U.S. patent application number 14/106149 was filed with the patent office on 2014-10-30 for methods, peptides and biosensors useful for detecting a broad spectrum of bacteria.
This patent application is currently assigned to WOUNDCHEK LABORATORIES. The applicant listed for this patent is WOUNDCHEK LABORATORIES. Invention is credited to Gerard J. Colpas, Diane L. Ellis-Busby, Jennifer M. Harvard, Mitchell C. Sanders, Shite Sebastian.
Application Number | 20140322790 14/106149 |
Document ID | / |
Family ID | 34556193 |
Filed Date | 2014-10-30 |
United States Patent
Application |
20140322790 |
Kind Code |
A1 |
Sebastian; Shite ; et
al. |
October 30, 2014 |
METHODS, PEPTIDES AND BIOSENSORS USEFUL FOR DETECTING A BROAD
SPECTRUM OF BACTERIA
Abstract
Described herein are methods of detecting a wound infection and
for detecting the presence or absence of bacteria, for example,
wound bacteria in a sample, by contacting a sample with a peptide
substrate derived from the modification of the reactive site loop
(RSL) domain of the .alpha.1-proteinase inhibitor. In the current
invention, we have demonstrated that these peptide substrates
without the alpha 1 protein can be efficiently used as peptide
substrates. The modification or the absence of modification of this
peptide substrate by the enzyme produced and/or secreted by the
bacteria, can serve as an indicator for the presence or absence of
the bacteria in the sample. The present invention also features a
biosensor for detecting the presence or absence of bacteria in a
sample.
Inventors: |
Sebastian; Shite;
(Somerville, MA) ; Colpas; Gerard J.; (Holden,
MA) ; Ellis-Busby; Diane L.; (Lancaster, MA) ;
Harvard; Jennifer M.; (Framingham, MA) ; Sanders;
Mitchell C.; (West Boylston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
WOUNDCHEK LABORATORIES |
Quincy |
MA |
US |
|
|
Assignee: |
WOUNDCHEK LABORATORIES
Quincy
MA
|
Family ID: |
34556193 |
Appl. No.: |
14/106149 |
Filed: |
December 13, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10576633 |
Nov 14, 2006 |
8609358 |
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PCT/US04/36600 |
Nov 3, 2004 |
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14106149 |
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60578811 |
Jun 9, 2004 |
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60516692 |
Nov 3, 2003 |
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Current U.S.
Class: |
435/188 ;
530/326; 530/327 |
Current CPC
Class: |
G01N 2333/8121 20130101;
G01N 33/56911 20130101; C12Q 1/04 20130101; C07K 14/001 20130101;
G01N 33/543 20130101; C12Q 1/37 20130101; C07K 7/08 20130101 |
Class at
Publication: |
435/188 ;
530/327; 530/326 |
International
Class: |
C12Q 1/04 20060101
C12Q001/04; C07K 14/00 20060101 C07K014/00; C07K 7/08 20060101
C07K007/08 |
Claims
1. An isolated peptide comprising a detectable label and an amino
acid sequence selected from the group consisting of EAAGAMFLEAIPK,
EGAMFLEAIPMSIPK, KGTEAAGAMFLEAIPMSIPPEVK, GAMFLEAIPMSIPPE, and
CGAMFLEAIPMSIPAAAHHHHH.
2. The peptide of claim 1 labeled with a fluorescent probe and a
quencher dye molecule.
3. The peptide of claim 1 wherein the detectable label is selected
from the group consisting of spin labels, antigen tags, epitope
tags, haptens, enzyme labels, prosthetic groups, fluorescent
materials, pH-sensitive materials, chemiluminescent materials,
colorimetric components, bioluminescent materials, and radioactive
materials.
Description
BACKGROUND OF THE INVENTION
[0001] Infection of wounds is a major source of healthcare
expenditure in the United States. Approximately 5% of all surgical
wounds become infected with microorganisms, and that figure is
considerably higher (10-20%) for patients undergoing abdominal
surgery. Bacterial species, such as Staphylococci are the most
frequently isolated organisms from infected wounds. This is
probably because humans are the natural reservoir for staphylococci
in the environment, with up to 50% of the population colonized at
any given time. Colonization rates are significantly higher in the
hospital setting, both among healthcare workers, and among
patients. Moreover, the colonizing organisms in the hospital
environment are likely to be resistant to many forms of
antimicrobial therapy, due to the strong selective pressure that
exists in the nosocomial environment, where antibiotics are
frequently used. Staphylococci are usually carried as harmless
commensals, however given a breach in the epidermis, they can cause
severe, even life threatening infection.
[0002] Staphylococci are the most common etiologic agents in
surgical wound infections; others include, but are not limited to
Streptococcus pyogenes, Pseudomonas aeruginosa, Enterococcus
faecalis, Serratia marcescens, Proteus mirabilis, Enterobacter
clocae, Acetinobacter anitratus, Klebsiella pneumoniae, and
Escherichia coli. Post-surgical infection due to any of the above
organisms is a significant concern of hospitals. The most common
way of preventing such infection is to administer prophylactic
antibiotic drugs. While generally effective, this strategy has the
unintended effect of breeding resistant strains of bacteria. The
routine use of prophylactic antibiotics should be discouraged for
the very reason that is encouraging the growth of resistant
strains.
[0003] Rather than using routine prophylaxis, a better approach is
to practice good wound management, i.e., keep the area free from
bacteria before, during, and after surgery, and carefully monitor
the site for infection during healing. Normal monitoring methods
include close observation of the wound site for slow healing, signs
of inflammation and pus, as well as measuring the patient's
temperature for signs of fever. Unfortunately, many symptoms are
only evident after the infection is already established.
Furthermore, after a patient is discharged from the hospital they
become responsible for monitoring their own healthcare, and the
symptoms of infection may not be evident to the unskilled
patient.
[0004] A system or biosensor that can detect a broad spectrum of
bacteria, especially during the early stages of infection before
symptoms develop, would be advantageous to both patients and
healthcare workers. If a patient can accurately monitor the
condition of a wound after hospital/clinic discharge, then
appropriate antimicrobial therapy can be initiated early enough to
prevent a more serious infection.
SUMMARY OF THE INVENTION
[0005] It has been found that a synthetic enzyme substrate, human
alpha-1 protease inhibitor, can be used in a test system to
identify multiple microorganism species, such as bacteria, that
commonly infect wounds.
[0006] Human alpha-1 proteinase inhibitor (.alpha.1-PI) is a member
of the serpin family. Serpins are a family of serine proteinase
inhibitors that function as irreversible suicide substrates
resulting in the inhibition of proteinases. Proteases are a common
virulence factor in pathogenic bacteria, and are sometimes used to
disable the host defenses. Of the serpin family (inhibitors is
already inherent in the name serpin), alpha-1-proteinase inhibitor
(.alpha.1-PI) is one of the major and most well-studied members.
.alpha.1-PI is involved in the regulation of elastases secreted
from activated neutrophils, which in turn control the degradation
of the host connective tissue (Salvesen, G S et al., "Human plasma
proteinase inhibitors", Ann. Rev. Biochem., 52: 655-709 (1983),
incorporated herein by reference). This unique category of serine
proteinase inhibitors possesses a characteristic exposed reactive
site loop (RSL) domain. Importantly, the RSL of .alpha.1-PI has
been demonstrated to be susceptible to cleavage by a number of
proteinases of both host and bacterial origin, resulting in the
inactivation of .alpha.1-PI.
[0007] The test system can be designed to simultaneously identify
multiple (for example, at least 2, at least 5, or at least 10)
different microorganism species, such as those that commonly infect
wounds. For example, it can identify those enzymes that are common
to certain classes of pathogenic bacteria, but which are not
present in non-pathogenic bacteria. Such enzymes can be identified,
for example, with a computer based bioinformatics screen of the
bacterial genomic databases. By using enzymes as the basis for
detection systems, very sensitive tests can be designed, since even
a very small amount of enzyme can catalyze the turnover of a
substantial amount of substrate.
[0008] Accordingly, the present invention features a method of
detecting the presence or absence of one or more microorganisms,
for example, a bacterium, in a sample by detecting the presence or
absence of a molecular marker for the bacterium in the sample.
[0009] A "molecule marker" is any molecule which can be used for
the detection of the presence or absence of a microorganism (e.g.,
a bacterium, fungus or virus) in a sample, such as a wound or body
fluid. In particular, the molecular markers to be detected include
proteins, such as proteins secreted by microorganisms, expressed on
the cell surface of microorganisms, or expressed on the surface of
a cell infected with a virus or prion. In one embodiment, the
enzyme is a bacterial protease.
[0010] In one aspect, the invention features a method for detecting
the presence or absence of one or more bacteria in a sample,
comprising the steps of contacting the sample with a detectably
labeled synthetic serpin reactive site loop (RSL) domain peptide
substrate, under conditions that result in modification of the
substrate by an enzyme produced and/or secreted by the bacterium;
and detecting the modification or the absence of the modification
of the substrate. Modification of the substrate indicates the
presence of the bacterium in the sample, and the absence of
modification of the substrate indicates the absence of the
bacterium in the sample.
[0011] The substrate can be synthetic. For example, it can be
derived from a native or non-naturally occurring RSL domain and can
have the same or equivalent activity as the RSL of a wild type
serpin. It can also comprise or consist of a variant, analog or
fragment of a serpin RSL domain. In one embodiment, the substrate
comprises SEQ ID NO: 1, 2 or 3. In one embodiment, the substrate is
CPI1, CPI2 or CPI3, as described below.
[0012] The bacterium can be, for example, a wound specific
bacterium, such as Staphylococcus aureus, Staphylococcus
epidermidis, Streptococcus pyogenes, Pseudomonas aeruginosa,
Enterococcus faecalis, Serratia marcescens, Proteus mirabilis,
Enterobacter clocae, Acetinobacter anitratus, Klebsiella pneumonia,
and Escherichia coli.
[0013] In another aspect, the present invention features a method
for detecting the presence or absence of a wound infection in a
subject, comprising the steps of a) contacting a sample obtained
from a wound in a subject with a detectably labeled synthetic
serpin RSL domain peptide substrate under conditions that result in
modification of the substrate by an enzyme produced and/or secreted
by a bacterium; and b) detecting a modification or the absence of a
modification of the substrate. Modification of the substrate
indicates the presence of a wound infection in the subject, and the
absence of modification of the substrate indicates the absence of
an infection in the subject.
[0014] In yet another aspect, the present invention features a
method for detecting the presence or absence of a wound infection
in a subject, comprising the steps of a) contacting a wound in a
subject with a detectably labeled synthetic serpin RSL domain
peptide substrate, for an enzyme produced and/or secreted by a
bacterium, under conditions that result in modification of the
substrate by an enzyme produced and/or secreted by the bacterium;
and b) detecting a modification or the absence of a modification of
the substrate. Modification of the substrate indicates the presence
of a wound infection in the subject, and the absence of
modification of the substrate indicates the absence of an infection
in the subject.
[0015] In another aspect, the invention features a biosensor for
detecting the presence or absence of a bacterium, for example, a
wound-specific bacteria in a sample, comprising a solid support and
a detectably labeled synthetic serpin RSL domain peptide substrate,
wherein the substrate is attached to the solid support. The peptide
may be labeled with fluorescent or colorimetric dyes for detection
of peptide cleavage by a bacterial protease.
[0016] In still another aspect, the present invention features a
kit for detecting a wound infection, comprising a biosensor for
detecting the presence or absence of a bacterium in a sample, and
one or more reagents for detecting the presence of the bacterium
that is the causative agent of the wound infection. For example,
the reagent can be used to detect an enzyme secreted by a
bacterium. In particular, the reagent can be used to detect the
modification of the substrate of the biosensor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIGS. 1A and 1B. FIG. 1A depicts the relative position of
the cleavage sites on the alpha-1-proteinase inhibitor
(.alpha.1-P1) RSL sequence (SEQ ID NO:4). FIG. 1B depicts the
sequences of the CPI1 peptide (SEQ ID NO: 1), the CPI2 peptide (SEQ
ID NO:2) and the alpha-1 proteinase inhibitor (human) (SEQ ID
NO:3).
[0018] FIGS. 2A through 2D. FIGS. 2A and 2B are graphs of the
relative fluorescence of samples containing overnight bacterial
culture, assay substrate (FIG. 2A, CPI1 Peptide; FIG. 2B, CPI2
Peptide), and reaction buffer (pH 7.2) with 150 mM NaCl over time
(in seconds). FIGS. 2C and 2D are graphs of the relative
fluorescence of samples containing overnight bacterial supernatant,
assay substrate (FIG. 2C, CPI1 Peptide;
[0019] FIG. 2D, CPI2 Peptide), and reaction buffer (pH 7.2) with
150 mM NaCl over time (in seconds).
[0020] FIGS. 3A through 3D. FIGS. 3A and 3B are graphs of the
relative fluorescence of samples containing 48 hour bacterial
culture, assay substrate (FIG. 3A, CPI1; FIG. 3B, CPI2), and
reaction buffer over time (in seconds). FIGS. 3C and 3D are graphs
of the relative fluorescence of samples containing 48 hour
bacterial supernatant, assay substrate (FIG. 3C, CPI1; FIG. 3D,
CPI2), and reaction buffer over time (in seconds).
[0021] FIGS. 4A and 4B illustrate sensor data for E. faecalis (with
a concentration series of: 10.sup.7, 10.sup.6, 10.sup.5 and
10.sup.4 cells/ml) with CPI2 peptide. FIG. 4A shows images of
sensors incubated with bacteria under 365 nm UV light source. FIG.
4B shows intensity data from each concentration obtained from
digitized photo.
[0022] FIGS. 5A and 5B illustrate sensor data for P. aeruginosa
(with a concentration series of: 10.sup.7, 10.sup.6, 10.sup.5 and
10.sup.4 cells/ml) with CPI2 peptide. FIG. 5A shows images of
sensors incubated with bacteria under 365 nm UV light source. FIG.
5B shows intensity data from each concentration obtained from
digitized photo.
[0023] FIGS. 6A and 6B illustrate sensor data for S. aureus (with a
concentration series of: 10.sup.7, 10.sup.6, 10.sup.5 and 10.sup.4
cells/ml) with CPI2 peptide. FIG. 6A shows images of sensors
incubated with bacteria under 365 nm UV light source. FIG. 6B shows
intensity data from each concentration obtained from digitized
photo.
[0024] FIGS. 7A and 7B illustrate sensor data for S. pyogenes (with
a concentration series of: 10.sup.7, 10.sup.6, 10.sup.5 and
10.sup.4 cells/ml) with CPI2 peptide. FIG. 7A shows images of
sensors incubated with bacteria under 365 nm UV light source. FIG.
7B shows intensity data from each concentration obtained from
digitized photo.
[0025] FIGS. 8A and 8B illustrate sensor data for S. marcescens
(with a concentration series of: 10.sup.7, 10.sup.6, 10.sup.5 and
10.sup.4 cells/ml) with CPI2 peptide. FIG. 8A shows images of
sensors incubated with bacteria under 365 nm UV light source. FIG.
8B shows intensity data from each concentration obtained from
digitized photo.
[0026] FIGS. 9A through 9C are bar graphs illustrating the relative
incubation for 40 hours with differing concentrations of bacteria.
(A=10.sup.6 CFU; B=10.sup.5 CFU; C=10.sup.4 CFU)
(Staph=Staphylococcus aureus; Serratia=Serraria marcescens;
Strep=Streptococcus salivarius; PA14=Pseudomonas aeruginosa;
Entero=Enterococcus faecalis).
[0027] FIGS. 10A through 10D are graphs illustrating the relative
fluorescence of bacteria extracted from wound dressings (peptide
substrate: CPI2) (reaction buffer: 1.times.PBS). (A: wound sample
nos. 1-10; B: wound sample nos. 11-20; C: wound sample nos. 20-30;
D: wound sample nos. 31-35).
[0028] FIG. 11 illustrates a graph of fluorescent measured from
various samples taken during a stability study.
[0029] FIG. 12 illustrates a graph of relative fluorescence
measured from a detectably labeled substrates after exposure to
various strains of a wound pathogen.
DETAILED DESCRIPTION OF THE INVENTION
[0030] As part of their normal growth processes, many
microorganisms secrete a number of enzymes into their growth
environment. These enzymes have numerous functions including, but
not limited to, the release of nutrients, protection against host
defenses, cell envelope synthesis (in bacteria) and/or maintenance,
and others as yet undetermined. Many microorganisms, such as
bacteria, also produce enzymes on the cell surface that are exposed
to (and interact with) the extracellular environment.
[0031] A system that can detect the presence of these enzymes that
are produced and/or secreted can equally serve to indicate the
presence of the producing/secreting bacteria. Alternatively, a
system that can detect the absence of these enzymes that are
produced and/or secreted can equally serve to indicate the absence
of the producing/secreting bacteria. Such a detection system is
useful for detecting or diagnosing an infection, for example, a
wound infection.
[0032] The present invention pertains to the use of substrates,
such as synthetic serpin RSL domain peptide substrates, to detect
the presence or absence of enzymes produced or secreted by a broad
spectrum of bacteria. As used herein, "synthetic" refers to a
non-naturally occurring peptide. The synthetic peptides can be
derived from (e.g., a variant, analog or fragment of) a full-length
or wild-type reactive site loop (RSL) of a member of the serpin
family (e.g., .alpha.1-PI). In one embodiment, the synthetic
peptide substrate is prepared according to the methods exemplified
herein. These synthetic substrates can be then labeled with a
detectable label such that under conditions wherein their
respective enzymes specifically react with them, they undergo a
modification, for example, a visible color change that is observed.
Substrates for use in the present invention include any molecule,
either synthetic or naturally-occurring, such as a molecule
comprising a cleavage site of the RSL sequence that can interact
with an enzyme of the present invention. The relative position of
the cleavage sites on the RSL sequence of the alpha-1-proteinase
inhibitor (.alpha.1-P1) has been previously reported (Nelson, D. et
al., "Inactivation of alpha 1-proteinase inhibitor as a broad
screen for detecting proteolytic activities in unknown samples,"
Anal. Biochem., 260(2):230-36 (1998)) and is represented in FIG.
1A. Examples of substrates include those substrates described
herein, as well as substrates for these enzymes that are known in
the art. In one embodiment, the substrate comprises the sequence
EAAGAMFLEAIPK (SEQ ID NO:1). In another embodiment, the substrate
comprises EGAMFLEAIPMSIPK (SEQ ID NO:2). Other examples include
.alpha.1-PI derived fluorescent peptides, for example,
Edans-EAAGAMFLEAIPK-Dabcyl (CPI1) and Edans-EGAMFLEAIPMSIPK-Dabcyl
(FIG. 1B).
[0033] Substrates for use in the present invention also include
colorimetric and or fluorometric components and a peptide, and
interact with at least one protein produced by and/or secreted by a
microorganism. In some embodiments, the peptide portion of the
substrate interacts with the protein of the microorganism. In other
embodiments, at least one colorimetric component portion of the
substrate interacts with the protein of the microorganism.
[0034] Examples of substrates are described in PCT application
PCT/US03/03,172 entitled "Method For Detecting Microorganisms" by
Mitchell C. Sanders, et al. filed on Jan. 31, 2003, and published
as WO 03/063693 on Aug. 7, 2003; and U.S. Application No.
60/578,502 entitled "Colorimetric Substrates, Colorimetric Sensors,
and Methods of Use," by Mitchell C. Sanders, et al. filed on Jun.
9, 2004. The entire teachings of these applications are
incorporated herein by reference.
[0035] The synthetic serpin RSL domain peptide substrate can be
used to detect multiple (i.e., more than one, e.g., at least 2, 3,
4, 5, 10, 15, 20, 25 or more) wound pathogens, such as bacteria.
The enzymes that are used in the bacteria detection method of the
present invention are preferably wound-specific enzymes. As used
herein, a wound-specific enzyme is an enzyme produced and/or
secreted by pathogenic bacteria, but is not produced and/or
secreted by non-pathogenic bacteria. Examples of pathogenic
bacteria include, but are not limited to, staphylococcus (for
example, Staphylococcus aureus, Staphylococcus epidermidis, or
Staphylococcus saprophyticus), streptococcus (for example,
Streptococcus pyogenes, Streptococcus pneumoniae, or Streptococcus
agalactiae), enterococcus (for example, Enterococcus faecalis, or
Enterococcus faecium), corynebacteria species (for example,
Corynebacterium diptheriae), bacillus (for example, Bacillus
anthracis), listeria (for example, Listeria monocytogenes),
Clostridium species (for example, Clostridium perfringens,
Clostridium tetanus, Clostridium botulinum, Clostridium difficile),
Neisseria species (for example, Neisseria meningitidis, or
Neisseria gonorrhoeae), E. coli, Shigella species, Salmonella
species, Yersinia species (for example, Yersinia pestis, Yersinia
pseudotuberculosis, or Yersinia enterocolitica), Vibrio cholerae.
Campylobacter species (for example, Campylobacterjejuni or
Campylobacter fetus), Helicobacter pylori, pseudomonas (for
example, Pseudomonas aeruginosa or Pseudomonas mallei), Haemophilus
influenzae, Bordetella pertussis, Mycoplasma pneumoniae, Ureaplasma
urealyticum, Legionella pneumophila, Treponema pallidum, Leptospira
interrogans, Borrelia burgdorferi, mycobacteria (for example,
Mycobacterium tuberculosis), Mycobacterium leprae, Actinoinyces
species, Nocardia species, chlamydia (for example, Chlamydia
psittaci, Chlamydia trachomatis, or Chilamydia pneumoniae),
Rickettsia (for example, Rickettsia ricketsii, Rickettsia
prowazekii or Rickettsia akari), brucella (for example, Brucella
abortus, Brucella melitensis, or Brucella suis), Proteus mirabilis,
Serratia marcescens, Enterobacter clocae, Acetinobacter anitratus,
Klebsiella pneumoniae and Francisella tularensis. Preferably, the
wound-specific bacteria is staphylococcus, streptococcus,
enterococcus, bacillus, Clostridium species, E. coli, yersinia,
pseudomonas, Proteus mirabilis, Serratia marcescens, Enterobacter
clocae, Acetinobacter anitratus, Klebsiella pneumoniae or
Mycobacterium leprae. For example, the wound-specific enzyme can be
produced and/or secreted by Staphylococcus aureus, Staphylococcus
epidermidis, Streptococcus pyogenes, Pseudomonas aeruginosa,
Enterococcus faecalis, Proteus mirabilis, Serratia marcescens,
Enterobacter clocae, Acetinobacter anitratus, Klebsiella pneumoniae
and/or Escherichia coli.
[0036] As used herein, "modification" refers to alteration of a
substrate, such as by cleavage or other directly or indirectly
detectable means. The enzymes of the present invention can modify
substrates, for example, proteins or polypeptides, by cleavage, and
such modification can be detected to determine the presence or
absence of a pathogen in a sample. One method for detecting
modification of a substrate by an enzyme is to label the substrate
with two different dyes, where one serves to quench fluorescence
resonance energy transfer (FRET) to the other when the molecules,
for example, dyes or colorimetric substances, are in close
proximity, and is measured by fluorescence.
[0037] FRET is the process of a distance dependent excited state
interaction in which the emission of one fluorescent molecule is
coupled to the excitation of another. A typical acceptor and donor
pair for resonance energy transfer consists of
4-[[-(dimethylamino)phenyl]azo]benzoic acid (DABCYL) and
5-[(2-aminoethylamino]naphthalene sulfonic acid (EDANS). EDANS is
excited by illumination with 336 nm light, and emits a photon with
wavelength 490 nm. If a DABCYL moiety is located within 20
angstroms of the EDANS, this photon will be efficiently absorbed.
DABCYL and EDANS will be attached to opposite ends of a peptide
substrate. If the substrate is intact, FRET will be very efficient.
If the peptide has been cleaved by an enzyme, the two dyes will no
longer be in close proximity and FRET will be inefficient. The
cleavage reaction can be followed by observing either a decrease in
DABCYL fluorescence or an increase in EDANS fluorescence (loss of
quenching).
[0038] If the substrate to be modified is a protein, peptide, or
polypeptide, the substrate can be produced using standard
recombinant protein techniques (see for example, Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley & Sons,
(1998), the entire teachings of which are incorporated by reference
herein). In addition, the enzymes of the present invention can also
be generated using recombinant techniques. Through an ample supply
of enzymes or their substrates, the exact site of modification can
be determined.
[0039] The substrates are labeled with a detectable label that is
used to monitor interactions between the enzyme and the substrate
and detect any substrate modifications, for example, cleavage of
the substrate or label resulting from such interactions. Examples
of detectable labels include various dyes that can be incorporated
into substrates, for example, those described herein, spin labels,
antigen tags, epitope tags, haptens, enzyme labels, prosthetic
groups, fluorescent materials, pH-sensitive materials,
chemiluminescent materials, chromogenic dyes, colorimetric
components, bioluminescent materials, and radioactive materials.
Examples of suitable enzyme labels include horseradish peroxidase,
alkaline phosphatase, .beta.-galactosidase, and
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride and
phycoerythrin; an example of a chemiluminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S, and .sup.3H. Other
examples of detectable labels include Bodipy, Pyrene, Texas Red,
IAEDANS, Dansyl Aziridine, IATR and fluorescein. Succimidyl esters,
isothiocyanates, and iodoacetamides of these labels are also
commercially available. In one embodiment, the substrate is labeled
with a fluorescent probe and a quencher dye molecule.
[0040] The substrates can be labeled with at least one colorimetric
component that is used to monitor interactions between the protein
and the substrate and detect any substrate modifications, for
example, cleavage of the peptide or label resulting from such
interactions. In this way, the colorimetric component acts as a
label or tag to indicate the presence or absence of the
modification in order to reveal the presence or absence of a
microorganism in a sample. In some embodiments the colorimetric
component is covalently attached to the peptide.
[0041] In some embodiments the protein cleaves at least a portion
of the substrate that includes a (at least one) colorimetric
component. For example, if the substrate includes a blue
colorimetric component and a yellow colorimetric component, the
uncleaved substrate can appear green. After the protein cleaves a
portion of the substrate that includes the yellow colorimetric
component, the substrate can appear blue.
[0042] In some embodiments, the modification of the substrate
includes hydrolyzing at least one peptide bond in the peptide and
results in at least a portion of the peptide being cleaved from the
substrate. The cleaved portion includes at least one colorimetric
component, resulting in a visible color change. In other
embodiments, the modification of substrate includes cleaving at
least one colorimetric compound from the peptide, resulting in a
visible color change.
[0043] The colorimetric component acts as a label or tag. In one
embodiment, at least one colorimetric component is a different
color from the other colorimetric component(s). Examples of
colorimetric components include horseradish peroxidase, alkaline
phosphatase, .beta.-galactosidase, and acetylcholinesterase;
examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin. In one embodiment of the
invention, the substrate comprises a peptide with at least two
colorimetric components, wherein each colorimetric component
comprises a different color, and wherein the substrate is attached
to a solid support. The modification of the substrate can comprise
cleaving at least a portion of the substrate, wherein the portion
includes one of the colorimetric components and the cleaving
results in a visible color change.
[0044] The sample in which the presence or absence of bacteria is
detected, or a wound infection is diagnosed, can be, for example, a
wound (e.g., a wound surface on a subject), a body fluid, such as
blood, urine, sputum, or wound fluid (for example, pus produced at
a wound site). The sample or solid support can also be any article
that bacteria may be contained on/in, for example, a catheter, a
bag (e.g., a urine collection bag, a blood collection bag or a
plasma collection bag), a disk, a polymer, a membrane, a resin, a
glass, a sponge, an article that collects the sample, an article
that contains the sample, a scope, a filter, a lens, a foam, a
cloth, a paper, a suture, a dipstick, a swab, a test tube, a well
of a microplate, contact lens solutions, or a swab from an area of
a room or building, for example, an examination room or operating
room of a healthcare facility, a bathroom, a kitchen, or a process
or manufacturing facility.
[0045] The present invention also features a biosensor for
detecting a (one or more, for example, at least 2, at least 5, at
least 10, at least 20, at least 30, at least 50, at least 75, or at
least 100) wound pathogens, e.g., bacteria described herein and for
notifying a consumer of the presence of the infection. The
biosensor can be used in healthcare settings or home-use to detect
infected wounds. It can comprise a (one or more) broad spectrum
substrate(s) that is coupled to a solid support that is proximal to
a wound or other body fluid that is being monitored for bacterial
contamination. Preferably, the substrate is a synthetic serpin RSL
domain peptide substrate covalently bound to a label and thus has a
detection signal that upon proteolysis of the substrate-label bond
indicates the presence of the bacteria.
[0046] The biosensor is made by first labeling a substrate of the
invention, such as a synthetic serpin RSL domain peptide substrate,
with one or more, and preferably, a plurality of detectable labels,
for example, chromatogenic or fluorescent leaving groups. Most
preferably, the labeling group provides a latent signal that is
activated only when the signal is proteolytically detached from the
substrate. Chromatogenic leaving groups include, for example,
para-nitroanalide groups. Should the substrate come into contact
with an enzyme secreted into a wound or other body fluid by
bacteria or presented on the surface of a bacterial cell, the
enzyme modifies the substrate in a manner that results in detection
of such a modification, for example, a change in absorbance, which
can be detected visually as a change in color (for example, on the
solid support, such as a wound dressing), or using
spectrophotometric techniques standard in the art.
[0047] The biosensor is a solid support, for example, a wound
dressing (such as a bandage, or gauze), any material that is
required to be sterile or free of microbial contamination
(contaminants), for example, a polymer, a membrane, a resin, a
glass, a sponge, a disk, a scope, a filter, a lens, a foam, a
cloth, a paper, a dipstick or a sutures, or an article that
contains or collects the sample (such as a container for holding
bodily fluids, e.g., a urine collection bag, blood or plasma
collection bag, test tube, catheter, swab, or well of a
microplate).
[0048] Typically, the solid support is made from materials suitable
for sterilization if the support directly contacts the wound or
sample. In one embodiment of the present invention, the biosensor
can be directly contacted with the wound. In some instances, a
sterile covering or layer is used to prevent contamination of the
wound or body fluid upon such direct contact. If such sterile
coverings are used, they will have properties that make them
suitable for sterilization, yet do not interfere with the
enzyme/substrate interaction. Preferably, the portion of the
biosensor that comes into contact with the wound is also
non-adherent to permit easy removal of the biosensor from the
sample surface. For example, if the biosensor comprises a wound
dressing, the dressing contacts the wound for a time sufficient for
the enzyme substrate to react and then the dressing is removed from
the wound without causing further damage to the wound or
surrounding tissue.
[0049] A broad spectrum substrate (e.g., a substrate suitable for
detection of more than one pathogen or bacterium), suitably labeled
with a detectable label, for example, a chromogenic dye, and
attached or incorporated into a sensor apparatus, can act as an
indicator of the presence or absence of multiple pathogenic
bacteria that secrete the aforementioned enzymes.
[0050] The biosensor of the present invention also can optionally
comprise one or more additional substrates (for example, at least
2, at least 5, at least 10, at least 20, at least 30, at least 50,
at least 75, or at least 100 substrates) for produced and/or
secreted enzymes of pathogenic bacteria. When more than one
substrate is utilized, each may be labeled so as to distinguish it
from another (for example, using different detectable labels)
and/or each may be localized in a particular region on the solid
support.
[0051] Substrates with hydrophobic leaving groups can be
non-covalently bound to hydrophobic surfaces. Alternatively,
hydrophilic or hydrophobic substrates can be coupled to surfaces by
disulfide or primary amine, carboxyl or hydroxyl groups. Methods
for coupling substrates to a solid support are known in the art.
For example, fluorescent and chromogenic substrates can be coupled
to solid substrates using non-essential reactive termini such as
free amines, carboxylic acids or SH groups that do not effect the
reaction with the wound pathogens. Free amines can be coupled to
carboxyl groups on the substrate using, for example, a 10 fold
molar excess of either N-ethyl-N'-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC) or
N-cyclohexyl-N'-2-(4'-methyl-morpholinium) ethyl
carbodiimide-p-toluene sulphonate (CMC) for 2 hrs at 4.degree. C.
in distilled water adjusted to pH 4.5 to stimulate the condensation
reaction to form a peptide linkage. SH groups can be reduced with
DTT or TCEP and then coupled to a free amino group on a surface
with N-e-Maleimidocaproic acid (EMCA, Griffith et al., Febs Lett.,
134:261-263 (1981), incorporated herein by reference).
[0052] The polypeptides of the invention can also comprise or
consist of fragments and variants of the broad spectrum peptide
substrates, e.g., serpin RSL domain peptide substrates, described
herein. Variants include a substantially homologous polypeptide
encoded by the same genetic locus as these peptide substrates,
e.g., the .alpha.1 RSL domain in an organism, i.e., an allelic
variant, as well as other variants. Variants also encompass
polypeptides derived from other genetic loci in an organism, but
having substantial homology to a peptide substrate described
herein. Variants also include polypeptides substantially homologous
or identical to these peptide substrates, but derived from another
organism, i.e., an ortholog. Variants also include polypeptides
that are substantially homologous or identical to these peptide
substrates, that are produced by chemical synthesis. Variants also
include polypeptides that are substantially homologous or identical
to these peptide substrates, that are produced by recombinant
methods.
[0053] The percent identity of two amino acid sequences can be
determined by aligning the sequences for optimal comparison
purposes (e.g., gaps can be introduced in the sequence of a first
sequence). The amino acids at corresponding positions are then
compared, and the percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % identity=# of identical positions/total # of
positions.times.100). In certain embodiments, the length of the
amino acid sequence aligned for comparison purposes is at least
30%, preferably, at least 40%, more preferably, at least 60%, and
even more preferably, at least 70%, 80%, 90%, or 100% of the length
of the peptide substrate sequence, e.g., the .alpha.1 RSL domain
sequence. The actual comparison of the two sequences can be
accomplished by well-known methods, for example, using a
mathematical algorithm. A preferred, non-limiting example of such a
mathematical algorithm is described in Karlin et al., Proc. Natl.
Acad. Sci. USA. 90:5873-5877 (1993), which is incorporated herein
by reference. Such an algorithm is incorporated into the BLAST
programs (version 2.2) as described in Schaffer et al. (Nucleic
Acids Res., 29:2994-3005 (2001), incorporated herein by reference).
When utilizing BLAST and Gapped BLAST programs, the default
parameters of the respective programs can be used. In one
embodiment, the database searched is a non-redundant (NR) database,
and parameters for sequence comparison can be set at: no filters:
Expect value of 10; Word Size of 3; the Matrix is BLOSUM62; and Gap
Costs have an Existence of 11 and an Extension of 1.
[0054] In another embodiment, the percent identity between two
amino acid sequences can be determined using the GAP program in the
GCG software package (Accelrys Inc., San Diego, Calif.) using
either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of
12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. In yet
another embodiment, the percent identity between two nucleic acid
sequences can be accomplished using the GAP program in the GCG
software package (Accelrys Inc.), using a gap weight of 50 and a
length weight of 3.
[0055] Other preferred sequence comparison methods are described
herein.
[0056] The invention also encompasses polypeptide substrates having
a lower degree of identity but having sufficient similarity so as
to perform one or more of the same functions performed by the
peptide substrate, e.g., the ability to act as a substrate for
enzymes produced or secreted by bacteria, for example,
wound-specific bacteria. Similarity is determined by conserved
amino acid substitution. Such substitutions are those that
substitute a given amino acid in a polypeptide by another amino
acid of like characteristics. Conservative substitutions are likely
to be phenotypically silent. Typically seen as conservative
substitutions are the replacements, one for another, among the
aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the
hydroxyl residues Ser and Thr: exchange of the acidic residues Asp
and Glu; substitution between the amide residues Asn and Gln;
exchange of the basic residues Lys and Arg; and replacements among
the aromatic residues Phe and Tyr. Guidance concerning which amino
acid changes are likely to be phenotypically silent are found in
Bowie et al., Science, 247: 1306-1310 (1990), incorporated herein
by reference).
[0057] Functional variants can also contain substitution of amino
acids similar to those in the .alpha.1 RSL domain that result in no
change or an insignificant change in function. Alternatively, such
substitutions may positively or negatively affect function to some
degree. Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region, such critical
regions include the proteolytic cleavage site for an
infection-specific protease.
[0058] Amino acids in a peptide substrate of the present invention
that are essential for cleavage by an enzyme, e.g., a protease, can
be identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science, 244: 1081-1085 (1989), incorporated herein by reference).
The latter procedure introduces a single alanine mutation at each
of the residues in the molecule (one mutation per molecule).
[0059] The invention also includes polypeptide fragments of the
peptide substrates or functional variants thereof, including
biologically active fragments with 60%, 70%, 80%, 90% or 95%
sequence homology to a synthetic or naturally-occurring peptide
substrate described herein, e.g., the .alpha.1 RSL domain sequence.
The present invention also encompasses fragments of the variants of
the polypeptides described herein. Biologically active fragments
include fragments that have retain the ability to act as substrates
for enzymes produced or secreted by bacteria, for example,
wound-specific bacteria
[0060] Fragments can be discrete (not fused to other amino acids or
polypeptides) or can be within a larger polypeptide. Further,
several fragments can be comprised within a single larger
polypeptide. In one embodiment a fragment designed for expression
in a host can have heterologous pre- and pro-polypeptide regions
fused to the amino terminus of the polypeptide fragment and an
additional region fused to the carboxyl terminus of the
fragment.
[0061] The biosensors and peptides of the present invention can be
used in any situation where it is desirable to detect the presence
or absence of bacteria, and in particular, pathogenic bacteria. For
example, bacteria that collects on work surfaces in health care
facilities, and in particular in operating rooms can be detected
with a biosensor as described herein. A substrate, or more than one
substrate, that can be modified by an enzyme secreted by or
presented on the surface of a bacteria is labeled and covalently
bound to a collector substrate, such as cotton fibers on the tip of
a swab. When more than one substrate is utilized, each may be
labeled so as to distinguish it from another (for example, using
different detectable labels) and/or each may be localized in a
particular region on the solid support. The swab tip is used to
wipe the surface suspected of being contaminated by bacteria. The
swab tip is placed in a medium and incubated using conditions that
allow modification of the labeled substrate if an enzyme specific
for the bound, labeled substrate(s) is present.
[0062] The present invention also features a kit for detecting
wound-specific bacteria as described herein. The kit can comprise a
solid support, for example, having a plurality of wells (e.g., a
microtiter plate), to which a detectably labeled substrate (such as
a serpin reactive site loop (RSL) domain peptide substrate) is
linked, coupled, or attached. A means for providing one or more
buffer solutions is provided. A negative control and/or a positive
control can also be provided. Suitable controls can easily be
derived by one of skill in the art. A sample suspected of being
contaminated by a pathogen (e.g., a bacterium described herein) is
prepared using the buffer solution(s). An aliquot of the sample,
negative control, and positive control is placed in its own well
and allowed to react. Those wells where modification of the
substrate, for example, a color change, is observed are determined
to contain a microbial pathogen. Such a kit is particularly useful
for detecting a wound infection in a subject.
[0063] Also encompassed by the present invention is a kit that
comprises a biosensor, such as a packaged sterilized wound
dressing, and any additional reagents necessary to perform the
detection assay.
EXAMPLES
[0064] The present invention will now be illustrated by the
following Examples, which are not intended to be limiting in any
way.
Example 1
Preparation of Bacteria for Detection of the Absence or Presence of
Bacteria in a Sample
[0065] A culture of each of the following bacterial species was
grown overnight (O/N) in Brain Heart Infusion (BHI) broth at
37.degree. C. with vigorous shaking (200 rpm), using methods that
are standard in the art: Staphylococcus aureus; Streptococcus
pyogenes; Serratia marcescens; Escherichia coli; Pseudomonas
aeruginosa (PA414); Pseudomonas aeruginosa (GSU3); and Enterococcus
faecalis. After overnight growth, a 1 ml sample of each culture was
obtained, and the cells were removed from the culture supernatant
by centrifugation at 12,000.times.g for 5 minutes. The remaining
culture supernatants were stored on ice until required (less than
one hour). The bacteria were assayed for the presence or absence of
enzymes as described below.
[0066] Alternatively, the bacterial cells are not separated from
the culture supernatant, but rather, the assay is carried out on a
sample containing the cells still in suspension in their culture
broth. After 48 hours (two days) of growth, the procedure was
repeated.
Example 2
Extraction of Bacteria from Wound Dressings for Determining the
Absence or Presence of Bacteria in a Wound Sample
[0067] Thirty-five fresh, frozen wound dressings were obtained from
Dr. Thomas Serena, medical director of the St. Vincent Wound
Clinic, Erie, Pa., and founder of the Penn North Centers for
Advanced Wound Care. The dressings were classified as medical waste
for this study and no information on the wounds or the patients was
obtained. The dressings were from random wounds and were collected
and shipped on the same day. The dressings were shipped on dry ice
and stored immediately at -80.degree. C. upon arrival.
Extraction
[0068] On the day of analysis, the dressings were removed from the
freezer and defrosted enough to stretch out the dressings for
cutting. All work was done under sterile conditions and following
the Blood-Borne Pathogen Guidelines. A wide variety of dressing
sizes and exudates were represented. Most of the dressings were
gauze-type with some including an absorbent center (i.e., it
appeared that no hydrocolloid or advanced wound management
dressings were included). A 2.times.3.5 cm rectangle was cut from
each dressing (7 cm.sup.2 area). The most representative portion of
the dressing was chosen in the case of large dressings.
[0069] A wide variety of dressing colors and exudates were
obtained. The dressings were placed in 15 ml sterile conical tubes
with 3 ml of sterile PBS and the extraction was performed overnight
at 4.degree. C. There were several samples that turned the PBS
cloudy white immediately upon immersion of the dressing (Samples 2,
3, 9, 14, 22, 26, and 27). We suspect that these dressing samples
contained silver from treatment of the wounds. After the dressing
extraction, the color of each of the solutions was recorded. Three
0.5 ml aliquots of each extract were collected for storage and
testing.
[0070] The cleavage reaction was carried out with 10 .mu.l of wound
sample extract, 3 .mu.l of CPI2 peptide substrate (5 mg/mL in
water/DMSO) in 100 .mu.l total volume at 37.degree. C. The reaction
was followed on a fluorimetric plate reader using an excitation
wavelength of 355 nm and an emission wavelength of 485 mn (FIGS.
10A-10D).
Example 3
Broad Spectrum Assay Using Variants of .alpha.1-PI RSL Sequence
[0071] Two peptide substrates, CPI1 and CPI2, were designed to
encompass all of the cleavage sites of the RSL sequence, as shown
in FIGS. 1A and 1B. The peptide substrates were labeled with the
fluorescent probe edans
(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid) and the
quencher dye molecule dabcyl
((4-(4-(dimethylamino)phenyl)azo)benzoic acid).
TABLE-US-00001 (CPI1) (SEQ ID NO: 1) Edans-EAAGAMFLEAIPK-Dabcyl
(CPI2) (SEQ ID NO: 2) Edans-EGAMFLEAIPMSIPK-Dabcyl
[0072] The bacteria were grown in an incubator overnight at
37.degree. C. in 5 mL BHI (Brain Heart Infusion) media. Each of the
resulting cultures was split into two samples. One was used as a
culture, and the other was spun down by centrifugation and the
supernatant was collected. The assays were run in 20 mM tris buffer
(pH 7.2) with 150 mM NaCl added (PBS). The cleavage reaction was
carried out with 7 .mu.L of culture or supernatant and 3 .mu.L of
peptide substrate (5 mg/mL in water/DMSO) in 100 .mu.L total volume
at 37.degree. C. The reaction was followed on a fluorimetric plate
reader using an excitation wavelength of 355 nm and an emission
wavelength of 485 nm.
[0073] The first set of experiments was performed by addition of
the bacterial culture (media and cells) directly into the assay
solution. The first assay used overnight (one day) growth cells and
supernatants and peptides CPI1 and CPI2 as substrates. As shown in
FIGS. 2A and 2B, protease activity was observed for a number of the
bacteria with the peptide substrates.
[0074] To determine which of the bacteria have secreted proteases,
a similar experiment was performed using bacterial supernatants
obtained from the overnight grown bacterial cultures with the
peptides CPI1 and CPI2. As shown in FIGS. 2C and 2D, the results
were similar to those bacterial culture, indicating that the
proteases are secreted.
[0075] In a second set of experiments, a similar assay was
performed using 48 hour (two day) growth cells and supernatants.
The assay used CPI1 and CPI2 as substrates. The assay was performed
as previously described for the first set of experiments, with the
exception of using 5 .mu.l of bacterial supernatant. The results
are shown in FIGS. 3A-D.
Example 4
Development of Biosensor Surfaces
[0076] The attachment of molecules to surfaces can be performed by
the use of several different types of interactions. Typically,
proteins can be attached to surfaces using hydrophobic,
electrostatic, or covalent interactions. There are many
commercially available membranes and resins with a variety of
surface properties. Surfaces can also be chemically modified to
provide the required surface properties.
[0077] Commercially available transfer membranes exist for protein
and peptide binding. They consist of positively and negatively
charged polymers such as ion exchange membrane disc filters and
resins. Nitrocellulose membranes offer hydrophobic and
electrostatic interactions. Glass fiber membranes offer a
hydrophobic surface that can easily be chemically modified to add
functional groups. There are also modified polymer membranes that
offer reactive functional groups that covalently bind proteins and
peptides.
[0078] It is also possible to utilize various functional groups on
membranes or resins and a crosslinking agent to covalently link to
proteins. Crosslinking reagents contain two reactive groups thereby
providing a means of covalently linking two target functional
groups. The most common functional groups to target on proteins are
amine, thiol, carboxylic acid, and alcohol groups that are used to
form intramolecular crosslinks. Crosslinking agents can be
homobifunctional or heterobifunctional and a selection of
crosslinking agents of various lengths are commercially
available.
[0079] Metal chelate (affinity binding) interactions can provide a
stronger bond to biological molecules. A his-tag built into the
peptide substrate can be used to allow linkage to a nickel binding
resin. Histidine-tagged peptides are purified based on the ability
of consecutive histidine residues to bind to a resin (Sepharose)
containing nickel ions immobilized by covalently attached
nitrilotriacetic acid (NTA). A CPI3 peptide comprising
CGAMFLEAIPMSIPAAAHHHHH (SEQ ID NO:5) was made based on CPI2 with
the addition of a histidine tag. CPI3 was labeled at the cysteine
group with a colorimetric dye. In this example, a remazol dye,
Reactive Black 5 (RB5), was used. This dye produces a dark blue
color with a peak absorption at 595 nm.
##STR00001##
[0080] The CPI3-Reactive Black 5 labeled peptide was bound to a NTA
resin through the histidine tag. The NTA resin gave a high level of
peptide binding without non-specific binding.
[0081] Labeled CPI3 on NTA resin was cleaved overnight at
37.degree. C. with Pseudomonas aeruginosa (PA14). Cleaved peptide
was collected with a positively charged membrane (Pall SB6407)
placed in the tube with the resin and bacteria. A control sample
consisted of CPI3 on NTA resin with a positively charged membrane
in phosphate buffered saline (PBS). A strong color change was
obtained on the positively charged membrane in the sample with
bacteria when compared to the control as shown in. The color change
on the membrane indicated that the CPI3 was cleaved and the cleaved
portion with dye has diffused onto the membrane creating a blue
color.
[0082] Lysine peptide tags can be used to link to a surface such as
UltraBind.TM. (Pall Gelman Laboratory, Ann Arbor, Mich.). UltraBind
is a polyethersulfone membrane that is modified with aldehyde
groups for covalent binding of proteins. Proteins are directly
reacted with the UltraBind surface. It is also possible to link
proteins or peptides to the surface using cross linker chains. For
example, the carbodiimide, EDC
(1-ethyl-3-(3-dimethylaninopropyl)carbodiimide, hydrochloride) is
commonly used to link carboxylic acid groups to amines. The lining
of the peptide with a cross linking agent allows the choice of a
linker chain to extend the peptide off the surface of the membrane
while still covalently binding it. The linking of the peptide
through a cross linker can be optimized to make the peptide
available to the bacterial enzymes. This allows for optimization of
the reaction time of the sensor since peptide availability is
directly related to this parameter.
Example 5
Surface Sensor Sensitivity
[0083] CPI2 was determined to be a good candidate for a
broad-spectrum sensor. Surface sensors were constructed as
follows:
[0084] Membrane Used: Pall SB6407
[0085] Peptide Used: CPI2
[0086] Amount Used: 8 .mu.g
[0087] The sensors were air dried and stored overnight at
-20.degree. C. Sensors were loaded into sterile 96 well plates for
the sensitivity study.
[0088] The bacteria E. faecalis, P. aeruginosa, S. aureus, S.
pyogenes, and S. marcescens were grown in an incubator overnight at
37.degree. C. in 5 ml BHI (Brain Heart Infusion) media and diluted
to the concentrations given for each experiment. Each strain of
bacteria was diluted with PBS (pH 7.4) to obtain an optical density
(OD) of approximately 0.52 at 550 nm. This OD was assumed to
represent approximately 10.sup.8 cells/ml of bacteria. The 10.sup.8
cells/ml stock of each bacteria was diluted with PBS to obtain a
concentration series of: 10.sup.7, 10.sup.6, 10.sup.5 and 10.sup.4
cells/ml. Sensitivity testing consisted of placing 100 .mu.L of
each of the concentration series of 10.sup.7, 10.sup.6, 10.sup.5
and 10.sup.4 cells/ml (cells) onto a row of sensors. Controls for
each sensitivity testing consisted of sensors exposed to 100 .mu.L
PBS.
[0089] Data was taken at 4, 24 and 48 hours for each plate. Data
consisted of color (fluorescent) images of each plate taken under
relatively uniform conditions (lighting, exposure time, etc.) using
a Kodak DC290 digital camera. Images were analyzed using NIH
ImageJ, a public domain image processing and analysis program
developed at the National Institutes of Health (NIH). Analysis
consisted of measuring the intensity value within each well (i.e.,
the sum of the intensity values of all the pixels in the selection
divided by the number of pixels). Pixels range in value from 0 to
1. ImageJ displays zero as white and those with a value of 1 as
black. All sensor sensitivity data is plotted on an index scale
from 0 to 1 versus time and the trends accurately reflect what is
seen in the images.
[0090] The results for the E. faecalis sensor tested with a
concentration series of: 10.sup.7, 10.sup.6, 10.sup.5, and 10.sup.4
cells/ml are shown in FIGS. 4A and 4B.
[0091] The results for the P. aeruginosa sensor tested with a
concentration series of: 10.sup.7, 10.sup.6, 10.sup.5, and 10.sup.4
cells/ml are shown in FIGS. 5A and 5B.
[0092] The results for the S. aureus sensor tested with a
concentration series of: 10.sup.7, 10.sup.6, 10.sup.5, and 10.sup.4
cells/ml are shown in FIGS. 6A and 6B.
[0093] The results for the S. pyogenes sensor tested with a
concentration series of: 10.sup.7, 10.sup.6, 10.sup.5, and 10.sup.4
cells/ml are shown in FIGS. 7A and 7B.
[0094] The results for the S. marcescens sensor tested with a
concentration series of: 10.sup.7, 10.sup.6, 10.sup.5, and 10.sup.4
cells/ml are shown in FIGS. 8A and 8B.
[0095] The CPI2 peptide substrate was efficiently cleaved by the
proteases of Enterococcus, Pseudomonas. Staphylococcus.
Streptococcus, and Serratia. CPI2 peptide substrate was not cleaved
when incubated with E. coli cells or supernatant. CPI2 peptide was
not cleaved in the presence of uninfected wound fluid. At the
highest concentrations of bacteria, many of the fluorescence assays
above show quenching which is evidenced by a drop in the
fluorescence level for some of the 10.sup.7 and 10.sup.6 samples.
The assay can be repeated with less substrate on the membrane to
reduce the fluorescence quenching.
[0096] The reactivity of each sensor was compared at the end of the
sensor study (40 hours) to determine the relative signal obtained
from each. The results are shown in FIGS. 9A-9C. The CPI2 peptide
was efficient in detecting most pathogens at 10.sup.5 CFU. All five
bacteria (but not E. coli) give a strong signal at a concentration
of 10.sup.5 bacteria/ml. However, at a concentration of 10.sup.4
bacteria/ml, Serratia is not observed to give a signal that can be
significantly measured above the background.
[0097] The cross reactivity of the CPI2 peptide with harmless
bacteria and with wound fluid can be analyzed more thoroughly using
appropriate controls. The applicability of the CPI2 peptide
substrate on a wound dressing and in a in vivo pig study can also
be determined.
[0098] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Example 6
Push-Through Assay
[0099] A CPI3 peptide is a 5-histidine tagged version of the broad
spectrum peptide CPI2 used for the detection of multiple pathogens.
A cysteine group is was added on the N-terminal end to allow for
labeling with dye:
TABLE-US-00002 CPI3 [Ac]-CGAMFLEAIPMSIPAAAHHHHH-[OH]
[0100] The CPI3 peptide was labeled with tetramethylrhodamine
iodoacetamide (TMRIA) dye (available from Molecular Probes, Eugene,
Oreg.) on the cysteine group. The labeling reaction was performed
in PBS pH 7.4 with an excess of TMRIA dye. The dye to peptide ratio
was calculated to be about 1.0.
[0101] Approximately 1 mg of CPI3 labeled with TMRIA dye was bound
to 1 ml nickel-nitrilotriacetic acid (Ni-NTA) agarose beads
(obtained from Qiagen, Valencia, Calif.) through the 5-histidine
tag on the peptide. Essentially all the CPI3 bound to the Ni-NTA
beads, as evidenced by the loss of color from the solution.
[0102] A 50 .mu.l bead volume of CPI3-TMRIA was placed in tubes and
200 .mu.l of 1.times.10.sup.4 or 1.times.10 cfu per mL Enterococcus
faecalis was added and allowed to incubate for 5 minutes at room
temperature. The bacterial proteases cleaved the CPI3 such that a
dye-peptide fragment was released from the Ni-NTA beads. The beads
were separated from solution through a short centrifugation with a
microfuge.
[0103] Corresponding volumes and bacterial concentrations (e.g. 100
.mu.l of 1.times.10.sup.5 cfu/.mu.l) to obtain 10.sup.5, 10.sup.6,
10.sup.7 cfu equivalents were removed from the tubes and placed in
the tip of a 1 ml syringe. Phosphate buffered saline with no added
bacteria was used as a control. The syringe was then placed on top
of a matching sized O-ring on a polyvinylidene fluoride (PVDF)
membrane backed by filter paper and the plunger depressed to force
the liquid through the membrane. Dye-peptide fragments were
retained at the surface of the PVDF membrane and only un-dyed
liquid passed through to the filter paper. After 5 minutes, the
PVDF membrane exposed to the 10.sup.7 cfu/mL liquid exhibited the
brightest color response, while the PVDF membrane exposed to the
10.sup.6 cfu/mL liquid exhibited less of a color response than the
membrane exposed to the 10.sup.7 cfu/mL liquid. After 5 minutes,
the PVDF membrane exposed to the 10.sup.5 cfu/mL liquid exhibited
less of a color response than the membrane exposed to the 10.sup.6
cfu/mL liquid, while the membrane exposed to the 0 cfu/mL liquid
exhibited no discernable color response.
Example 7
Stability of Detectably Labeled Substrates
[0104] To evaluate the long-term stability or viability of
detectably labeled substrates, CPI2 substrates were detectably
labeled with HRP and attached to AffiGel beads. The beads were aged
at 40.degree. C. for a total of 35 days. At various intervals along
the 35-day ageing process, samples of the beads were taken and
exposed to a solution containing 10.sup.6 CFU/ml of P. aeruginosa
or a control buffer of PBS. The beads exposed to the bacterial gave
a dark blue signal, while those exposed to the control did not.
FIG. 11 illustrates a graph of the color measured of the various
samples taken during the stability study. The data shows that the
beads are very stable and do not have any problems with
deterioration of the detectable signal over time, indicating that
the beads are very durable.
Example 8
Inter-Strain Operability
[0105] A CIP2 fluorescent resonance entergy transfer (FRET) peptide
was exposed to five different clinical strains of E. faecalis
(obtained from the University of Colorado). The peptide reacted
strongly with all five strains. FIG. 12 illustrates a graph of the
relative fluorescence measured after exposure to the various
strains. The data indicates that the detectably labeled peptide
will detect the presence of different strains of a wound
pathogen.
Example 9
Reactivity of CPI2 Peptide Conjugated with HRP
[0106] The CPI2 peptide was conjugated with horse radish peroxidase
(HRP), and then coupled to various beads (e.g., affigel and agarose
beads) for liquid and solid phase assays. Some of the beads were
exposed to a solution containing 10.sup.6 CFU/ml of Pseudomonas
aeruginosa. A dark blue color formed in the presence of ABTS and
hydrogen peroxide, indicating the presence of the bacteria. The
detectable color signal worked in less than four minutes,
suggesting that the detectably labeled beads could be used as a
rapid broad spectrum point of care test useful for detecting
harmful pathogens.
Example 10
Clinical Swab Sample Tests
[0107] The CPI2 peptide was conjugated with horse radish peroxidase
(HRP), and then coupled to various beads (e.g., affigel and agarose
beads) for liquid and solid phase assays. Swab samples that had
been exposed to patient wounds were obtained from the University of
Massachusetts Medical School. The swab samples were tested for the
presence of bacteria with the following protocol:
[0108] 1) The HRP-beads were diluted 1:10 with PBS to form a bead
slurry. 10 microliters of the slurry was added to each well of a
filter plate.
[0109] 2) 10 microliters of each frozen wound sample were added to
80 microliters of PBS. The resulting solution was then added to a
well in the filter plate.
[0110] 3) The plate was incubated for four minutes, and then the
filter was spun into another plate containing US Biological stable
liquid substrate (1.46 mM ABTS and H.sub.2O.sub.2).
[0111] 4) After one minute of development time, the microplate
reader was read at 405 nm.
[0112] Samples that contained wound pathogens turned dark blue in
the presence of ABTS and hydrogen peroxide.
Sequence CWU 1
1
5113PRTArtificial SequenceSynthetic peptide sequence 1Glu Ala Ala
Gly Ala Met Phe Leu Glu Ala Ile Pro Lys 1 5 10 215PRTArtificial
SequenceSynthetic peptide sequence 2Glu Gly Ala Met Phe Leu Glu Ala
Ile Pro Met Ser Ile Pro Lys 1 5 10 15 323PRTHomo sapiens 3Lys Gly
Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met 1 5 10 15
Ser Ile Pro Pro Glu Val Lys 20 415PRTArtificial SequenceSynthetic
peptide sequence 4Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile
Pro Pro Glu 1 5 10 15 522PRTArtificial SequenceSynthetic peptide
sequence 5Cys Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile Pro
Ala Ala 1 5 10 15 Ala His His His His His 20
* * * * *