expression of a sortase enzyme (e.g. lactobacillus acidophilus, or soil bacteria). Examples of negative controls include solutions that do not contain any proteins, lyophilized or solubilized enzymes or other proteins that do not cleave sortase substrates, as well as non-pathogenic live bacteria that do not express sortase enzymes. For example, a negative control can be lyophilized or solubilized bacterial proteases that do not cleave sortase substrates (e.g. thermolysin). Alternatively, a microorganism lacking sortase may be used (e.g., isogenic S. aureus).
EXAMPLES
The present compositions, methods and kits, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present methods and kits. The following is a description of the materials and experimental procedures used in the Examples.
Example 1
Creation of a Silicon Two-Layer Porous Biosensor
A silicon (Si) two-layer porous biosensor is fabricated using electrochemical etching, with a short period of high applied current (11 seconds at 500 mA/cm2; producing layer 1 pores of about 50 to 100 nm) followed by a longer period at low current (55 seconds at 167 mA/cm2; producing layer 2 pores of less than 20 nm). The current is applied to a doped (10−3Ω-cm) p-type (100)-oriented single crystal Si wafer in ethanolic HF solution. To prevent corrosion in aqueous solution, siloxy-terminated porous Si surfaces were prepared by thermal oxidation. The oxidation increases the hydrophilicity of porous Si, allowing water to effectively infiltrate the pores.
Example 2
Detecting Bacteria Using a Pentapeptide Adsorbed to a Biosensor
A biosensor having the nanostructure described in Example 1 has a surface coating of peptides having the sequence LPKTGD (SEQ ID NO: 126), a sequence recognized and cleaved by sortase A in Listeria monocytogenes. The peptides are adsorbed to the surface of the biosensor. The biosensor is exposed to a light source, and the reflected light beams from layer 1 and layer 2 are measured.
Fluid from a food item is then applied to the biosensor and allowed to incubate. During the incubation, living Listeria monocytogenes present in the fluid cleave the LPKTGD (SEQ ID NO: 126) peptides on the surface of the biosensor, producing a small, soluble GD peptide (glycine-aspartic acid). The soluble GD peptide enters the pores of layer 1.
The surface of the biosensor is then re-exposed to a light source and the reflected light beams from layer 1 and layer 2 are measured again. The reflectance data before and after adding the fluid sample are analyzed using fast Fourier transformation, and then compared to determine the presence or absence of GD peptide fragments. The presence of GD peptides in layer 1 indicates the presence of Listeria monocytogenes in the fluid.
Example 3
Detecting Bacteria Using a Shortened Internalin A Protein Adsorbed to a Biosensor
A biosensor having the nanostructure described in Example 1 has a surface coating of a shortened Internalin A protein (SEQ ID NO:132). The protein has the sequence of residues 1-771 of GenBank Acc. No. NP—463962.1 and terminates at the carboxy terminus with the amino acid sequence LPTTG (SEQ ID NO: 91), a sequence recognized and cleaved by sortase A in Listeria monocytogenes. The protein is adsorbed to the surface of the biosensor. The biosensor is exposed to a light source, and the reflected light beams from layer 1 and layer 2 are measured. The sequence of GenBank Acc. No. NP—463962.1 is set forth in SEQ ID NO:134.
Fluid from a food item is then applied to the biosensor and allowed to incubate. During the incubation, living Listeria monocytogenes present in the fluid cleave the shortened Internalin A protein on the surface of the biosensor, producing soluble glycine residues that enter the pores of layer 1.
The surface of the biosensor is then re-exposed to a light source and the reflected light beams from layer 1 and layer 2 are measured again. The reflectance data before and after adding the fluid sample are analyzed using fast Fourier transformation, and then compared to determine the presence or absence of glycine residues. The presence of glycine in layer 1 indicates the presence of Listeria monocytogenes in the fluid.
Example 4
Detecting Bacteria Using a Modified Internalin A Protein Adsorbed to a Biosensor
A biosensor having the nanostructure described in Example 1 has a surface coating of a modified Internalin A protein (SEQ ID NO:0133). The protein has the sequence of residues 1-771 of GenBank Acc. No. NP—463962.1, with the addition of five lysine residues at the C-terminus. The protein terminates at the carboxy terminus with the amino acid sequence LPTTGKKKKK (Leu-Pro-Thr-Thr-Gly-Lys-Lys-Lys-Lys-Lys; SEQ ID NO: 127). The protein is adsorbed to the surface of the biosensor. The biosensor is then exposed to a light source, and the reflected light beams from layer 1 and layer 2 are measured.
Fluid from a food item is then applied to the biosensor and allowed to incubate. During the incubation, living Listeria monocytogenes present in the fluid cleave the modified Internalin A on the surface of the biosensor, producing a small, soluble peptide of sequence GKKKKK (Gly-Lys-Lys-Lys-Lys-Lys; SEQ ID NO: 128). The soluble GKKKKK peptide (SEQ ID NO: 128) enters the pores of layer 1.
The surface of the biosensor is then re-exposed to a light source and the reflected light beams from layer 1 and layer 2 are measured again. The reflectance data before and after adding the fluid sample are analyzed using fast Fourier transformation, and then compared to determine the presence or absence of GKKKKK peptides (SEQ ID NO: 128). The presence of GKKKKK peptides (SEQ ID NO: 128) in layer 1 indicates the presence of Listeria monocytogenes in the fluid.
The present disclosure is not to be limited in terms of the particular embodiments described in this application. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 particles refers to groups having 1, 2, or 3 particles. Similarly, a group having 1-5 particles refers to groups having 1, 2, 3, 4, or 5 particles, and so forth.
While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

## Claims

1. A method for detecting Listeria monocytogenes in a food matrix, the method comprising:
contacting the food matrix with one or more bacterial sortase substrates, wherein the one or more sortase substrates consist of one or more peptides selected from the group consisting of the amino acid sequence set forth in SEQ ID NO:126, SEQ ID NO:132, or SEQ ID NO:133, and a biosensor comprising a porous membrane, wherein the porous membrane excludes intact sortase substrates and admits the product of a cleaved sortase substrate;
measuring the reflectivity spectrum of the biosensor; and
comparing the reflectivity spectrum to that of a biosensor not contacted with the food matrix,
wherein an altered reflectivity spectrum indicates the presence of Listeria monocytogenes in the food matrix.

2. The method of claim 1, wherein measuring the reflectivity spectrum of the biosensor is accomplished using one or more of Reflective Interferometric Fourier Transform Spectroscopy (RIFTS), grazing angle x-ray reflectivity, gonioreflectometry, or spectral reflectivity.

3. The method of claim 1, wherein the biosensor comprises a self-compensating interferometric biosensor.

4. The method of claim 1 wherein the porous membrane comprises silicon dioxide or aluminum oxide.

5. The method of claim 1, wherein the porous membrane comprises at least a base layer and a surface layer, each layer comprising interconnected pores, wherein the base layer comprises pores of a smaller diameter than the surface layer and excludes the product of a cleaved sortase substrate, and wherein the base layer contacts a solid support.

6. The method of claim 1, wherein the one or more sortase substrates are attached to the porous membrane.

7. The method of claim 1, wherein the one or more sortase substrates further comprise one or more peptides comprising the amino acid sequence Leu-Pro-X-Thr-Gly (SEQ ID NO. 1), wherein X may be any proteinogenic amino acid.

8. The method of claim 1, wherein the one or more sortase substrates further comprise one or more peptides comprising the amino acid sequence Leu-Pro-Asn-Thr-Ala (SEQ ID NO: 3).

9. The method of claim 7, wherein the proteinogenic amino acid is selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, and tyrosine.

10. The method of claim 1, wherein the presence of Listeria monocytogenes in the food matrix results in proteolytic cleavage of the one or more peptides, thereby releasing the product of a cleaved sortase substrate.

11. The method of claim 10, wherein the product of a cleaved sortase substrate is Gly-Asp or Gly-Lys-Lys-Lys-Lys-Lys (SEQ ID NO: 128).

12. The method of claim 10, wherein the product of a cleaved sortase substrate enters the surface layer of the porous membrane, thereby causing an alteration in the reflectivity spectrum of the surface layer.

13. The method of claim 1, wherein the food matrix comprises fluids associated with dairy, meat, fish, fruit, or vegetables.

14. A kit for detecting Listeria monocytogenes in a food matrix, the kit comprising:
one or more bacterial sortase substrates, wherein the one or more sortase substrates consist of one or more peptides selected from the group consisting of the amino acid sequence set forth in SEQ ID NO:126, SEQ ID NO:132, or SEQ ID NO:133; and
a biosensor comprising a porous membrane, wherein the porous membrane excludes intact sortase substrates and admits the product of a cleaved sortase substrate.

15. The kit of claim 14, further comprising one or more positive controls comprising one or more bacterial sortase enzymes specific for the one or more bacterial sortase substrates.

16. The kit of claim 14, wherein the biosensor comprises a self-compensating interferometric biosensor.

17. The kit of claim 14, wherein the porous membrane comprises silicon dioxide or aluminum oxide.

18. The kit of claim 14, wherein the porous membrane comprises at least a base layer and a surface layer, each layer comprising interconnected pores, wherein the base layer comprises pores of a smaller diameter than the surface layer and excludes the product of a cleaved sortase substrate, and wherein the base layer contacts a solid support.

19. The kit of claim 14, wherein the one or more sortase substrates are attached to the porous membrane.

20. The kit of claim 14, wherein the one or more sortase substrates further comprise one or more peptides comprising the amino acid sequence Leu-Pro-X-Thr-Gly (SEQ ID NO. 1), wherein X may be any proteinogenic amino acid.

21. The kit of claim 14, wherein the one or more sortase substrates further comprise one or more peptides comprising the amino acid sequence Leu-Pro-Asn-Thr-Ala (SEQ ID NO: 3).

22. The kit of claim 20, wherein the proteinogenic amino acid is selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, and tyrosine.

23. The method of claim 1, wherein the one or more sortase substrates further comprise one or more peptides comprising the amino acid sequence Asn-X-Thr-Asn (SEQ ID NO: 2), wherein X may be any proteinogenic amino acid.

24. The method of claim 23, wherein the proteinogenic amino acid is selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, and tyrosine.

25. The kit of claim 14, wherein the one or more sortase substrates further comprise one or more peptides comprising the amino acid sequence Asn-X-Thr-Asn (SEQ ID NO: 2), wherein X may be any proteinogenic amino acid.

26. The kit of claim 25, wherein the proteinogenic amino acid is selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, and tyrosine.