U.S. patent application number 11/934057 was filed with the patent office on 2008-05-08 for identification of bacterial species and subspecies using lipids.
This patent application is currently assigned to COLORADO STATE UNIVERSITY RESEARCH FOUNDATION. Invention is credited to Julia Mitsue Inamine Eckstein, Torsten Manfred Eckstein.
Application Number | 20080108104 11/934057 |
Document ID | / |
Family ID | 39360170 |
Filed Date | 2008-05-08 |
United States Patent
Application |
20080108104 |
Kind Code |
A1 |
Eckstein; Torsten Manfred ;
et al. |
May 8, 2008 |
IDENTIFICATION OF BACTERIAL SPECIES AND SUBSPECIES USING LIPIDS
Abstract
The use of free, extractable lipids found in bacteria for
identification of bacterial species and subspecies is described.
Bacteria have been found to differ sufficiently in their extracted
lipid compositions to effect identification using thin layer
chromatographic techniques. Burkholderia pseudomallei, Burkholderia
thailandensis, and Burkholderia mallei have been distinguished in
this manner. Lipopeptides specific to Mycobacterium avium
subspecies paratuberculosis, but not to the closely related
bacterium Mycobacterium avium subspecies avium have also been used
as a basis for bacterial subspecies identification using mass
spectrometry and seroreactivity. Mass spectrometric analysis of
total bacterial lipids of Burkholderia pseudomallei, Burkholderia
thailandensis, and Burkholderia mallei, and mass spectrometric
analysis of total bacterial lipids for Mycobacterium avium
subspecies paratuberculosis and Mycobacterium avium subspecies
avium, without further lipid separation, has shown that species and
subspecies of bacteria may be identified using such analysis.
Inventors: |
Eckstein; Torsten Manfred;
(Fort Collins, CO) ; Eckstein; Julia Mitsue Inamine;
(Fort Collins, CO) |
Correspondence
Address: |
COCHRAN FREUND & YOUNG LLC
2026 CARIBOU DR
SUITE 201
FORT COLLINS
CO
80525
US
|
Assignee: |
COLORADO STATE UNIVERSITY RESEARCH
FOUNDATION
601 S. Howes (410 University Services Center)
Fort Collins
CO
80521
|
Family ID: |
39360170 |
Appl. No.: |
11/934057 |
Filed: |
November 1, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60856118 |
Nov 2, 2006 |
|
|
|
Current U.S.
Class: |
435/34 |
Current CPC
Class: |
G01N 2333/32 20130101;
C12Q 1/04 20130101; G01N 2333/35 20130101 |
Class at
Publication: |
435/034 |
International
Class: |
C12Q 1/04 20060101
C12Q001/04 |
Goverment Interests
STATEMENT REGARDING FEDERAL RIGHTS
[0002] This invention was made with government support under
Contract Numbers: P01-AI-046393; P01-AI-057836; R01-AI-033706;
R01-AI-051283; R01-AI-053796; U54-AI-065357; and R37-AI-018357,
awarded by the National Institute of Allergy and Infectious
Diseases of the U.S. National Institutes of Health to Colorado
State University; Contract Number 2004-35605-14243 USDA/CSREES,
awarded by the U.S. Department of Agriculture Coop State Research
and Extension Service to Colorado State University; and Contract
Number Q6286224112 from the Johne's Disease Integrated Program
(JDIP) awarded as a subcontract from the University of Minnesota to
Colorado State University, awarded to the University of Minnesota
by the U.S. Department of Agriculture (CREESE-NRI). The government
has certain rights in the invention.
Claims
1. A method for identifying a single bacterial species containing
at least one extractable lipid, comprising the steps of: extracting
said at least one bacterial lipid using an organic solvent; and
identifying said at least one lipid by its polarity.
2. The method of claim 1, further comprising the steps of: removing
the organic solvent; dissolving the at least one lipid in a solvent
having a chosen polarity; and identifying said at least one lipid
using two-dimensional thin-layer chromatography.
3. The method of claim 2, wherein the two-dimensional thin-layer
chromatography comprises silica-based gels.
4. The method of claim 2, wherein the solvent having a chosen
polarity is selected from the group consisting of chloroform,
methanol, water, petroleum-ether, acetone, ethyl acetate, toluene,
and mixtures thereof.
5. The method of claim 1, further comprising the step of
lyophilizing the bacterial cells before said step of extracting
said at least one lipid.
6. The method of claim 1, wherein said bacterial species is
selected from the group consisting of Burkholderia pseudomallei,
Burkholderia thailandensis, and Burkholderia mallei.
7. A method for identifying a single bacterial subspecies
containing at least one extractable lipid, comprising the steps of:
extracting said at least one bacterial lipid using an organic
solvent; and identifying said at least one lipid by its
polarity.
8. The method of claim 7, further comprising the steps of: removing
the organic solvent; dissolving said at least one lipid in a
solvent having a chosen polarity; and identifying said at least one
lipid using two-dimensional thin-layer chromatography.
9. The method of claim 8, wherein the two-dimensional thin-layer
chromatography comprises silica-based gels.
10. The method of claim 8, wherein the solvent having a chosen
polarity is selected from the group consisting of chloroform,
methanol, water, petroleum-ether, acetone, ethyl acetate, toluene,
and mixtures thereof.
11. The method of claim 7, further comprising the step of
lyophilizing the bacterial cells before said step of extracting
said at least one lipid.
12. The method of claim 7, wherein said bacterial subspecies is
selected from the group consisting of Mycobacterium avium
subspecies paratuberculosis, Mycobacterium avium subsp. avium, and
Mycobacterium avium subsp. hominissuis.
13. A method for identifying a single bacterial species containing
at least one extractable lipid, comprising the steps of: extracting
said at least one bacterial lipid using an organic solvent; and
identifying said at least one lipid by its molecular weight.
14. The method of claim 13, wherein said step of identifying said
at least one lipid by its molecular weight is accomplished using
mass spectrometry.
15. The method of claim 14, wherein the mass peak corresponding to
said at least one lipid and the fragmentation pattern thereof are
observed.
16. A method for identifying a single bacterial subspecies
containing at least one extractable lipid, comprising the steps of:
extracting said at least one bacterial lipid using an organic
solvent; and identifying said at least one lipid by its molecular
weight.
17. The method of claim 16, wherein said step of identifying said
at least one lipid by its molecular weight is accomplished using
mass spectrometry.
18. The method of claim 17, wherein the mass peak corresponding to
said at least one lipid and the fragmentation pattern thereof are
observed.
19. The method of claim 16, wherein said bacteria subspecies is
selected from the group consisting of Mycobacterium avium
subspecies paratuberculosis, Mycobacterium avium subsp. avium, and
Mycobacterium avium subsp. hominissuis.
20. The method of claim 16, wherein said at least one lipid
comprises a lipopeptide selected from the group consisting of
Para-LP-01 and Para-LP-02, whereby the presence of either of said
Para-LP-01 or Para-LP-02 confirms the presence of the bacterial
subspecies Mycobacterium avium subspecies paratuberculosis.
21. A method for identifying a single bacterial subspecies
containing at least one extractable lipid, comprising the steps of:
extracting said at least one bacterial lipid using an organic
solvent; and identifying said at least one lipid by its
immunological properties.
22. The method of claim 21, further comprising the steps of:
removing the organic solvent; dissolving said at least one lipid in
a solvent having a chosen polarity; and separating said at least
one lipid using two-dimensional thin-layer chromatography.
23. The method of claim 22, wherein said step of identifying said
at least one lipid is accomplished using an enzyme-linked
immunosorbent assay for said separated at least one lipid.
24. The method of claim 23, wherein said at least one lipid
comprises a lipopeptide selected from the group consisting of
Para-LP-01 and Para-LP-02, whereby the presence of either of said
Para-LP-01 or Para-LP-02 confirms the presence of the bacterial
subspecies Mycobacterium avium subspecies paratuberculosis.
25. The method of claim 21, wherein said step of identifying said
at least one lipid is accomplished using thin layer chromatography,
enzyme-linked immunosorbent assay.
Description
RELATED CASES
[0001] The present patent application claims the benefit of
Provisional Patent Application Ser. No. 60/856,188 filed on Nov. 2,
2006 and entitled "Major Cell Wall Lipopeptide Of Mycobacterium
Avium Subspecies" by Torsten M. Eckstein et al., which application
is hereby incorporated by reference herein for all that it
discloses and teaches.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the
identification of bacterial species and, more particularly, to the
use of lipids found in bacteria for the identification of bacterial
species and subspecies.
BACKGROUND OF THE INVENTION
[0004] Bacteria are often detected in the context of the disease
they cause, but few infectious diseases are sufficiently specific
that a physician can treat them directly. However, even for those
diseases proof is necessary. The most common identification method
is by direct culture of the specific pathogen, which is for many
pathogens the "gold-standard" of detection and determination of the
infectious disease. The second most used method for defining an
infectious disease and its causative agent is indirect
identification through antibody detection either to the whole
bacterium, crude extracts, fractions of the pathogen, or single
molecules. Indirect identification methods have the disadvantage of
identifying other bacteria instead of the actual pathogen due to
cross-reactivity of the extracts, fraction, or whole bacteria.
Therefore, such tests only provide a first step for identification.
Of particular interest are tests that focus on single molecules
such as proteins. However, such tests have not been proven to be
species-, subspecies-, or type-specific, and thus, share the same
disadvantages as exist for whole extracts or fractions of whole
bacteria (that is, cross-reactivity, as an example).
[0005] All bacteria consist of nucleic acids (RNA, DNA), proteins,
saccharides, and lipids. Although these molecules may provide
information for identifying bacterial species, subspecies and
types, the most commonly used are nucleic acids and proteins.
Comparative methods for chromosomal DNA and proteins have been
developed to distinguish these species, but these molecules are not
generally useful because of their low efficiency in specific
techniques (DNA in PCR amplification, or cross-reactivity in
hybridization techniques), or due to the large number of similar
molecules within the proteome (several thousand molecules may have
to be compared as a result of separation techniques having poor
efficiency). There are fewer molecules belonging to the group of
saccharides and lipids. However, methods for successful separation
of saccharides are not yet available.
[0006] Despite these shortcomings, DNA sequences are presently used
as amplification templates for identification of the bacterial
species, subspecies, and types. Such DNA sequences are principally
repetitive sequences for increasing the positive outcome of the PCR
amplification procedure. Since these tests are multi-factorial,
false-negative data that cannot be proven to be correct may result,
and the tests do not have a "backup" target to verify a negative
result.
[0007] The small subunit (SSU) rRNA has been found to be useful for
distinguishing species from one another with a high degree of
certainty. However, this marker can define an unknown strain of a
species only if there is a certain taxonomical distance between
this species and another. Classification/taxonomy defines the
differences between bacteria. To determine a classification or
taxonomic relationship, at least three bacteria are required,
whereas to distinguish bacteria a minimum of two bacteria are
required. Closely related species (for example, Mycobacterium avium
and Mycobacterium intracellulare) cannot be distinguished with
certainty by this method alone, and use of the SSU 16S rRNA
sequence cannot distinguish bacterial subspecies and types since
these subspecies have the same sequence. Furthermore, determining
the SSU rRNA sequence requires multifactorial amplification by
polymerase chain reaction.
[0008] Additional biological tests including DNA G+C content and
chemotaxonomic methods, such as analysis of prominent cell wall
molecules, may be required to obtain high confidence for species
identification. Other investigations use the fatty acid composition
to determine species. However, fatty acids must be generated by
chemical reactions to release them from lipid molecules.
Identification of bacterial subspecies and types may require other
biochemical, enzymatic, and/or physiological methods, including
information as to where those bacterial subspecies and types were
obtained.
[0009] Species identification begins with isolation and growth of
the bacterium either in-vitro or, if not possible, in-vivo,
followed by extraction of chromosomal DNA and PCR amplification of
the SSU 16S rRNA. However, in many situations the original sequence
is not known and amplification processes cannot be performed. To
reduce the number of possibilities, multiple standard biochemical,
enzymatic, and physiological tests allow the determination of the
bacterial species with high certainty for most bacterial species.
However, to determine bacterial subspecies and types additional
microbiological aspects must be considered, such as growth time,
growth supplements, colony morphology, as examples.
SUMMARY OF THE INVENTION
[0010] Accordingly, it is an object of the present invention to
provide a method for identifying bacterial species.
[0011] Another object of the invention is to provide a method for
identifying bacterial subspecies.
[0012] Yet another object of the present invention is to provide a
method for identifying bacterial species without the use of DNA
amplification procedures.
[0013] Still another object of the invention is to provide a method
for identifying bacterial species with a reduced number of false
negatives.
[0014] Additional objects, advantages and novel features of the
invention will be set forth in part in the description which
follows, and in part will become apparent to those skilled in the
art upon examination of the following or may be learned by practice
of the invention. The objects and advantages of the invention may
be realized and attained by means of the instrumentalities and
combinations particularly pointed out in the appended claims.
[0015] To achieve the foregoing and other objects, and in
accordance with the purposes of the present invention, as embodied
and broadly described herein, the method for identifying a single
bacterial species containing at least one extractable lipid,
hereof, includes the steps of: extracting at least one bacterial
lipid using an organic solvent; and identifying at least one lipid
by its polarity.
[0016] In another aspect of the invention, and in accordance with
its objects and purposes, the method for identifying a single
bacterial subspecies containing at least one extractable lipid,
hereof, includes the steps of: extracting at least one bacterial
lipid using an organic solvent; and identifying at least one lipid
by its polarity.
[0017] In still another aspect of the invention, and in accordance
with its objects an purposes, the method for identifying a single
bacterial species containing at least one extractable lipid,
hereof, includes the steps of: extracting at least one bacterial
lipid using an organic solvent; and identifying at least one lipid
by its molecular weight.
[0018] In yet another aspect of the invention, and in accordance
with its objects and purposes, the method for identifying a single
bacterial subspecies containing at least one extractable lipid,
hereof, includes the steps of: extracting at least one bacterial
lipid using an organic solvent; and identifying at least one lipid
by its molecular weight.
[0019] In a further aspect of the invention, and in accordance with
its objects and purposes, the method for identifying a single
bacterial subspecies containing at least one extractable lipid,
hereof, includes the steps of: extracting at least one bacterial
lipid using an organic solvent; and identifying at least one lipid
by its immunological properties.
[0020] Benefits and advantages of the present invention include,
but are not limited to, providing a method for identifying
bacterial species and subspecies without the use of amplification
procedures, and with a minimum of false negatives.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The accompanying drawings, which are incorporated in and
form a part of the specification, illustrate the embodiments of the
present invention and, together with the description, serve to
explain the principles of the invention. In the drawings:
[0022] FIGS. 1A-1F are thin layer chromatograms of Burkholderia
thailandensis (A and D), B. pseudomallei (B and E), and B. mallei
(C and F) for non-polar solvent system E (A, B, and C) and polar
solvent system A (D, E and F) of TABLE 1 hereof.
[0023] FIG. 2A illustrates a two-dimensional thin layer
chromatography plate for lipids extracted from Mycobacterium avium
subspecies paratuberculosis in non-polar solvent system E of TABLE
1 hereof, while FIG. 2B illustrates a two-dimensional thin layer
chromatography plate for lipids extracted from Mycobacterium avium
subspecies hominissuis in the same solvent system.
[0024] FIG. 3A shows the structure identified for the lipopeptide,
Para-LP-01 (lipid 1 of M. avium subsp. paratuberculosis
[(N-terminal to C-terminal) C20:0 fatty acyl
D-Phe-N-Me-L-Val-L-Ile-L-Phe-L-Ala methyl ester]), while the
characteristic ion thereof, 940.6, is clearly visible in the mass
spectrum shown in FIG. 3B.
[0025] FIG. 4A shows the structure identified for the lipopeptide
Para-LP-02 (lipid 2 of M. avium subsp. paratuberculosis
[(N-terminal to C-terminal) 6-hydroxyeicosanoic fatty acyl
D-Phe-N-Me-L-Val-L-Ile-L-Phe-L-Ala methyl ester]), while the
characteristic ion thereof, 956.7, is visible in the mass spectrum
shown in FIG. 4B.
[0026] FIG. 5 shows matrix-assisted laser desorption ionization
time-of-flight mass spectra of total bacterial lipids for
Burkholderia thailandensis, Burkholderia mallei (middle); and
Burkholderia pseudomallei (bottom).
[0027] FIG. 6 shows matrix-assisted laser desorption ionization
time-of-flight mass spectra of total bacterial lipids of
Mycobacterium avium subspecies hominissuis (top); and Mycobacterium
avium subspecies paratuberculosis (bottom).
DETAILED DESCRIPTION OF THE INVENTION
[0028] Briefly, the present invention includes the use of free,
extractable lipids found in bacteria for identification of
bacterial species and subspecies. In what follows the term "lipid"
refers to molecules that are soluble in non-polar (organic)
solvents, but are barely or insoluble in water. Lipids are
therefore termed lipophilic, which refers to the ability of a
chemical compound to dissolve in fats, oils, lipids, and non-polar
solvents or mixtures of non-polar and polar solvents. Further, the
term "bacterial lipids" refers to lipid molecules generated by
bacteria and found either within the cell itself or as molecules
released into the environment surrounding the bacteria, known as
supernatant or culture filtrate, when the bacteria are grown in a
liquid medium.
[0029] The data presented below are from lipids from bacterial
cells and not from the culture filtrates. However, lipids may be
extracted from the culture filtrate which is initially lipid free,
and are important for bacterial identification.
[0030] Lipids have a variety of polarities and compositions, and
examining lipid profiles are shown to characterize bacterial
species, subspecies, and types, and to distinguish them from other
bacterial species, and subspecies (a type in some bacteria is
similar to a subspecies; in others, a type is a sub-subspecies.).
Additionally, some bacterial species and groups include more lipid
moieties than the average bacterium, making lipids valuable for
directly identifying pathogens by detecting antibodies from the
host of the infectious disease.
[0031] Lipids are molecules that can be extracted from cell samples
of bacterial species, subspecies and types using various organic
solvents (chloroform, methanol, petroleum ether, and acetone, as
examples) and mixtures of these species with water, acids, and/or
bases, as examples. The Folch wash using chloroform/methanol/water
in the ratios 6:4:1 by volume may be employed as a purification
step. Additional fractionation may be useful for enhancing the
presence of certain lipids. Lipid molecules may then be separated
by two-dimensional thin layer chromatography using a solvent system
which spans the polarity range for these molecules. As will be set
forth below, five mixtures of solvents were employed for this step.
This is usually defined as a range for which the liquids having the
strongest polarity will migrate away from the loading location, and
for the least polar system, none of the lipids will reach the final
solvent front. Lipids may be visualized using known standard
general or specific spraying methods or by UV-light detection.
[0032] Thus, lipids obtained from different bacterial species,
subspecies, and types may be separated by their polarities and, as
will be described in more detail below, it is possible to
distinguish bacterial species, subspecies and types of closely
related bacteria, by comparing their lipid profiles with those for
other species.
[0033] In accordance with one embodiment of the present invention,
specific lipid molecules have been found in certain bacteria, but
not in related species, subspecies and types. The determination of
the chemical structure and/or the characteristic profiles for each
of those molecules generated using mass spectrometric analyses and
the ionization fragmentation pattern of these molecules will
definitively identify the bacterial species, subspecies, and type.
Thus, the knowledge of the specific lipid profile, the knowledge of
the chemical structure of species-specific and subspecies-specific
lipids, and the knowledge of their ionization and fragmentation
patterns in mass spectrometric analyses provide a method for
identifying a bacterium in a mixture of bacteria, in an unknown
sample, cells, and/or tissues by analyzing the lipid extracts.
[0034] More specifically, lipids may be extracted from lyophilized
cells using chloroform/methanol (2:1 by volume), in contact with
(30 ml/g) dried cells for several hours at 55.degree. C. for 3 h.
After centrifugation at about 3500 rpm for 5 min., the supernatant
may be separated from cell debris by transfer to a new tube.
Additional lipid extraction may be performed on dried debris with
chloroform/methanol/water (10:10:3 by volume) at room temperature
for several hours (up to over night). Debris may be separated from
the supernatant by centrifugation at approximately 3500 rpm for 5
min. and transferred to the first supernatant. Supernatant may be
dried under constant flow of nitrogen gas. A Folch wash may be
performed on the crude lipid extract to remove potential salt
contamination, if necessary. The Folch wash may be performed by
adding about 6 ml of chloroform/methanol solution (2:1 by volume)
and approximately 1 ml of water to the dried crude lipid extract
with vigorous mixing. The organic layer (bottom) may be transferred
to a new tube and dried using flowing nitrogen gas. Lipids may be
extracted from culture filtrates as described for lyophilized
bacterial cells.
[0035] Lipid extraction from wet cells may be performed with slight
modification. Extraction from wet cells may be performed with
chloroform/methanol (1:1 by volume) (7 ml/1 ml wet cells) at room
temperature for about 3 h. After centrifugation at about 3500 rpm
for approximately 5 min., the supernatant may be transferred to a
new tube. Dried debris may be further extracted with
chloroform/methanol (2:1 by volume) (30 ml/g dried debris) at
55.degree. C. for about 3 h. Supernatant may be transferred to the
tube containing the first lipid extraction supernatant after
centrifugation at about 3500 rpm for approximately 5 min., and
dried under a constant flow of nitrogen gas. If necessary, a Folch
wash can be performed as described above.
[0036] Lipid separation may be accomplished using two-dimensional
thin layer chromatography (2-D TLC) on silica-based gels/plates
using five different solvent systems spanning a range of polarity
(see TABLE 1). The most polar system should allow all lipids to
move away from the loading location, and the least polar system
should not have lipids moving with the solvent front. Ideally, 200
.mu.g total lipids will be spotted onto a 2-D TLC; however, as
little as 20 .mu.g total lipids may be sufficient. After
separation, lipid spots may be visualized by spraying with a
general stain (for example, 10% CuSO.sub.4 in 8% H.sub.3PO.sub.4),
followed by heating until the spots appear. More specific stains
may be used to detect specific lipid groups such as glycolipids,
lipids containing free amino groups, phospholipids and others.
Approximately 400 .mu.g (the equivalent of a single colony) is
generally required for a single analysis. TABLE-US-00001 TABLE 1
2D-TLC solvent systems used for lipid analysis. Polarity Name
1.sup.st Dimension 2.sup.nd Dimension Polar B
Chloroform/methanol/water (60:30:6) Chloroform/acetic
acid/methanol/water (40:25:3:6) .dwnarw. A
Chloroform/methanol/water (100:14:0.8)
Chloroform/acetone/methanol/water (50:60:2.5:3) Apolar E
Chloroform/methanol (96:4) Toluene/acetone (80:20) C
Petroleum-ether/acetone (92:8) Toluene/acetone (95:1) D
Petroleum-ether/ethyl acetate (98:2) Petroleum-ether/acetone
(98:2)
[0037] Thus, the lipid profile of a bacterial species, subspecies
and type is used to distinguish a specific bacterial species or
subspecies from another bacterial species or subspecies (see
EXAMPLE 1 below).
[0038] Lipids specific to a bacterial species, subspecies and type
are used to distinguish a particular bacterial species or
subspecies from others by direct detection using mass spectrometry
(see EXAMPLE 2 below).
[0039] Some bacterial lipids are useful as diagnostic markers;
recent studies have demonstrated that bacterial lipids are
processed by CD1+ bearing cells. This permits lipids to be
recognized as specific antigens by CD1 restricted T cells, and
targeted as T cell vaccine candidates or used to develop
cellular-based immuno-detection strategies with lipids for
diagnostic purposes. Lipids also serve as B cell antigens that
generate antibodies within the host. Immunogenic lipids specific to
a bacterial species or subspecies are indirectly used for bacterial
detection by methods such as ELISA (see EXAMPLE 3 below).
[0040] Mass spectra of total bacterial lipids have proven valuable
in the identification of bacterial species and subspecies (see
EXAMPLE 4 below).
[0041] Having generally described the invention, the following
EXAMPLES provide additional details:
EXAMPLE 1
Identification of Related Bacterial Species
Burkholderia pseudomallei Versus B. thailandensis and B. mallei
[0042] Burkholderia pseudomallei is the causative agent of
melioidosis, a chronic disease in humans. The disease manifests
itself in two distinct forms: an acute infection or as an acute
bloodstream infection, and as a chronic infection. In chronic or
recurrent melioidosis, the lungs are most commonly affected.
Mortality is high--up to 86%. This disease is very common in
South-East Asia and Northern Australia. A closely related
non-pathogenic bacterium, Burkholderia thailandensis, is commonly
found in the environment. Another related bacterial pathogen is
Burkholderia mallei, the causative agent of glanders, mainly a
disease in horses, but can also affect humans. The standard method
for laboratory diagnosis of melioidosis is the isolation of the
pathogen. This generally takes at least 3 days. Humans with severe
infections, especially those with septicemia, often die before
results become available. In addition, for patients with affected
visceral organs (liver, lung, and spleen) it is almost impossible
to obtain material for culturing the pathogen. The detection of
antibodies in the sera to species-specific antigens is helpful in
the diagnosis of melioidosis. The most common serological test used
in endemic regions, the indirect hemagglutination assay (IHA) using
crude bacterial antigen-coated erythrocytes, is difficult to
perform and often has poor specificity. Newer ELISA tests use purer
B. pseudomallei antigen preparations (for example, the 200 kDa
antigen, LPS, crude culture filtrate (CCF) containing a 30 kDa
antigen, and recombinant Bps-1 antigen) with demonstrated increased
sensitivities and specificities up to 80%. However, for routine
diagnosis these methodologies have not been proven to be superior
to the IHA test.
[0043] Reference will now be made in detail to the present
embodiments of the invention, examples of which are illustrated in
the accompanying drawings. Identical numbers are used to represent
identical lipids on the two-dimensional chromatograms. Turning now
to FIG. 1, thin layer chromatograms of Burkholderia thailandensis
(A and D), B. pseudomallei (B and E), and B. mallei (C and F) are
illustrated for non-polar solvent system E (A, B, and C) and polar
solvent system A (D, E and F) described in TABLE 1 hereof.
[0044] The three related bacterial species can readily be
distinguished from one another by comparing the lipid profiles in
the two solvent systems utilized, as may be observed in TABLES 2-5
which show the Rf values of each lipid within the given system
(where the Rf value is the percentage of the distance the lipid has
moved when compared to the movement of the solvent front
representing 100% or unity), and FIGS. 1A-1F. TABLE-US-00002 TABLE
2 B. pseudomallei, FIG. 1B. Solvent System E Lipid 1.sup.st
dimension 2.sup.nd dimension 1 0.12 0.24 2 0.29 0.24 3 0.15 0.61 4
0.39 0.62
[0045] TABLE-US-00003 TABLE 3 B. mallei, FIG. 1C, Solvent System E
Lipid 1.sup.st dimension 2.sup.nd dimension A 0.45 0.62
[0046] TABLE-US-00004 TABLE 4 B. pseudomallei, FIG. 1E. Solvent
System A Lipid 1.sup.st dimension 2.sup.nd dimension 5 0.05 0.12 6
0.05 0.7 7 0.17 0.9
[0047] TABLE-US-00005 TABLE 5 B. mallei, FIG. 1F, Solvent System A
Lipid 1.sup.st dimension 2.sup.nd dimension B 0.12 0.06
[0048] The thin layer chromatograms for B. thailandensis illustrate
that there are no lipids dissolved in either solvent system E (FIG.
1A) or solvent system A (FIG. 1D).
[0049] Clearly, B. mallei presents one lipid spot in each solvent
system, while B. pseudomallei presents 4 readily observable lipids
and 3 readily observable lipids in the solvent systems E and A,
respectively. Lipid spots can be distinguished if their edges
differ by about 2%.
EXAMPLE 2
Identification of Related Bacterial Subspecies (Mycobacterium avium
Subspecies Paratuberculosis (MAP) Versus M. avium subsp. avium
(MAA) and M. avium subsp. hominissuis (MAH)) by Lipid Profiling and
by Mass Spectroscopy
A. Lipid Profiling:
[0050] Mycobacterium avium subspecies paratuberculosis is the
causative agent of Johne's disease, a chronic enteritis in
ruminants (cattle, sheep), while M. avium subsp. hominissuis and M.
avium subsp. avium are closely related subspecies of the same
species M. avium that are often found in the environment.
[0051] M. avium subsp. avium (MAA) and M. avium subsp. hominissuis
(MAH) are environmental opportunistic pathogen that cause
respiratory diseases in the general population which can be
distinguished only by the presence or absence of IS901.
Environmental mycobacteria are ubiquitous in municipal as well as
natural waters, and the primary source of human infection is
thought to be water. MAP can be distinguished from MAA/MAH by the
presence of IS900 (although similar elements are present in MAA/MAH
and other mycobacteria, and it is now being questioned whether this
is a true MAP-specific IS element), and by its growth
characteristics (MAP has the slowest growth rate, with a generation
time of 22-26 h compared to 10-12 h for MAA/MAH, and requires the
siderophore mycobactin to grow). Potential transmission mechanisms
from cattle to humans have been identified including dairy
products, meat, and contaminated water sources. If there is an
association between MAP and Crohn's disease, and if dairy products
(particularly milk) and beef are involved in transmission, the
human health concerns may be considerable.
[0052] The bacterial culturing of MAP from feces is still
considered the "gold standard" for the diagnosis of infected
animals. However, this procedure is time consuming (up to 16 weeks)
and expensive. Most of the attention is going now to the detection
of serum antibodies against MAP products and these are the bases of
commercially available test kits. However, these tests suffer from
very modest sensitivity. The overall specificities for four tests
for bovine paratuberculosis were higher than 95% but the
sensitivities were only 38.4, 26.6, 58.8, and 43.4% for the
complement fixation test, the agarose gel immunodiffusion test, an
ELISA test from Allied Laboratories, and an ELISA test from CSL,
Limited, respectively. This is a disadvantage for those tests since
their specificities are less than that for the gold standard of
fecal cultures.
[0053] As stated, the subspecies MAP and MAA/MAH can be presently
distinguished only by growth rate, colony morphology, and growth
supplements within a sample (blood, feces, tissue, saliva, milk, as
examples). FIG. 2A illustrates a two-dimensional thin layer
chromatography plate for lipids extracted from Mycobacterium avium
subspecies paratuberculosis in non-polar solvent system E, while
FIG. 2B illustrates a two-dimensional thin layer chromatography
plate for lipids extracted from Mycobacterium avium subspecies
hominissuis in the same solvent system. By analyzing the extracted
lipid profile using one of the five solvent systems described
above, M. avium subsp. paratuberculosis can clearly be
distinguished from M. avium subsp. hominissuis by lipids 1 to 3,
and by the presence or absence of lipid 1, respectively. See also
TABLES 6 and 7 which show the Rf values for the various lipids.
TABLE-US-00006 TABLE 6 Mycobacterium avium subspecies
paratuberculosis, In Solvent System 3 Lipid 1.sup.st dimension
2.sup.nd dimension 1 0.7 0.27 2 0.38 0.42 3 0.35 0.12 4 0.63 0.5 5
0.8 0.38 6 0.68 0.69 7 0.7 0.74 8 0.8 0.72
[0054] TABLE-US-00007 TABLE 7 Mycobacterium avium subspecies
hominissuis, In Solvent System 3 Lipid 1.sup.st dimension 2.sup.nd
dimension A 0.11 0 B 0.19 0.18 C 0.28 0.3 D 0.43 0.52 E 0.46 0.54 F
0.5 0.54 G 0.59 0.6 H 0.06 0.68 I 0.72 0.48 J 0.81 0.69 K 0.77
0.78
[0055] As stated above, lipid spots can be distinguished if their
edges differ by about 2%.
B. Identification of Mycobacterium avium Subsp. Paratuberculosis
from a Putative Sample by Direct Detection of Specific Lipids Using
Mass Spectrometry:
[0056] The lipid, Para-LP-01 was selected for further
characterization because it is a major cell wall-associated lipid,
was well-isolated when the total lipids were separated by 2D-TLC
using an apolar solvent system, and was present only in MAP
bacterial species.
[0057] MAP strain K-10 is a bovine isolate grown on Middlebrook
7H11 agar plates supplemented with 10% OADC (oleic acid, albumin,
dextrose, catalase) and Mycobactin J (2 .mu.g/ml). After 12 weeks
of growth at 37.degree. C., cells were harvested by scraping
colonies from the plates into PBS, pH 7.0, and centrifuging at
3,500 rpm for 30 min. Cell pellets were then lyophilized.
[0058] Total lipids were extracted from lyophilized MAP cells in
accordance with the procedure identified above, and separated by
2-dimensional (2-D) thin layer chromatography (TLC) on aluminum
backed silica 60 F.sub.254 gel plates using chloroform/methanol
(96:4) in the first dimension and toluene/acetone (80:20) in the
second dimension. Plates were sprayed with 10% copper sulfate in 8%
phosphoric acid, and the lipids visualized by heating. Total lipids
were further analyzed by differential spraying of the plates to
detect carbohydrates (using .alpha.-naphthol), free amino groups
(using ninhydrin), and phosphates (using ceric ammonium molybdate)
after heating. Several lipids were found to be specific to MAP;
two, in particular were found in significant quantities: Para-LP-01
(lipid 1 in FIG. 2A) and Para-LP-02 (lipid 2 in FIG. 2A).
[0059] Para-LP-01 was purified by preparative TLC. It was first
scraped from plates run in chloroform/methanol 96:4 by volume, and
then the lipopeptide was subjected to a second preparative TLC
purification using the solvent system of toluene/acetone 80:20 by
volume. For extraction from the silica, chloroform/methanol (2:1 by
volume) solution was used in an incubation at 4.degree. C.
overnight. Extracts were dried under nitrogen and purified by Folch
wash, and the organic layer was transferred to a fresh tube. The
dried organic layer was suspended in chloroform/methanol (2:1) at a
concentration of 10 mg/ml. Amino acids were identified following
the hydrolysis of 100 .mu.g of lipid Para-LP-01 with 6N HCl
overnight at 110.degree. C. by gas chromatography coupled to mass
spectrometry (GC/MS). Specifically, the analyte was introduced into
a DB-5 column (10 m.times.0.18 mm internal diameter, 0.18 .mu.m
film thickness) on a trace gas chromatograph connected to a mass
detector at an initial temperature of 60.degree. C. for 1 min.,
increasing to 130.degree. C. at 30.degree. C./min and finally to
280.degree. C. at 5.degree. C./min. Fatty acid analysis was
conducted by hydrolyzing the Para-LP-01 with 6 N HCl overnight at
110.degree. C. followed by 3N HCl in methanol at 85.degree. C. for
16 h. The sample was treated with TRI-SIL reagent for 20 min. at
70.degree. C. Analysis of the trimethylsilylated compounds was
performed by GC/MS using the same column and temperature program
described for amino acids. To determine the enantiometric form of
amino acids an (R)-(-)-2-butanol and an (S)-(+)-2-butanol
derivatization was performed, and the resulting O-butyl,
N-heptafluorobutyryl amino acid butyl derivatives were analyzed by
GC/MS as described above.
[0060] GC/MS analysis of the derivatized amino acids yielded three
major components corresponding to standards for alanine,
isoleucine, and phenylalanine, respectively. A minor component that
correlated with a valine standard was also identified. Details of
the gas chromatography/mass spectroscopy and NMR analyses of the
Para-LP-01 may be found in "A Major Cell Wall Lipopeptide of
Mycobacterium avium subspecies paratuberculosis," by Torsten M.
Eckstein et al., J. Biol. Chem. 281, pp. 5209-5215 (2006), the
teachings of which are hereby incorporated by reference herein.
[0061] The enantiomeric forms of the amino acids were determined by
analyzing the O-butyl, N-heptafluorobutyryl amino acid butyl
derivatives. GC analysis demonstrated that the amino acids alanine,
valine, and isoleucine were present only in the L-configuration
whereas phenylalanine was detected in both the L- and
D-configurations.
[0062] Positive ion FAB-MS (Cesium-ion Fast Atom Bombardment Mass
Spectrometry) analysis of Para-LP-01 identified the masses of
alanine (methyl ester), phenylalanine, and isoleucine, along with
seven fatty acyl chains linked to a pentapeptide core. MALDI-TOF
(Matrix-Assisted Laser Desorption Ionization Time-Of-Flight)
analysis identified seven saturated fatty acyl chains linked to the
peptide core: hexadecanoic acid, heptadecanoic acid, octadecanoic
acid, nonadecanoic acid, eicosanoic acid, heneicosanoic acid, and
docosanoic acid.
[0063] The above analyses indicate that Para-LP-01 is a lipopeptide
complex or family in which a pentapeptide core is attached to a
series of saturated fatty acids dominated by C20. From component
analysis, the calculated mass (890.6) differed from the major ion
identified by FAB-MS (918.6) by 28 amu. The nature of this
difference was examined by .sup.1H NMR and .sup.1H-.sup.13C NMR
demonstrated the presence of two phenylalanines, one alanine, one
isoleucine, and one valine, and it also provided strong evidence
for a saturated fatty acid. In addition, the calculated difference
in the mass of the lipopeptide of 28 amu could be accounted for by
N-linked and O-linked methyl groups. Further structural analysis of
the native lipopeptide and its deuteromethylated derivative using
MALDI-TOF indicated that four deuteromethyl groups were
incorporated into the lipopeptide, and that the replacement of one
methyl group by a deuteromethyl group, which had to come from an
ester, was consistent with a methyl ester at the carboxyl group.
Since five amide bonds were expected according to the data reported
above, the MALDI-TOF data showed that one amide bond was naturally
N-methylated.
[0064] ES/MS (Electrospray Mass Spectrometry) was used to determine
the location of the N-methylation, and demonstrated that the
peptide sequence is comprised of
N-Me-valine-isoleucine-phenylalanine-alanine methyl ester. Thus,
the structure of the lipopeptide consists of pentapeptide
Phe-N-Me-L-Val-L-Ile-Phe-L-Ala methyl ester N-linked to a C20 fatty
acid that was identified as the major fatty acid. As stated above,
amino acid analysis revealed the presence of both the D- and
L-isomers of phenylalanine. In silico analyses of the MAP genome
revealed two single genes and three gene clusters encoding putative
peptide synthetases. The one most likely to be involved in the
biosynthesis of the lipopeptide Para-LP-01, MAP1420, consists of
five modules, one for each of the five amino acids. The first
module contains the motif for the incorporation of an epimerized
amino acid as the first amino acid. The second module contains the
motif for N-methylation of a non-epimerized amino acid, while the
final three modules would also direct the incorporation of L-amino
acids. In addition, the first module exhibits a high degree of
homology (63% identity; 75% similarity) to the first module of the
pstA gene product in MAA that is responsible for the incorporation
of a D-phenylalanine into the lipopeptide core of the highly
immunogenic glycopeptidolipids. Thus, the structure of Para-LP-01
was determined to be: C20:0 fatty acyl
D-Phe-N-Me-L-Val-L-Ile-L-Phe-L-Ala methyl ester (N-terminal to
C-terminal), as is shown in FIG. 3A hereof.
[0065] With the identification of lipids from M. avium subsp.
paratuberculosis that are specific to this subspecies and the
identification of the chemical structure of those lipids (here
lipid 1 from FIG. 2), the presence of this subspecies has been
identified by mass spectrometry of the total lipid extract of the
sample. The characteristic ion of lipid 1 (940.6) can be clearly
identified in FIG. 3B. Further fragmentation of this ion
illustrates that the ion 940.6 belongs to lipid 1 of M. avium
subsp. paratuberculosis since those fragmentation ions and ions of
yet additional subfragmentation require a specific structure from
lipid 1 of M. avium subsp. paratuberculosis.
[0066] A similar analysis shows that the lipopeptide Para-LP-02 has
the structure shown in FIG. 4A (lipid 2 of M. avium subsp.
paratuberculosis [(N-terminal to C-terminal) 6-hydroxyeicosanoic
fatty acyl D-Phe-N-Me-L-Val-L-Ile-L-Phe-L-Ala methyl ester]), while
the characteristic ion thereof, 956.7, is visible in FIG. 4B.
Para-LP-02 differs from Para-LP-01 by its fatty acyl chain.
EXAMPLE 3
Host Immune Response to Mycobacterial Lipids
A. Microtiter Plate Elisa:
[0067] Immunogenic species, such as members of the M. avium
complex, exhibit seroreactivity of the glycopeptidolipids, which
are immunogenic molecules in the outer part of the cell envelope.
Structurally, they consist of a lipopeptide core made out of a
tetrapeptide (three amino acids and one amino alcohol) that is
N-linked to a mono- or di-unsaturated long fatty acid. O-linked to
this core molecule are mono- and oligosaccharides, which may be
further modified (for example, by methylation or acylation). These
sugar moieties are responsible for the 28 different serovars within
this complex. One characteristic of MAP is the absence of those
highly immunogenic glycopeptidolipids due to gene decay. Several
Studies have focused on the seroreactivities of lipid moieties of
mycobacteria, but also lipoglycans (lipoarabinomannan, lipomannan)
and oligosaccharides. The seroreactivities of single lipid
molecules: phenolic glycolipids (PGLs), 2,3 diacyl trehalose, and
polar lipooligosaccharides (LOS) in patients with tuberculosis have
been examined, and only the LOS antigen appears to be a potential
marker for detecting the development of tuberculosis in HIV
patients. However, in another study PGLs were used in combination
with trehalosedimycolate (TDM) and sulfolipids (SLs) to analyze the
seroreactivity with sera from patients with tuberculosis. This
study reported a sensitivity of 81.1% with specificity of
95.7%.
[0068] Enzyme-linked immunosorbent assay (ELISA) was performed at
room temperature in a 96-well microtiter plate with bovine sera and
tested by a Mycobacterium paratuberculosis Test Kit for Johne's
disease. All sera and antibody dilutions were made with 10% fetal
bovine serum in phosphate buffered saline (PBS; pH 7.4). Para-LP-01
was suspended in hexane, sonicated for 3 min., and 1 .mu.g was
loaded into each well and air-dried. Blocking was performed with
200 .mu.l of blocking buffer (3% bovine serum albumin (BSA) in PBS,
pH 7.4) for 1 h. After removing the blocking solution, serial
dilutions of the bovine sera (200 .mu.l) were added to duplicate
wells and incubated for 2 h. Wells were washed five times with 200
.mu.l blocking buffer and then 100 .mu.l of the secondary antibody
(sheep anti-bovine IgG coupled to horseradish peroxidase diluted
1:2000 was added and incubated for 2 h. The wells were washed five
times with 200 .mu.l PBS (pH 7.4) before 100 .mu.l of
3,3',5,5'-tetramethylbenzidine was added. After 5 min., the
reaction was stopped using 100 .mu.l of 2N sulfuric acid and the
OD.sub.450 was determined using a plate reader.
[0069] Para-LP-02 was found to cross-react with antibodies
generated in chicken towards Para-LP-01. Both lipids were found to
react with bovine sera from cattle with Johne's disease.
B. TLC-ELISA:
[0070] Lipids may be identified as immunogenic and thus as
diagnostic markers by thin layer chromatography enzyme-linked
immunosorbent assay (TLC-ELISA). Lipids may be separated using
two-dimensional thin layer chromatography (2D-TLC) in many solvent
systems (see Table 1, hereof, for examples). After lipid
separation, the plates may be dried at room temperature by constant
or intermittent airflow, as examples. Dry TLC plates containing
separated lipids may be blocked with 0.25% bovine serum albumin
(BSA) in phosphate buffered saline (PBS) (pH7.4) for one hour.
After removal of the blocking solution, the plates may be incubated
with specific antibodies or a host serum of interest (in 10% fetal
bovine serum, if necessary) overnight at 4.degree. C. or at room
temperature for a few hours. After several wash steps with PBS,
plates may be incubated with secondary antibodies specific for the
primary antibodies of interest or the host serum for about 1 h. The
plates may then be subjected to several washes using PBS. Color
detection may be performed by contacting the plates with TMB
(3,3',5,5'-tetramethylbenzidine) for at least 5 min., or until blue
spots appear on a light blue background. The color detection step
is then stopped by washing the plates several times with PBS.
Plates may be dried overnight for further image analyses. Dark blue
spots turn yellow to whitish color overnight, but may remain as
dark blue spots. Background color may change to light or darker
green. The TLC plates may be further analyzed by additional
staining for detection of all lipids separated by this solvent
system. Briefly, dried plates may quickly be dampened in PBS and
quickly incubated in 2N sulfuric acid. The color of the plate will
turn yellow. Lipid spots may be visualized by careful heating of
the plates until spots appear.
[0071] In summary, two major nonpolar lipids, termed Para-LP-01 and
Para-LP-02, were identified to be present only in MAP but not in
MAA. These lipids were purified and subjected to structural
analyses. The fatty acids associated with the Para-LP-01 lipid were
saturated and ranged from C16 to C22, while the fatty acids
associated with the Para-LP-02 lipid were hydroxylated at the 6
position of the saturated fatty acids ranging in size from C16 to
C22.
[0072] Para-LP-01 is a major lipid in the cell wall and is likely a
major component of the outer part of the cell envelope, just as the
glycopeptidolipids (GPLs) of MAA are surface exposed. Although MAP
is technically a member of the M. avium complex, it lacks GPLs and
is missing some of the genes responsible for their biosynthesis.
Para-LP-01 and Para-LP-02 consist of a peptide core with five amino
acids that are distinct from those found in the GPL core.
Furthermore, an additional modification of an amino acid
(N-methylation) was identified within the Para-LP-01 and Para-LP-02
that is not found in the GPLs. Finding an N-methylated valine in
the lipopeptide structure of Para-LP-01 and of Para-LP-02 was
unexpected.
[0073] Many lipid components of the cell envelope of mycobacteria
demonstrate seroreactivity. The best examples are the highly
immunogenic GPLs of the M. avium complex localized to the outer
part of the cell envelope. Although many studies have demonstrated
the seroreactivities of different lipid molecules none have
identified a lipopeptide as the target molecule. Thus, the present
MAP-specific molecules are the first described mycobacterial
lipopeptides exhibiting biological activity through its
seroreactivity with sera from cattle with Johne's disease.
EXAMPLE 4
Mass Spectra of Total Bacterial Lipids
[0074] Total lipids of the cells of single bacterial species or
subspecies or of the culture filtrate the bacteria were grown in
were extracted as described above. Mass spectra of the total
bacterial lipids were performed by MALDI-TOF using DHB
(dihydrobenzoate) as the matrix for co-crystallization. Ions appear
as a mass over charge plus sodium in general, or with hydrogen.
[0075] FIG. 5 shows MALDI-TOF mass spectra of total bacterial
lipids for Burkholderia thailandensis, Burkholderia mallei
(middle); and Burkholderia pseudomallei (bottom). The three
bacterial species can clearly be identified using the mass spectra
thereof.
[0076] FIG. 6 shows MALDI-TOF mass spectra of total bacterial
lipids of Mycobacterium avium subspecies hominissuis (top); and
Mycobacterium avium subspecies paratuberculosis (bottom). The two
bacterial subspecies can clearly be identified using the mass
spectra thereof.
[0077] The foregoing description of the invention has been
presented for purposes of illustration and description and is not
intended to be exhaustive or to limit the invention to the precise
form disclosed, and obviously many modifications and variations are
possible in light of the above teaching. The embodiments were
chosen and described in order to best explain the principles of the
invention and its practical application to thereby enable others
skilled in the art to best utilize the invention in various
embodiments and with various modifications as are suited to the
particular use contemplated. It is intended that the scope of the
invention be defined by the claims appended hereto.
* * * * *