U.S. patent application number 14/395815 was filed with the patent office on 2015-07-09 for compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof.
The applicant listed for this patent is Indiana University and Technology Corporation. Invention is credited to Pamela Brown, Yves Brun, Edward Hall, Velocity Hughes, Erkin Kuru, Srinivas Tekkam, Michael Van Nieuwenhze.
Application Number | 20150191763 14/395815 |
Document ID | / |
Family ID | 48191043 |
Filed Date | 2015-07-09 |
United States Patent
Application |
20150191763 |
Kind Code |
A1 |
Van Nieuwenhze; Michael ; et
al. |
July 9, 2015 |
COMPOSITIONS FOR IN SITU LABELING OF BACTERIAL CELL WALLS WITH
FLUOROPHORES AND METHODS OF USE THEREOF
Abstract
Disclosed herein are compositions for assessing peptidoglycan
biosynthesis in bacteria, for identifying bacteria, and for
screening for bacterial cell wall-acting and/or cell
wall-disrupting agents via modified D-amino acids and methods of
use thereof. Also disclosed are live bacteria having one or more
modified D-amino acids as described herein incorporated into
peptidoglycan of a bacterial cell wall.
Inventors: |
Van Nieuwenhze; Michael;
(Bloomington, IN) ; Hall; Edward; (Bloomington,
IN) ; Kuru; Erkin; (Bloomington, IN) ; Brown;
Pamela; (Columbia, MO) ; Tekkam; Srinivas;
(Bloomington, IN) ; Hughes; Velocity;
(Bloomington, IN) ; Brun; Yves; (Bloomington,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Indiana University and Technology Corporation |
Indianapolis |
IN |
US |
|
|
Family ID: |
48191043 |
Appl. No.: |
14/395815 |
Filed: |
April 21, 2013 |
PCT Filed: |
April 21, 2013 |
PCT NO: |
PCT/US2013/037504 |
371 Date: |
October 20, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61636640 |
Apr 21, 2012 |
|
|
|
61718048 |
Oct 24, 2012 |
|
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Current U.S.
Class: |
435/32 ;
435/252.1; 435/34; 530/322; 549/399; 560/115 |
Current CPC
Class: |
C07K 9/003 20130101;
C07D 311/74 20130101; C12Q 1/045 20130101; C07C 271/20 20130101;
C12Q 1/025 20130101; C12Q 1/18 20130101 |
International
Class: |
C12Q 1/04 20060101
C12Q001/04; C07K 9/00 20060101 C07K009/00; C07C 271/20 20060101
C07C271/20; C12Q 1/02 20060101 C12Q001/02; C07D 311/74 20060101
C07D311/74 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
A1059327 awarded by the National Institutes of Health. The United
States Government has certain rights in the invention.
Claims
1. A modified amino acid comprising a D-amino acid covalently
attached to a fluorescent label.
2. The modified amino acid of claim 1, wherein the fluorescent
label is 7-hydroxycoumarin 3-carboxylic acid (HCC--OH),
7-nitrobenzofurazan (NBD), 4-chloro-7-nitrobenzofurazan (NBD-Cl),
ADA, Cy.sub.3BADA, AF.sub.647ADA, AF.sub.350ADA, BADA, FADA, HADA,
NADA, TADA, YADA, FDL, HDL, NDL, TDL, AHA, EDA, ETDA, fluorescein
(F) or carboxytetramethylrhodamine (T).
3. The modified amino acid of claim 1, wherein the D-amino acid is
selected from the group consisting of 3-amino-D-Ala, D-Ala, D-Asp,
D-Cys, D-Glu and D-Lys.
4. A muramylpentapeptide precursor unit comprising an N-acetyl
muramic acid (NAM) moiety having a stem peptide of three to five
amino acids, wherein one or more of the amino acids in the stem
peptide comprises a modified amino acid of any one of the preceding
claims and optionally an additional modified amino acid, wherein
the additional modified amino acid comprises a clickable D-amino
acid.
5. A peptidoglycan unit comprising the muramylpentapeptide
precursor unit of claim 4 covalently linked to an N-acetyl
glucosamine (NAG) moiety.
6. A live bacterial organism comprising a bacterium having a
modified cell wall comprising modified peptidoglycan containing at
least one modified amino acid according to claim 1, and optionally
at least one additional modified amino acid, wherein the at least
one additional amino acid comprises a clickable D-amino acid.
7. A method of assessing bacterial cell wall synthesis in real
time, the method comprising: providing live bacteria with a first
amount of at least one modified amino acid of claim 1, and
optionally a second amount of at least one additional modified
amino acid comprising a clickable D-amino acid, under conditions
sufficient for bacterial cell wall synthesis, wherein the bacteria
covalently incorporate the at least one modified amino acid and
optionally the at least one additional modified amino acid into a
stem peptide of peptidoglycan of the bacterial cell wall.
8. The method of claim 7, wherein the first amount and second
amount comprise a first concentration and a second concentration,
respectively, wherein the first and second concentrations range
from about and including 0.10 .mu.M to about and including 1
mM.
9. The method of claim 7, further comprising detecting the at least
one modified amino acid, and optionally the at least one additional
modified amino acid incorporated into the stem peptide.
10. The method of claim 7, wherein the bacteria are Gram-positive
bacteria or Gram-negative bacteria.
11. A method of screening for a putative cell wall-acting agent,
the method comprising: co-contacting bacteria with an effective
amount of an agent and an amount of at least one modified amino
acid of claim 1, and optionally an amount at least one additional
modified amino acid comprising a clickable D-amino acid, under
conditions sufficient to permit ongoing peptidoglycan biosynthesis
in a bacterial cell wall, wherein the agent comprises a cell
wall-acting agent if the agent interferes with ongoing
peptidoglycan biosynthesis in the bacterial cell wall.
12. The method of claim 11, further comprising: detecting one or
more D-amino acids incorporated in the bacterial cell wall.
13. The method of claim 12, further comprising: comparing the
amount and/or identity of incorporated D-amino acids in the
bacterial cell wall resulting from contacting the bacteria with the
agent with the corresponding amount and/or identity of incorporated
D-amino acids in a bacterial cell wall resulting from contacting
the bacteria with a known cell wall-acting agent.
14. A method of screening for a putative cell wall-disrupting
agent, the method comprising: contacting modified bacteria with an
amount of an agent, wherein the agent comprises a cell
wall-disrupting agent if the agent weakens integrity of
peptidoglycan in an existing bacterial cell wall, and wherein the
modified bacteria have a modified cell wall containing modified
peptidoglycan having at least one stem peptide containing at least
one modified amino acid of claim 1, and optionally at least one
additional modified amino acid comprising a clickable D-amino
acid.
15. The method of claim 14, further comprising: detecting one or
more D-amino acids disrupted in the bacterial cell wall.
16. The method of claim 15, further comprising: comparing the
amount and/or identity of disrupted D-amino acids in the bacterial
cell wall resulting from contacting the bacteria with the agent
with the corresponding amount and/or identity of disrupted D-amino
acids in a bacterial cell wall resulting from contacting the
bacteria with a known cell wall-disrupting agent.
17. The method of claim 11, wherein the bacteria are Gram-positive
bacteria or Gram-negative bacteria.
18. A method of identifying bacteria, the method comprising:
contacting live bacteria with an amount of at least one modified
amino acid of claim 1, and optionally an amount of at least one
additional modified amino acid comprising a clickable D-amino acid,
under conditions sufficient for ongoing bacterial cell wall
synthesis, wherein the bacteria covalently incorporate into
peptidoglycan of a bacterial cell wall the at least one modified
amino acid, and optionally the at least one additional modified
amino acid, wherein each of the least one modified amino acid and
optionally the at least one additional modified amino acid
comprises spectrally distinct fluorescent label; and visualizing
the spectrally distinct fluorescent labels to determine an
incorporation pattern of the at least one modified amino acid, and
optionally the at least one additional modified amino acid, wherein
the incorporation pattern identifies the bacteria.
19. A kit for incorporating labeled D-amino acids into live
bacteria, the kit comprising: at least one modified amino acid of
claim 1; and a positive bacterial control and optionally a negative
bacterial control, wherein the positive bacterial control has at
least one modified amino acid of claim 1 incorporated into a stem
peptide of peptidoglycan of the bacterial cell wall, wherein the
optional negative bacterial control, if included, does not have the
modified amino acid of claim 1 incorporated into a stem peptide of
peptidoglycan of the bacterial cell wall.
20. The kit of claim 17, further comprising at least one clickable
D-amino acid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority under 35 U.S.C.
119 to U.S. provisional patent application Ser. No. 61/636,640,
filed Apr. 21, 2012, and entitled "COMPOSITIONS FOR COVALENTLY
LABELING BACTERIAL CELL WALLS WITH FLUOROPHORES AND METHODS OF
USE," and U.S. provisional patent application Ser. No. 61/718,048,
filed Oct. 24, 2012, and entitled "COMPOSITIONS FOR IN SITU
LABELING OF BACTERIAL CELL WALLS WITH FLUOROPHORES AND METHODS OF
USE THEREOF," the contents of both which are herein incorporated by
reference in their entireties.
FIELD OF THE INVENTION
[0003] The invention relates generally to microbiology, and more
particularly to compositions and methods for assessing cell wall
synthesis in bacteria, for identifying bacteria, and for screening
for cell wall-acting/-disrupting agents.
BACKGROUND
[0004] Bacterial growth is controlled by the domain-specific
peptidoglycan (PG) cell wall, a rigid and essential structure
composed of glycan strands cross-linked by D-amino acid
(DAA)-containing short peptides, whose biosynthesis machinery is a
target for antibiotics.
[0005] Despite the importance of PG, knowledge of its dynamics has
been severely hampered by lack of a strategy for direct imaging of
sites of PG biosynthesis in live cells. Significant limitations of
current labeling methods, such as toxic effects and poor membrane
permeability of the probes, have limited their applicability to
only a small set of bacterial species. Moreover, these methods are
labor-intensive and their sensitivity suffers from their indirect
and multiple-step nature.
[0006] Methods relying on fluorescently labeled antibiotics to
study bacterial cell wall synthesis and to discover new antibiotics
to which bacteria remain susceptible have had a profound impact on
the field. The current methods, however, have at least two inherent
limitations. First, antibiotic concentration needs to be carefully
controlled to avoid damage to the cell. Second, because these
agents bind to specific sites on cell surfaces, they only will
appear at sites of active PG biosynthesis.
[0007] For the foregoing reasons, there is a need for additional
compositions and methods for assessing PG biosynthesis in bacteria
and for discovering bacterial cell wall-acting/-disrupting agents
that can be used to treat infections caused by multidrug-resistant
bacteria.
BRIEF SUMMARY
[0008] In a first respect, a modified amino acid is disclosed that
includes a D-amino acid covalently attached to a fluorescent
label.
[0009] In a second respect, a muramylpentapeptide precursor unit is
disclosed that includes an N-acetyl muramic acid (NAM) moiety
having a stem peptide of three to five amino acids. One or more of
the amino acids in the stem peptide includes a modified amino acid
that includes a D-amino acid covalently attached to a fluorescent
label and optionally an additional modified amino acid. The
additional modified amino acid includes a clickable D-amino
acid.
[0010] In a third respect, a peptidoglycan unit is disclosed that
includes a muramylpentapeptide precursor unit as described above in
the second respect that is covalently linked to an N-acetyl
glucosamine (NAG) moiety.
[0011] In a fourth respect, a method of assessing bacterial cell
wall synthesis in real time is described. The method includes the
step of providing live bacteria with a first amount of at least one
modified amino acid comprising a D-amino acid covalently attached
to a fluorescent label, and optionally a second amount of at least
one additional modified amino acid comprising a clickable D-amino
acid, under conditions sufficient for bacterial cell wall
synthesis. The bacteria covalently incorporate the at least one
modified amino acid and optionally the at least one additional
modified amino acid into a stem peptide of peptidoglycan of the
bacterial cell wall.
[0012] In a fifth respect, a method of screening for a putative
cell wall-acting agent is disclosed. The method includes the step
of co-contacting bacteria with an effective amount of an agent and
an amount of at least one modified amino acid comprising a D-amino
acid covalently attached to a fluorescent label, and optionally an
amount at least one additional modified amino acid comprising a
clickable D-amino acid, under conditions sufficient to permit
ongoing peptidoglycan biosynthesis in a bacterial cell wall. The
agent comprises a cell wall-acting agent if the agent interferes
with ongoing peptidoglycan biosynthesis in the bacterial cell
wall.
[0013] In a sixth respect, a method of screening for a putative
cell wall-disrupting agent is disclosed. The method includes the
step contacting modified bacteria with an amount of an agent. The
agent is a cell wall-disrupting agent if the agent weakens
integrity of peptidoglycan in an existing bacterial cell wall. In
this method, the modified bacteria have a modified cell wall
containing modified peptidoglycan having at least one stem peptide
containing at least one modified amino acid comprising a D-amino
acid covalently attached to a fluorescent label, and optionally at
least one additional modified amino acid comprising a clickable
D-amino acid.
[0014] In a seventh respect, a method of identifying bacteria is
disclosed. The method includes two steps. The first step includes
contacting live bacteria with an amount of at least one modified
amino acid comprising a D-amino acid covalently attached to a
fluorescent label, and optionally an amount of at least one
additional modified amino acid comprising a clickable D-amino acid,
under conditions sufficient for ongoing bacterial cell wall
synthesis. The bacteria covalently incorporate into peptidoglycan
of a bacterial cell wall the at least one modified amino acid, and
optionally the at least one additional modified amino acid. Each of
the least one modified amino acid and optionally the at least one
additional modified amino acid comprises a spectrally distinct
fluorescent label. The second step includes visualizing the
spectrally distinct fluorescent labels to determine an
incorporation pattern of the at least one modified amino acid, and
optionally the at least one additional modified amino acid, wherein
the incorporation pattern identifies the bacteria.
[0015] In an eighth respect, a kit for incorporating labeled
D-amino acids into live bacteria is disclosed. The kit includes at
least one modified amino acid comprising a D-amino acid covalently
attached to a fluorescent label and a positive bacterial control.
The kit can include an optional negative bacterial control. The
positive bacterial control has at least one modified amino acid
comprising a D-amino acid covalently attached to a fluorescent
label incorporated into a stem peptide of peptidoglycan of the
bacterial cell wall. The optional negative bacterial control, if
included, does not have the modified amino acid comprising a
D-amino acid covalently attached to a fluorescent label
incorporated into a stem peptide of peptidoglycan of the bacterial
cell wall.
[0016] These and other features, objects and advantages of the
present invention will become better understood from the
description that follows. In the description, reference is made to
the accompanying drawings, which form a part hereof and in which
there is shown by way of illustration, not limitation, embodiments
of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The features, objects and advantages other than those set
forth above will become more readily apparent when consideration is
given to the detailed description below. Such detailed description
makes reference to the following drawings.
[0018] FIG. 1 shows the three general stages of PG biosynthesis and
general structures of the NAM and NAG units of PG.
[0019] FIG. 2 shows exemplary D-Ala-based FDAAs. D-NBD and D-HCC
(based on (R)-diaminopropionic acid) emit in the green and blue
regions, respectively.
[0020] FIG. 3 shows results of control experiments in which the
cell walls of Agrobacterium tumefaciens (top row), Bacillus
subtilis (middle row) and Escherichia coli (bottom row) were
fluorescently labeled with fluorescent D-Ala (D-HCC) or fluorescent
L-Ala (L-HCC). An exemplary structure for D-Ala (D-HCC) also is
shown in the bottom row.
[0021] FIG. 4 shows results of a pulse chase experiment with a
fluorescent D-Ala in B. subtilis (top row) or A. tumefaciens
(bottom row).
[0022] FIG. 5 shows results of a short pulse experiment with
fluorescent D-Ala in B. subtilis.
[0023] FIG. 6 shows results of a fluorescent D-Ala derivative in a
dual-labeling format.
[0024] FIG. 7 shows exemplary structures for FDAAs, such as
HCC--OH-labeled 3-amino-D-Ala (HADA), NBD-Cl-labeled 3-amino-D-Ala
(NADA), F-labeled D-Lys (FDL) and T-labeled D-Lys (TDL), as well as
exemplary structures for CDAAs, such as EDA and ADA.
[0025] FIG. 8 shows that long labeling pulses with HADA uniformly
label PG in live E. coli (left), B. subtilis (center) and A.
tumefaciens (left). The FDAA fluorescence was retained in isolated
sacculi, which also stained with a NAG-specific wheat germ
agglutinin (WAG) lectin conjugated to Alexa Fluor.RTM. 594 (red).
Scale bars, 2 .mu.m.
[0026] FIGS. 9A-D show FDAA incorporation into the stem peptide of
the PG unit.
[0027] FIG. 9A shows a schematic representing the
muramylpentapeptide precursor as incorporated into a nascent PG
unit and a modified D-amino acid (FDAA).
[0028] FIG. 9B shows HPLC detection of modified muropeptides in E.
coli incubated with HADA, HALA and NADA, or NALA. Samples were
monitored using a dual wavelength UV monitor set for general
muropeptide detection and for FDAA-specific wavelengths. Peaks
HEC-1 and NEC-1 correspond to the HADA- or NADA-modified
muropeptides in E. coli that were further characterized by
electrospray ionization MS/MS (ESI-MS/MS).
[0029] FIG. 9C shows percentage of FDAA incorporation into the
total muropeptides varies among bacteria as revealed by HPLC
analysis.
[0030] FIG. 9D shows a schematic representing MS/MS analyses of
FDAAs exclusively incorporated into the 4th position of
muropeptides in E. coli and A. tumefaciens and the 5th position in
B. subtilis.
[0031] FIGS. 10A-F show FDAAs label diverse bacterial growth
patterns. Arrows in the triple labeling panels indicate the
sequence of labeling. White scale bars, 2 .mu.m, red scale bars, 1
.mu.m.
[0032] FIG. 10A shows time-lapse microscopy of HADA-labeled E. coli
and B. subtilis .DELTA.dacA cells imaged during growth on LB
agarose pads.
[0033] FIG. 10B shows super-resolution microscopy of E. coli after
short pulses with HADA.
[0034] FIG. 10C show super-resolution microscopy of A. tumefaciens
after short pulses with HADA.
[0035] FIG. 10D shows super-resolution microscopy of S. aureus
after a short pulse with HADA. Autofluorescence is shown in
red.
[0036] FIG. 10E shows triple labeling of A. tumefaciens with HADA
(blue), EDA (clicked with red sulfo-Cy3-azide) and NADA
(green).
[0037] FIG. 10F shows triple labeling of S. venezuelae with NADA
(green), TDL (red) and HADA (blue).
[0038] FIG. 11 shows that short pulses of HADA label distinct modes
of growth in diverse bacteria. Strains were labeled for
.about.2%-8% of the doubling time: E. coli (30 seconds), A.
tumefaciens (2 minutes), B. subtilis .DELTA.dacA (30 seconds), S.
aureus (2 minutes), L. lactis (2 minutes), S. pneumoniae (4
minutes), C. crescentus (5 minutes), Synechocystis sp. PCC 6803 (1
hour), S. venezuelae (2 minutes), B. congloneratum (8 minutes), B.
phytofirmans (20 minutes), V. Spinosum (10 minutes). Scale bars, 2
.mu.m.
[0039] FIG. 12 shows a schematic for sequentially incorporating
distinct FDAAs, such as NADA, TDL and HADA, into newly synthesized
PG in live bacteria.
[0040] While the present invention is amenable to various
modifications and alternative forms, exemplary embodiments thereof
are shown by way of example in the drawings and are herein
described in detail. It should be understood, however, that the
description of exemplary embodiments is not intended to limit the
invention to the particular forms disclosed, but on the contrary,
the intention is to cover all modifications, equivalents and
alternatives falling within the spirit and scope of the invention
as defined by the embodiments above and the claims below. Reference
should therefore be made to the embodiments and claims herein for
interpreting the scope of the invention.
DETAILED DESCRIPTION
[0041] The compositions and methods now will be described more
fully hereinafter with reference to the accompanying drawings, in
which some, but not all permutations and variations of embodiments
of the invention are shown. Indeed, the invention may be embodied
in many different forms and should not be construed as limited to
the embodiments set forth herein. These embodiments are provided in
sufficient written detail to describe and enable one skilled in the
art to make and use the invention, along with disclosure of the
best mode for practicing the invention, as defined by the claims
and equivalents thereof.
[0042] Likewise, many modifications and other embodiments of the
compositions and methods described herein will come to mind to one
of skill in the art to which the invention pertains having the
benefit of the teachings presented in the foregoing descriptions
and the associated drawings. Therefore, it is to be understood that
the invention is not to be limited to the specific embodiments
disclosed and that modifications and other embodiments are intended
to be included within the scope of the appended claims. Although
specific terms are employed herein, they are used in a generic and
descriptive sense only and not for purposes of limitation.
[0043] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
skill in the art to which the invention pertains. Although any
methods and materials similar to or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described
herein.
[0044] Moreover, reference to an element by the indefinite article
"a" or "an" does not exclude the possibility that more than one
element is present, unless the context clearly requires that there
be one and only one element. The indefinite article "a" or "an"
thus usually means "at least one."
[0045] As used herein, "about" means within a statistically
meaningful range of a value or values such as a stated
concentration, length, molecular weight, pH, sequence identity,
time frame, temperature or volume. Such a value or range can be
within an order of magnitude, typically within 20%, more typically
within 10%, and even more typically within 5% of a given value or
range. The allowable variation encompassed by "about" will depend
upon the particular system under study, and can be readily
appreciated by one of skill in the art.
Overview
[0046] Previous efforts to label PG in live bacteria principally
have relied upon cell wall-active antibiotics (e.g., vancomycin,
ramoplanin) modified with fluorophores or cell wall
precursors/substrates covalently modified with fluorescent reporter
groups. The compositions and methods described herein, however,
take advantage of mechanisms for incorporating labeled DAAs into
the stem peptides displayed on a bacterial cell wall surface.
[0047] The work described herein demonstrates how to make
derivatized DAA having a suitable label, such as an appropriate
fluorophore and how such derivatized compounds can be visualized in
live cells by fluorescence microscopy following the incorporation
of the derivatized compounds into PG and thus the cell wall. In the
range of physiologically relevant concentrations, the incorporated
FDAAs do not appear to be toxic to bacteria. Unlike previous
methods that employ covalently modified cell wall precursors, the
methods described herein do not appear to adversely affect cell
morphology. In addition, the methods described herein enable
pulse-chase experiments that cannot be easily executed in the
presence of fluorescently-modified cell wall active drugs. Because
the disclosed derivatized compounds have low or minimal toxicity to
live cells, they are ideal markers to evaluate and screen
microbiostatic or microbiotoxic compounds that do adversely affect
microorganism growth and viability, such as studies directed to
development of novel antibiotics.
[0048] Studies disclosed herein demonstrate that the compositions
and methods are applicable to a wide array of Gram-positive and
Gram-negative bacteria and provides significant utility for probing
PG biosynthesis, cell wall morphogenesis and the response of the PG
biosynthetic machinery to cell wall-active agents and/or cell
wall-disrupting agents. The present disclosure therefore provides
compositions and methods for studying bacterial cell wall PG
biosynthesis and for discovering bacterial cell wall-acting and/or
cell wall-disrupting agents.
Compositions
[0049] Fluorescent D-Amino Acids (FDAAs)
[0050] Compositions of the invention include labeled D-amino acids
(DAAs), especially fluorescent D-amino acids (FDAAs). As used
herein, "amino acid" or "amino acid residue" are used
interchangeably to mean a molecule containing a first, or alpha,
carbon attached to an amine group, a carboxylic acid group and a
side-chain that is specific to each amino acid. A natural amino
acid can include conventional elements such as carbon, hydrogen,
oxygen, nitrogen and sulfur. An amino acid may be a naturally
occurring amino acid or artificially-created unnaturally occurring
amino acid. Preferably, the amino acid is naturally occurring, and,
unless otherwise limited, may encompass known analogues/synthetics
of natural amino acids that can function in a similar manner as
naturally occurring amino acids. With the exception of glycine, the
natural amino acids all contain at least one chiral carbon atom.
These amino acids therefore exist as pairs of stereoisomers (D- and
L-isomers). Of particular interest herein are D-isomers or D-amino
acids, particularly D-Ala, D-Asp, D-Cys, D-Glu and D-Lys, which are
frequently found in the stem peptide of the PG unit.
[0051] It is well known in the art that amino acids within the same
conservative group typically can substitute for one another without
substantially affecting the function of a protein. For the purpose
of the present disclosure, such conservative groups are set forth
in Table 1 and are based preferably on shared properties, as
readily appreciated to those skilled in the art. See also, Alberts
et al., "Small molecules, energy, and biosynthesis" 56-57 In:
Molecular Biology of the Cell (Garland Publishing Inc. 3.sup.rd ed.
1994).
TABLE-US-00001 TABLE 1 Amino Acids and Their Conservative
Substitutions. Side Side Hydrop- Preferred Chain Chain athy
Conservative Residue Polarity pH Index Substitutions Ala (A)
Non-polar Neutral 1.8 Ser Arg (R) Polar Basic (strongly) -4.5 Lys,
Gln Asn (N) Polar Neutral -3.5 Gln, His Asp (D) Polar Acidic -3.5
Glu Cys (C) Non-polar Neutral 2.5 Ser Gln (Q) Polar Neutral -3.5
Asn, Lys Glu (E) Polar Acidic -3.5 Asp Gly (G) Non-polar Neutral
-0.4 Pro His (H) Polar Basic (weakly) -3.2 Asn, Gln Ile (I)
Non-polar Neutral 4.5 Leu, Val Leu (L) Non-polar Neutral 3.8 Ile,
Val Lys (K) Polar Basic -3.9 Arg, Gln Met (M) Non-polar Neutral 1.9
Leu, Ile Phe (F) Non-polar Neutral 2.8 Met, Leu, Tyr Pro (P)
Non-polar Neutral -1.6 Gly Ser (S) Polar Neutral -0.8 Thr Thr (T)
Polar Neutral -0.7 Ser Trp (W) Non-polar Neutral -0.9 Tyr Tyr (Y)
Polar Neutral -1.3 Trp, Phe Val (V) Non-polar Neutral 4.2 Ile,
Leu
[0052] The following six groups each contain amino acids that are
typical but not necessarily exclusive conservative substitutions
for one another: 1) Alanine (A), Serine (S) and Threonine (T); 2)
Aspartic acid (D) and Glutamic acid (E); 3) Asparagine (N) and
Glutamine (Q); 4) Arginine (R) and Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), and Valine (V); and 6) Phenylalanine
(F), Tyrosine (Y) and Tryptophan (W).
[0053] Examples of suitable labels for the DAAs include, but are
not limited to, radiolabels, biotin (which may be detected by
avidin or streptavidin conjugated to peroxidase), lanthanides,
alkaline phosphatase and fluorescent labels (e.g., coumarins,
fluoresceins, cyanines, bodipy dyes, green fluorescent protein,
quantum dots rhodamine, especially the Alexa Fluor.RTM. family of
fluorescent dyes available from Invitrogen/Molecular Probes). Other
labels amenable for use in the modified D-amino acids disclosed
herein include metals and isotopic labels.
[0054] Labeling of DAAs can be carried out by covalently attaching
the label to a free amine group, such as free amine groups present
on the side-chain that is specific to each amino acid. If the side
chain lacks a free amine group, one of skill in the art understands
how to add such groups, as is the case of adding such a group to
D-Ala to obtain 3-amino-D-Ala. Some labels can be detected by using
a labeled counter suitable for the detection of the label in
question. In the Examples below, 7-hydroxycoumarin 3-carboxylic
acid (HCC--OH), 7-nitrobenzofurazan (NBD),
4-chloro-7-nitrobenzofurazan (NBD-Cl), fluorescein (F) and
carboxytetramethylrhodamine (T) were covalently attached to DAAs as
labels.
[0055] Other coupling chemistries are known in the art that can be
used for introducing labels into amino acids having functional
groups other than an amine. Such amino acids include a functional
alcohol group (e.g., serine and tyrosine), thiol group (e.g.,
cysteine), or carbonyl or carboxylate group (e.g., aspartate and
glutamate). Such functional groups can be derivatized or reacted
with suitably modified, activated coupling agents having labels of
the types disclosed herein.
[0056] An example of a FDAA includes HADA, which is a
HCC--OH-labeled 3-amino-D-Ala. Another example of a FDAA includes
NADA, which is a NBD-Cl-labeled 3-amino-D-Ala. Another example
includes FDL, which is a F-labeled D-Lys. Another example is TDL,
which is a T-labeled D-Lys. Another example includes HDL, which is
a HCC--OH-labeled D-Lys. Another example includes NDL, which is a
NBD-Cl-labeled D-Lys. Another example includes FADA, which is a
F-labeled 3-amino-D-Ala. Another example includes TADA, which is a
T-labeled 3-amino-D-Ala. Other FDAAs can include a D-Glu having its
side chain modified to include a free amine group linked to any of
the fluorescent labels above (e.g., HADG, NADG, FADG and TADG).
[0057] See, e.g., FIG. 7, for other examples of preferred labels
and modified FDAAs.
[0058] Methods of fluorescently labeling and detecting amino acids
are well known in the art. See, Braun & Dittrich (2010)
Beilstein J. Org. Chem. 6:69; Katritzky & Narindoshvili (2009)
Org. Biomol. Chem. 7:627-634; Merkel et al. (2010) Chembiochem.
11:305-314; Cava et al. (2011) Cell Mol. Life Sci. 68:817-831; Lam
et al. (2009) Science 325:1552-1555; Cava et al. (2011) EMBO J.
30:3442-3453; and Lupoli et al. (2011) J. Am. Chem. Soc.
133:10748-10751.
[0059] Clickable D-Amino Acids (CDAA's)
[0060] Compositions of the invention also include clickable D-amino
acids (CDAAs). The CDAAs have a DAA backbone that includes, for
example, an alkyne or azide functional group present on the
side-chain that is specific to each amino acid that can be captured
in situ by a labeled, detecting agent carrying a conjugate
functional group via click-chemistry. Functional groups in a DAA
backbone that can be targeted by the labeled, detecting agent
include, but are not limited to, primary amines, carboxyls,
sulfhydryls, carbohydrates and carboxylic acids.
[0061] One of skill in the art is familiar with "click" chemistry,
which utilizes chemical cross-linking agents to add functional
groups to molecules. See, Kolb et al. (2001) Angew. Chem. Int. Ed.
40:2004-2021; and Evans (2007) Aust. J. Chem. 60:384-395.
[0062] Cross-linking and enrichment strategies for separating a
cross-linking reaction from enrichment steps have been developed
based on bioorthogonal chemistries including the azide-alkyne
"click" cycloaddition and Staudinger ligation using alkyne- or
azide-labeled cross-linking agents (e.g., fluorescent labels).
Azides and alkynes are not naturally found in proteins, peptides,
nucleic acids or glycans; therefore, these moieties can be
engineered onto the DAAs and labeled, detecting agent to generate
azide-containing molecules and alkyne-containing molecules that are
reactive with one another. As such, the orthogonality of azides and
alkynes to biological processes (e.g., competing reactions) is a
significant advantage of these methods. Moreover, "click"
cycloadditions can be performed under aqueous conditions, allowing
enrichment by conjugation of an appropriate affinity or labeling
tag. See, generally, Rostovtsev et al. (2002) Angew. Chem. Int. Ed.
41:2596-2599; Tornoe et al. (2002) J. Org. Chem. 67:3057-3064;
Baskin et al. (2007) Proc. Natl. Acad. Sci. USA 104:16793-16797;
Saxon et al. (2000) Science 287:2007-2010; Chowdhury et al. (2009)
Anal. Chem. 81:5524-5532; Tmka & Burlingame (2010) Mol. Cell.
Proteomics 9:2306-2317; Nessen et al. (2009) J. Proteome Res.
8:3702-3711; Vellucci et al. (2010) J. Am. Soc. Mass Spectrom.
21:1432-1445; and Jewett & Bertozzi (2010) Chem. Soc. Rev.
39:1272-1279. See also, Int'l Patent Application Publication No. WO
2012/006603. As used herein, "click chemistry" and "clickable"
therefore mean a reaction between azide-containing molecules and
alkyne-containing molecules to yield a covalent
product-1,5-disubstituted 1,2,3-triazole. The reaction can be a
copper(I)-catalyzed alkyne azide cycloaddition (CuAAC) or, in cases
where copper toxicity may be an issue, can be a
copper(I)-free-catalyzed alkyne azide cycloaddition.
[0063] Examples of functional groups for use on azide-containing
molecules and alkyne-containing molecules include, but are not
limited to, hexynyl groups, pentynyl groups, heptynyl groups,
azido-propyl groups, azido-butyl groups and azido-pentyl groups.
When the functional group of alkyne-containing molecules (e.g.,
DAAs) is an alkynyl group (e.g., hexynyl, pentynyl or heptynyl),
the functional group of azide-containing molecules (e.g., labeled,
detecting agents) has the corresponding clickable azido group.
Likewise, when the functional group of azide-containing molecules
(e.g., DAAs) is an azide group (e.g., azido-propyl, azido-butyl, or
azido-pentyl), the functional group of alkyne-containing molecules
(e.g., labeled, detecting agents) has the corresponding clickable
alkynyl group.
[0064] As such, various labeling designs for detecting agents are
known including, but not limited to, biotinylated agents,
isotope-coded agents, fluorophore-labeled agents, mass-tag-labeled
agents and chromophore-labeled agents. It is also known that the
addition of functional groups can cause the cross-linker to become
very bulky or less cell-permeable, and thus not very effective for
in vivo and/or in situ cross-linking. To reduce the total size of
the cross-linker, separation of the cross-linking step from
conjugation of affinity tags can be one effective strategy. See,
Trester-Zedlitz et al. (2003) J. Am. Chem. Soc. 125:2416-2425; Tang
et al. (2005) Anal. Chem. 77:311; Kang et al. (2009) Rapid Commun.
Mass Spectrom. 23:1719-1726; Chu et al. (2006) J. Am. Chem. Soc.
128:10362-10636; Muller et al. (2001) Anal. Chem. 73:1927-1934;
Collins et al. (2003) Bioorg. Med. Chem. Lett. 13:4023-4026;
Petrotchenko et al. (2005) Mol. Cell. Proteomics 4:1167-1179; Wine
et al. (2002) Anal. Chem. 74:1939-1945; Sinz et al. (2001)
Biochemistry 40:7903-7913; and Sinz & Wang (2004) Anal.
Biochem. 331:27-32.
[0065] In some instances, the detecting agents, whether having an
alkynyl or azido functional group, can be fluorophore-labeled.
Examples of fluorophores include, but are not limited to, Alexa
Fluor.RTM. dyes, BODIPY.RTM. dyes, fluorescein, Oregon Green.RTM.
488 and Oregon Green.RTM. 514 dyes, Rhodamine Green and Rhodamine
Green-X dyes, eosin, tetramethylrhodamine, Lissamine Rhodamine B
and Rhodamine Red-X dyes, X-Rhodamine, Texas Red.RTM. and Texas
Red.RTM.-X dyes, naphthofluorescein, Carboxyrhodamine 6G, QSY dyes:
fluorescence quenchers, nonfluorescent malachite green, coumarin
derivatives, Pacific Orange dye, cascade blue and other pyrene
derivatives, cascade yellow and other pyridyloxazole derivatives,
naphthalenes (e.g., dansyl chloride), dapoxyl dye, bimane,
1-dimethylamine-N(2-azido-ethyl) naphthalene-5-sulfonamide,
6-(6-amino-2-(2-azidoethyl)
1,3-dioxo-1H-benzo(de)-2(3H)isoquinoline, 6-(6-amino-2-(2-propinyl)
1,3-dioxo-1H-benzo(de)-2(3H)isoquinoline,
8-(4-azidoethyloxyphenyl)-2,6-diethyl-1,3,5,7-tetramethyl-4,4-difluoro-4--
bora-3a,4a-diaza-s-indacene,
8-(4-propynyloxyphenyl)-2,6-diethyl-1,3,5,7-tetramethyl-4,4-difluoro-4-bo-
ra-3a,4a-diaza-s-indacene,
1-(3-azido-propoxy)-7-methylamino-phenoxazin-3-one,
1-(2-propynyl)-7-methylamino-phenoxazin-3-one,
N-(5-(3-azidopropylamino)-9H-benzo(a)-phenoxazin-9-ylidene)-N-methyl-meth-
anaminium chloride,
N-(5-(3-propynyl-amino)-9H-benzo(a)-phenoxazin-9-ylidene)-N-methyl-methan-
aminium chloride,
(9-(3-azido-propoxy)-7-piperidin-1-yl-phenoxazin-3-ylidene)-dimethyl-ammo-
nium perchlorate. See also, Kele et al. (2009) Org. Biomol. Chem.
7:3486-3490; Nagy et al. (2010) Chem. Asian J. 5:773-777; Filnov et
al. (2011) Nat. Biotechnol. 29:757-761; Subach et al. (2011) Nature
Methods 8:771-777; Yang et al. (2011) J. Am. Chem. Soc.
133:9964-9967; Zin (2011) Nature Methods 8:726-728; and
"Fluorophores and their amine-reactive derivatives," Chapter 1 and
"Click Chemistry and other functional group modifications," Chapter
3 in The Molecular Probes.RTM. Handbook (available on the World
Wide Web at
invitrogen.com/site/us/cn/home/References/Molecular-Probes-The-Handbook.h-
tml). A variety of clickable fluorophores are commercially
available from, for example Sigma Aldrich, Active Motif Chromeon
and Invitrogen/Molecular Probes. In the examples below, CDAAs were
clicked with red sulfo-Cy3-azide.
[0066] An example of a CDAA includes EDA. Another example of a CDAA
includes ADA. See, e.g., FIG. 7.
[0067] Fluorescent Muramylpentapeptide Precursor Units (FMPUs)
[0068] Compositions of the invention also include fluorescent
muramylpentapeptide precursor units (FMPUs) having an NAM moiety
with a peptide chain of three to five amino acids in which one or
more of the amino acids in the stem peptide are FDAAs and/or CDAAs
as described herein. See, e.g., FIGS. 9A and 9D.
[0069] Fluorescent Peptidoglycan Units (FPGUs)
[0070] Compositions of the invention also include fluorescent
peptidoglycan units (FPGUs). The FPGUs have a FMPU as described
herein linked to a NAG moiety. See, e.g., FIGS. 9A and 9D.
[0071] Bacterial Cells Having Fluorescent D-Amino Acids
[0072] Compositions of the invention also include live bacteria
having FDAAs, CDAAs, FMPUs and/or FPGUs as described herein
incorporated into PG in a cell wall.
[0073] While Gram-positive bacteria tend to have a thicker PG
layer, it is intended that the bacteria can be Gram-positive
bacteria or Gram-negative bacteria. Examples of suitable
Gram-positive bacteria include, but are not limited to, Actinomyces
spp., Bacillus spp., Brachybacterium spp., Clostridium spp.,
Corynebacterium spp., Diplococcus spp., Enterococcus spp.,
Lactococcus spp., Listeria spp., Nocardia spp., Propionibacterium
spp., Staphylococcus spp., Streptococcus spp. Streptomyces spp. In
the examples below, live B. subtilis, B. conglomeratum, L. lactis,
S. aureus, S. pneumnoniae, S. venezuelae, were grown in the
presence of FDAAs and/or CDAAs.
[0074] Examples of suitable Gram-negative bacteria include, but are
not limited to, Acinetobacter spp., Agrobacterium spp., Bordetella
spp., Borrelia spp., Brucella spp., Burkholderia spp.,
Campylobacter spp., Caulobacter spp., Chlamydia spp., Enterobacter
spp., Escherichia spp., Helicobacter spp., Hemophilus spp.,
Klebsiella spp., Legionella spp., Neisseria spp., Proteus spp.,
Pseudomonas spp, Salmonella spp., Shigella spp., Synechocystis
spp., Verrucomicrobia spp., Vibrio spp. and Yersina spp. In the
examples below, live A. tumefaciens, B. phytofirmans, C.
crescentus, E. coli, Synechocystis sp. PCC 6803 and V. spinosum
were grown in the presence of FDAAs and/or CDAAs.
Kits
[0075] Compositions of the invention also include kits having one
or more FDAA, CDAA, FMPU and/or FPGU as described herein and
optionally one or more labeled detecting agents (if CDAAs are
included in the kits) for use in in situ labeling/probing of PG
during biosynthesis, as well as for screening for bacterial cell
wall-acting and/or cell wall-disrupting agents. The kits also can
include additional reagents such as unlabeled DAAs, unlabeled
L-amino acids (LAAs) and/or labeled LAAs. The kits also can include
positive and/or negative bacterial controls, where the controls
have unlabeled DAAs, CDAAs and LAAs or labeled DAAs and LAAs
incorporated into PG in a cell wall.
[0076] As used herein, "kit" means any manufacture (e.g., a package
or a container) having, for example, at least one FDAA and/or CDAA
and a positive and/or negative control. The kit may be promoted,
distributed, or sold as a unit for performing any of the methods
described herein.
[0077] Though not necessarily required, kits preferably include
instructions, procedures and/or directions that guide users or ones
skilled in the art how to use the agents, reagents, and/or other
components for their intended purpose. For example, kits can
include a package insert describing procedures for carrying out any
one of the methods described herein or analytical information for
correlating the level of expression measured in live bacteria.
Likewise, the package insert can include representative images of
positive or negative samples with low or high levels of
incorporation as compared to an appropriate control. The kits can
be promoted, distributed or sold as units for performing the
methods described below.
[0078] The kits also can include a receptacle or other means for
holding a sample to be evaluated for FDAA and/or CDAA
incorporation, and means for determining the presence and/or
quantity of FDAA and/or CDAA incorporation in live bacteria.
[0079] The kits also can include at least one buffer. Examples of
buffers include, but are not limited to, cell isolation buffers,
fixation buffers, lysis buffers, permeabilization buffers,
sonication buffers, separation buffers, stabilization buffers and
wash buffers. Though not limited, buffers include strong acids in
combination with weak bases, strong bases in combination with weak
acids, a combination of weak acids and bases, or even a small or
low concentration (e.g., within the range from about 0.1 mM to
about 10 mM) of an acid or base, in the absence of a conventional
conjugate base or acid, respectively; typically, however another
component of the mixture may provide such conjugate acid or base
function. Examples of acids and bases, both in terms of
ionization/dissociation strength (i.e., strong or weak) and type
(i.e., inorganic or organic), are well known in the art.
[0080] Any or all of the kit components can be provided within
containers that protect them from the external environment, such as
in sealed containers.
Methods
[0081] Methods include assessing bacterial cell wall biosynthesis
(and PG recycling) in real time. As shown in FIG. 1, bacterial cell
wall biosynthesis typically involves three steps: translocation,
transglycosylation and transpeptidation. In the translocation and
transglycosylation steps, carbohydrate backbone is formed by
polymerization via glycosidic bond formation between the
C(4)-hydroxyl of a membrane-bound lipid II intermediate and the
anomeric center of a membrane-bound glycan strand. Bacterial
transpeptidases mediate crosslinking of the resulting elongated
glycan strand. The cross-link is installed via attack of an amino
group, either from the Lys residue itself or from a short peptide
chain appended to the Lys residue, onto the penultimate D-Ala
residue of an adjacent pentapeptide strand and results in cleavage
of the terminal D-Ala residue. This rigid macromolecular structure,
essential to both Gram-negative and Gram-positive bacteria, enables
bacterial cells to resist lysis and, subsequently, cell death
resulting from high internal osmotic pressure.
[0082] These methods typically begin by providing live
Gram-positive or Gram-negative bacteria with FDAAs and/or CDAAs as
described herein under conditions where the bacteria can covalently
incorporate the FDAAs and/or CDAAs into PG of a bacterial cell
wall. The FDAAs and/or CDAAs can be provided to organisms
preferably within a given range of concentrations, for example,
from about 0.1 .mu.M to about 1 mM, as well as in any whole integer
or fractional integer concentration thereof within this preferred
range. The FDAAs and/or CDAAs can also be provided to organisms at
preferred concentrations, for example, at about 0.1 .mu.M and about
1 mM. Other ranges are also possible besides this preferred range
and fall within the scope of this disclosure, the specific
identification of which depends upon the particular biological
organism or system under study, as well as upon the nature of the
FDAAs and/or CDAAs used, their physiochemical properties and uptake
by the particular biological organism or system under study, as
well as the experimental set-up and purpose of the study at hand,
as one of skill in the art would understand.
[0083] Determination of the optimal concentration (or amount) of
FDAAs and/or CDAAs and the preferred ranges thereof for a
particular organism is the subject of routine experimentation well
within the purview of those skilled in the art. A typical route to
ascertaining the optimal concentrations and preferred ranges of the
FDAAs and/or CDAAs described herein is to perform a dose response
experiment, wherein the parallel populations of a given organism
are contacted with different concentrations (or amounts) of a given
FDAA and/or CDAA, and the extent of incorporation of the
compound(s) is assessed by biochemical assay (e.g., extent of
compound labeling in PG fractions) and/or by visualization methods
(e.g., fluorescence microscopy). Other approaches to selecting the
optimal concentration (of amount) of FDAAs and/or CDAAs and the
preferred ranges thereof for a particular organism are viable as
well, as one skilled in the art would readily appreciate based upon
this disclosure.
[0084] The methods also can include detecting the FDAAs and/or
CDAAs in the bacterial cell wall to verify that they have been
incorporated. The FDAAs and/or CDAAs (after being clicked) can be
detected via fluorescence microscopy and other methods, depending
upon the type of label or reporter used.
Screening Methodologies
[0085] The cell wall biosynthetic pathway is unique to bacterial
cells; therefore, agents that inhibit steps within this pathway are
anticipated to show selective toxicity toward bacterial cells. As
such, methods of the invention also can include screening for
putative cell wall-acting or cell wall-disrupting agents. As used
herein, "cell wall-acting" means an ability of an agent to
interfere with PG biosynthesis in a bacterial cell wall, especially
at the transglycosylation step, as this step takes place on the
outer leaflet of the cell membrane so cellular penetration is not a
prerequisite for the agent to manifest its biological activity. As
used herein, "cell wall-disrupting" means an ability of an agent to
disrupt or weaken the integrity of PG in an existing bacterial cell
wall.
[0086] The methods can begin by contacting bacteria with a putative
cell wall-acting agent or putative cell wall-disrupting agent,
where the agent is cell wall-acting if the agent interferes with
ongoing peptidoglycan biosynthesis in a bacterial cell wall or is
cell wall-disrupting if the agent weakens integrity of
peptidoglycan in an existing bacterial cell wall. When screening
for putative cell wall-acting agents, the bacteria can be
co-contacted with FDAAs and/or CDAAs as described herein
simultaneously with the putative agent. When screening for putative
cell wall-disrupting agents, the bacteria can have FDAAs and/or
CDAAs as described herein covalently incorporated into PG of the
cell wall prior to being contacted with the putative agent.
[0087] The methods also can include detecting whether the FDAAs
and/or CDAAs have been incorporated in the bacterial cell wall or
whether the FDAAs and/or CDAAs remain in the bacterial cell wall.
As noted above, the FDAAs and/or CDAAs (after being clicked) can be
detected via fluorescence microscopy and other methods, depending
upon the type of label or reporter used. The pattern and/or
location of FDAAs and/or CDAAs incorporation can be used to
identify the bacteria (see, e.g., FIGS. 10-12).
[0088] The methods also can include comparing the results from the
putative cell wall-acting agent or cell wall-disrupting agent with
a known cell wall-acting agent or known cell wall-disrupting
agent.
[0089] The compounds of the present disclosure have utility for
identifying bacteria. As demonstrated in the Examples set forth
herein, certain bacterial species display unique specificity for
incorporating certain D-amino acids in PG and the bacteria cell
wall. Thus, the use of the disclosed modified D-amino acids of the
present disclosure enable identification of bacterial species by
virtue of the pattern of labeling observed in the bacteria as a
result of incorporation of the modified D-amino acids into PG of
the bacterial cell wall.
EXAMPLES
[0090] The invention will be more fully understood upon
consideration of the following non-limiting examples, which are
offered for purposes of illustration, not limitation.
Example 1
[0091] Control experiments were carried out in A. tumefaciens, B.
subtilis and E. coli with fluorescent D-Ala (D-HCC) and fluorescent
L-Ala (L-HCC). For example, experiments in A. tumefaciens revealed
that only D-HCC was incorporated into the cell wall. This
observation was true for all strains tested. Likewise, experiments
with B. subtilis revealed predominant labeling at the septum, a
result consistent with this being the site of active cell wall
synthesis. Subsequent isolation of peptidoglycan from these cells
also revealed that isolated sacculi retained the fluorescent label
(FIG. 3).
[0092] In addition, experiments with a B. subtilis dacA mutant
(DacA is a D,D-carboxypeptidase that cleaves the terminal D-Ala
from the peptide stem) resulted in uniform labeling of the cell
wall, which suggested the dominant mode of labeling in B. subtilis
is at the terminal position of the peptide stem.
[0093] A series of pulse-chase experiments were performed in which
exponentially growing cells were diluted and treated with one of
the D-Ala probes (250 .mu.M-500 .mu.M), The cells were incubated
until saturation, washed, and placed on LB-containing agar pads and
imaged at 5-minute intervals for 12-18 hours (FIG. 4).
[0094] An experiment in B. subtilis revealed that fluorescence
persisted at the cell poles, an observation that is consistent with
the notion that there is not active cell wall synthesis taking
place in these regions (FIG. 4). Interestingly, the labeling
pattern observed with A. tumefaciens provides supporting evidence
for a mode of growth that involves budding as no signal dilution
from the mother cell is observed as recently shown.
[0095] More significantly, short exposures to FDAA derivatives have
proven to be optimal for imaging the sites of active cell wall
biosynthesis. For example, when a culture of exponentially growing
cells is contacted with either D-NBD or D-HCC, and the cells were
pulsed for 2%-8% of their usual generation time and immediately
fixed, sites of active synthesis were clearly visible in
evolutionarily distinct bacteria such as E. coli, B. subtilis, A.
tumefaciens, L. lactis, M. conglomeratus, C. crescentus and S.
aureus. Significantly, with experiments conducted in B. subtilis,
these short pulses result in a staining pattern that appears to be
consistent with the helical pattern that has been observed with
fluorescently modified vancomycin/ramoplanin (FIG. 5).
[0096] Finally, experiments were performed with the labeled D-Ala
derivative in a dual-labeling format. For example, A. tumefaciens
cells were incubated for a period of 4 minutes in 500 .mu.M D-NBD,
followed by washing and incubation for 4 minutes in 500 .mu.M
D-HCC. The excess dye was removed and the cells were pelleted and
fixed. The fluorescence micrographs reveal distinct patterns of
growth, in terms of polar growth and septal synthesis, based on the
age of the daughter cell (FIG. 6).
Example 2
Methods
[0097] Synthesis of Fluorescent D-Amino Amino Acids (FDAAs)
[0098] HADA/HALA: To a flame-dried flask,
7-hydroxycoumarin-3-carboxylic acid (HCC) was added in anhydrous
DMF (14.5 mL, 0.1 M) under an atmosphere of argon.
Carbonyldiimidazole (236 mg, 1.455 mmol) was added in one portion
and stirred at room temperature (RT) for 2 hours.
[0099] Boc-D-2,3-diaminopropionic acid (for HADA) or
Boc-L-2,3-diaminopropionic acid (for HALA) (297 mg, 1.455 mmol) was
added in one portion and the reaction mixture was allowed to stir
at RT overnight (17 hours). The majority of the solvent was removed
in vacuo, and the product was diluted with EtOAc (100 ml) and
washed with 1 N HCl (50 ml) and water (100 ml). The water layers
were combined and back-extracted with EtOAc (50 ml) to prevent loss
of product due to an emulsion. The organic layers were combined,
washed with brine (50 ml), dried over Na.sub.2SO.sub.4, filtered,
and the solvent was removed in vacuo. Without further purification
the crude product was treated with trifluoroacetic
acid/dichloromethane (50:50, 10 ml) for 30 minutes at RT, and the
solvent was removed in vacuo. The product was purified via
reverse-phase HPLC with 10%-90% McCN/H.sub.2O. The pure fractions
were concentrated in vacuo, and the product was redissolved in 1 N
HCl/MeCN and lyophilized to yield the desired product as a pale
yellow solid (297 mg. 62% for HADA and 277 mg, 58% for HALA).
[.alpha.].sup.20.sub.D=-21.8.degree. (c 2.2, DMSO-d6); HRMS-ESI-TOF
m/z calc'd for C.sub.13H.sub.12O.sub.6N.sub.2 ([M+H].sup.+):
293.0774. Found 293.0774; HPLC: tR=5.96 min (10-90% MeCN/H.sub.2O
over 10 minutes); .sup.1H NMR (400 MHz, DMSO-d6) .delta.=2.46 (s,
1H), 3.69-3.77 (m, 1H), 3.79-3.87 (m, 1H), 4.07 (t, J=5.6 Hz, 1H),
6.85 (d, J=2.0 Hz, 1H), 6.89 (dd, J=2.0, 8.4 Hz, 1H), 7.89 (d,
J=8.8 Hz, 1H), 8.43 (br s, 3H), 8.76 (s, 1H), 8.86 (t, J=6.2 Hz,
1H), 11.34 (br s, 1H); .sup.13C NMR (100 MHz, DMSO-d6):
.delta.=52.1, 102.3, 111.3, 113.5, 115.0, 132.5, 148.8, 156.8,
161.2, 163.1, 164.6, 169.7; the signal for one carbon was
overlapping with the solvent peak.
[0100] NADA/NALA: Boc-D-2,3-diaminopropionic acid (for NADA) or
Boc-L-2,3-diaminopropionic acid (for NALA) (100 mg, 0.49 mmol) and
sodium bicarbonate (123 mg, 1.47 mmol) were dissolved in water (1.8
ml) and heated to 55.degree. C. in water bath. A solution of
4-chloro-7-nitrobenzofurazan (NBD, 108 mg, 0.539 mmol) in methanol
(8.5 ml) was added dropwise over 10 minutes. Care was taken at all
times to avoid excessive exposure to light during the reaction and
workup. The reaction was allowed to stir at 55.degree. C. for 1
hour. The solvent was removed in vacuo and acidified with 1 N HCl.
The aqueous mixture was extracted with dichloromethane (50 ml per
extraction.times.3 extractions) and the organic extracts were
washed with brine (50 ml), dried over Na.sub.2SO.sub.4, filtered,
and the solvent was removed in vacuo. Without further purification
the crude product was treated with 4 N HCl/dioxane (10 ml) for 1
hour at RT, and the solvent was removed in vacuo. The product was
purified via reverse-phase HPLC with 20%-90% MeCN/H.sub.2O. The
pure fractions were concentrated in vacuo, and the product was
redissolved in 1 N HCl/MeCN and lyophilized to yield the desired
product as a bright orange solid (105 mg. 71% for both NADA and
NALA). [.alpha.].sup.20.sub.D=-32.degree. (c 1.1, DMSO-d6)
HRMS-ESI-TOF m/z calc'd for C.sub.9H.sub.9O.sub.5N.sub.5 ([M+H]+):
268.0682. Found 268.0680; HPLC: tR=5.02 minutes (20-90%
MeCN/H.sub.2O over 10 minutes). .sup.1H NMR (400 MHz, DMSO-d6):
.delta.=4.06 (m. 2H), 4.29 (m, 1H), 6.61 (d. J=8.0 Hz, 1H), 8.56
(d, J=8.0 Hz, 1H), 8.66 (br s, 3H), 9.32 (br s, 1H); .sup.13C NMR
(100 MHz, DMSO-d6): .delta.=43.4, 51.5, 100.5, 122.5, 138.1, 144.4,
144.9, 145.2, 169.2.
[0101] FDL: To a flame dried flask was added N.alpha.-Boc-D-Lys-OH
(19.3 mg, 0.078 mmol) and fluorescein isothiocyanate (25 mg, 0.065
mmol) in dry DMF (0.65 ml). The reaction was stirred under argon at
room temperature for 4 hours. The solvent was removed in vacuo. The
residue was redissolved in ethyl acetate (10 ml), washed with 1 N
HCl (10 ml) and brine (10 ml), and dried over anhydrous sodium
sulfate. The solvent was again removed in vacuo. and the crude
product was treated with trifluoroacetic acid/dichloromethane (1:1)
for 0.5 hours.
[0102] The acid was removed in vacuo, and the product was purified
by reverse phase HPLC with 30%-45% McCN/H.sub.2O. The pure
fractions were lyophilized to yield the product as a dark yellow
solid (25.0 mg, 72%/). [.alpha.].sup.20.sub.D=-7.1.degree. (c 0.72,
MeOH-d4); HMRS-ESI-TOF m/z calc'd for
C.sub.27H.sub.26N.sub.3O.sub.7S ([M+H]+): 536.1492. Found 536.1470;
HPLC: tR=5.08 min (30%-45% MeCN/H.sub.2O over 10 minutes); .sup.1H
NMR (400 MHz, MeOD-d4): .delta.=1.50-1.63 (m, 2H), 1.78 (quintet,
J=7.0 Hz, 2H), 1.90-1.99 (m, 1H), 2.00-2.10 (m, 1H), 3.66 (br s,
2H), 4.00 (t, J=6.3 Hz, 1H), 6.59 (dd, J=8.8 Hz, 2H), 6.73 (s, 2H),
6.74 (d, J=8.90 Hz, 2H), 7.18 (d, J=8.2 Hz, 1H), 7.76 (d, J=8.2 Hz,
1H), 8.17 (s, 1H); .sup.13C NMR (100 MHz, MeOD-d4): .delta.=23.3,
29.5, 31.3, 45.0, 53.9, 103.5, 111.9, 114.1, 120.5, 126.1, 129.2,
130.5, 131.8, 142.5, 154.6, 162.1, 171.0, 171.9, 182.9.
[0103] TDL: To a flame dried flask was added N.alpha.-Boc-D-Lys-OH
(3.3 mg. 0.0134 mmol), 5- (and 6-) carboxytetramethylrhodamine
succinimidyl ester (5 mg, 0.0095 mmol), and diisopropylethylamine
(2.5 .mu.l, 0.0143 mmol) in dry DMF (0.2 ml). The reaction was
stirred under argon at room temperature overnight. The solvent was
removed in vacuo, and the crude mixture was treated with
trifluoroacetic acid/dichloromethane (1:1) for 0.5 hours. The
reaction was dried in vacuo, and purified by reverse-phase HPLC
with 20%-40% MeCN/H.sub.2O. The pure fractions were lyophilized to
yield the product as a deep red solid (4.6 mg, 61%).
[.alpha.].sup.20.sub.D=-210.degree. (c 0.20, MeOH-d4); HRMS-ESI-TOF
m/z calc'd for C.sub.13H.sub.35N.sub.4O.sub.6 ([M+H]): 599.2557.
Found 599.2559; HPLC: tR=7.86 and 9.06 minutes (2 isomers isolated
results from mixed isomer starting material) (20%-40% MeCN/H.sub.2O
over 10 minutes); .sup.1H NMR (400 MHz, MeOD-d4): .delta.=1.25-1.35
(m, 2H), 1.46-1.62 (m, 2H), 1.68 (quintet, J=7.2 Hz, 2H), 1.75-2.05
(m, 2H), 3.41 (t, J=7 Hz, 1H), 3.92 (t, J=Hz, 1H), 7.01 (d, J=2 Hz,
2H), 7.05 (dd, J=2.0 Hz, 9.4 Hz, 2H), 7.13 (d, J=9.4 Hz, 2H), 7.81
(d, J=1.5 Hz, 1H), 8.19 (dd, J=1.5 Hz, 8.6 Hz, 1H), 8.39 (d, J=8.6
Hz, 1H).
[0104] Spectral Characteristics of FDAAs
[0105] Excitation and emission spectra of FDAAs (500 .mu.M in 100
mM Tris pH 7.0) were determined in black 96-well polystyrene plates
(Corning) using top-read function of a Spectra Max M2 plate reader.
The excitation and emission spectra were measured in separate runs
within a range of 200 nm and with increments of 1 nm.
[0106] Click Chemistry
[0107] EDA and ELA, and Sulfo-Cy3-Azide were gifts from Boaopharma
and Lumiprobe, respectively. AZA and "clickable" Alexa 488 Fluors
were purchased from Iris Biotech GmbH and Invitrogen, respectively.
The Cu(I) catalyzed click chemistry was performed using the
chemicals supplied by Invitrogen following their standard protocol
once the cells had been fixed with EtOH (70% v/v) and permeabilized
with methanol (100% v/v).
[0108] Growth Conditions
[0109] Strain characteristics and growth conditions are described
in Table 2.
TABLE-US-00002 TABLE 2 Strains, their predicted PG chemotypes and
conditions for growth and labeling. PG [HADA] Temperature Species
Strain Source Gram Chemotype Media (mM)* (.degree. C.) Aeration
Bacillus subtilis PY76 Daniel Kearns + A1.gamma.b LB N/A 37 Y IUB
Bacillus subtilis PY76 Daniel Kearns + A1.gamma.b LB 1 37 Y
dacA::cam IUB Bacillus subtilis CU1065 John Helmann + A1.gamma.b LB
N/A 37 Y CU Bacillus subtilis HB0048 John Helmann + A1.gamma.b LB
N/A 37 Y CU1065 CU ditA::spc Brachybacterium IUB culture +
A4.gamma. LB 1 30 Y conglomeratum collection Lactococcus lactis IUB
culture + A4.alpha. LB 1 37 Y collection Staphylococcus IUB culture
+ A3.alpha. LB 1 37 N aureus collection Streptococcus IU1945
Malcolm + A1.alpha. & BHI 0.5 37 N pneumoniae Winkler IUB
A3.alpha. Streptomyces Justin Nodwell + A3.gamma. LB 0.5 30 Y
venezuelae MU Agrobacterium Yves Brun - A1.gamma. LB 1 26 Y
tumefaciens IUB Burkholderia Ann Hirsch - A1.gamma. LB 0.5 30 Y
phytofirmans UCLA Caulobacter YB5630 - Yves Brun - A1.gamma. PYE
0.5 30 Y crescentus CB15 .DELTA.rsa IUB .DELTA.hfsA Escherichia
coli MG1655 Patricia Foster - A1.gamma. LB 1 37 Y IUB Synechocystis
sp. David Kehoe - A1.gamma. BG-11 1 26 CO.sub.2 + PCC 6803 IUB
artificial sunlight Verrucomicrobium Naomi Ward - A1.gamma. VM 0.25
30 Y spinosum UW Key: IUB = Indiana University Bloomington CU =
Cornell University MU = McMaster University UCLA = University of
California, Los Angeles UW = University of Wyoming *Short pulse
concentrations. By default, a normalized 1% (v/v) DMSO
concentration was used for all labeling experiments. indicates data
missing or illegible when filed
[0110] For any experiment involving FDAAs and/or CDAAs, DMSO was
added to the growth media to a final concentration of 1% to help
solubilize the FDAAs and/or CDAAs. Presence of 1% DMSO did not
affect labeling or growth in bacteria tested. When necessary,
chloramphenicol or spectinomycin was added to the growth media at 5
.mu.g/ml or 100 .mu.g/ml, respectively. Strains were maintained on
plates containing growth media with 1.5% agar.
[0111] Growth Curves
[0112] For growth curves, exponentially growing E. coli, A.
tumefaciens and A. tumefaciens and B. subtilis .DELTA.dacA were
diluted to OD.sub.600 0.05 into wells of polystyrene 24-well plates
(Falcon) containing 750 .mu.l LB with 1% DMSO or 1% DMSO+FDAAs (250
.mu.M-1 mM). The absorbance at 600 nm was read every 5 minutes for
18 hours in a BIO-TEK Synergy HT Plate Reader (30.degree. C.,
static).
[0113] Short Labeling Pulses and Fluorescence Microscopy
[0114] For short labeling pulses, exponentially growing cells were
screened for the minimum concentration of FDAAs or CDAAs (250
.mu.M-1 mM) and minimum amount of exposure duration to identify the
optimal conditions for each bacterium, as shown in FIG. 11 and
Table 2. To image growth patterns in different species,
exponentially growing cells (OD.sub.600 .about.0.3) were labeled,
fixed, washed, "clicked" if appropriate, and imaged.
[0115] For most strains, excess dye was removed by washing the
cells three to four times with 1 ml 1.times.PBS (NaCl 8 g/L, KCl
0.2 g/L, Na.sub.2HPO.sub.4-2H.sub.2O 1.78 g/L, KH.sub.2PO.sub.4
0.27 g/L, pH 7.4) and pelleting for 2-5 minutes at
10,000-16,000.times.g in a microfuge. When required, cells were
fixed with EtOH (70%, ice-cold, 20-minute incubation). Exceptions
included S. pneumnoniae (1% gluteraldehyde, 20 minutes incubation)
and B. subtilis (cold PBS treatment).
[0116] For dual labeling, the same procedure was followed with the
addition of a second round of a short labeling pulse using the
second FDAA prior to fixation. Cells were washed before, between
and after each FDAA treatment with pre-warmed medium in order
ensure similar labeling conditions. For triple labeling, a third
round of labeling involving CDAAs was added. For dual and triple
labeling of A. tumefaciens, the incubation times with each label
were 5 minutes and 7 minutes, respectively.
[0117] Phase and fluorescence microscopy was performed with a
Nikon.RTM. 90i Fluorescence Microscope equipped with a Plan Apo
100.times./1.40 Oil Ph3 DM Objective and a Chroma 83700 triple
filter cube with corresponding excitation and emission filters
(DAPI for HADA/HALA; FITC for NADA/NALA; and Alexa Fluor.RTM. 488s
and Texas Red.RTM. for Sulfo-Cy3 or WGA-594). All images were
captured using NIS software from Nikon.RTM. and a Photometrics
Cascade 1K cooled charge-coupled device camera, and were processed
and analyzed using ImageJ. When a comparison was made, cultures
were treated in exactly the same manner and the same parameters
were applied for collecting and post processing of the microscopy
data.
[0118] Long Labeling Pulses and Time-Lapse Microscopy
[0119] Exponentially growing cells were diluted to OD.sub.600 0.05
in media containing half of the optimal FDAA concentration used for
short labeling pulses and were grown until late exponential phase.
The cells were fixed, washed and then imaged using the Nikon.RTM.
90i as described previously. For time-lapse microscopy the cells
were washed with media and mounted onto LB+1% (w/v) agarose pads on
25-mm by 75-mm glass slides, sealed with 1:1:1 mixture of vasoline,
lanolin and paraffin and imaged with intervals of 4 minutes (B.
subtilis .DELTA.dacA), 5 minutes (E. coli) or 10 minutes (A.
tumefaciens) using a Nikon.RTM. Ti-E Inverted Fluorescence
Microscope equipped with a Plan Apo 60.times./1.40 Oil Ph3 DM
Objective and a CFP/YFP filter cube and an Andor DU885 EMCCD Camera
using CFP settings for detection of HADA.
[0120] Super-Resolution Microscopy
[0121] Structured illumination microscopy was performed using a
DeltaVision.RTM. OMX Imaging System equipped with an Olympus.RTM.
UPlanSApo 100.times./1.40 Oil PSF Objective and a Photometrics
Cascade II EMCCD Camera. The samples were excited with a laser at
405 nm and the emission was detected through a 419-465 emission
filter.
[0122] Environmental Samples
[0123] HADA (500 .mu.M+1% DMSO) and/or NADA (500 .mu.M+1% DMSO) was
added to either a 1.5 ml saliva sample from a 26-year old male or
to a 1.5 ml concentrated (.about.10 times) fresh water sample
collected from Indiana University and incubated for 2 hours (HADA)
at 37.degree. C. or for 2 hours (HADA) and 2 hours (NADA) at
26.degree. C., respectively. The samples were then fixed, washed
and imaged.
[0124] Live-Dead Staining
[0125] E. coli cells were labeled with HADA in 0.1% DMSO. Cells
were subsequently stained using the LIVE-DEAD BacLight Kit
(Invitrogen) according to the manufacturer's standard protocol.
[0126] Sacculi Purification
[0127] Sacculi from cells were purified as described in Litzinger
et al. (2010) J. Bacteriol. 192:3122-3143 with following
modifications. Exponentially growing cells were diluted to
OD.sub.600 0.05 in 10 ml LB containing half of the optimum FDAA
concentration+1% (v/v) DMSO and grown to late exponential phase.
After aliquots were taken for whole cell imaging, cells were
collected by centrifugation at 25,000.times.g for 15 minutes at RT
and resuspended in 0.8 ml water. The suspension was added to
boiling sodium dodecyl sulfate (SDS, 5% w/v) drop-wise and
incubated with stirring for 30 minutes. SDS insoluble material was
collected by ultracentrifugation at 39,000.times.g for 10 minutes
at 30.degree. C. and was resuspended in 1 ml water and boiled again
in SDS (4% w/v) with stirring for 30 minutes. Samples were then
washed four times in 1.5 ml water and resuspended in 1 ml 10 mM
Tris-HCl pH 7.0+10 mM NaCl+0.32 M immidazole+.alpha.-amylase (100
.mu.m/ml)+DNase I (50 .mu.g/ml)+MgSO.sub.4 (1 mM) and incubated for
2 hours at 37.degree. C. Samples were pelleted and resuspended in
0.05 M Tris-HCl pH 7.8+1.4 mg/ml pronase (type XXV from
Streptomyces griseus) and incubated 2 hours at 60.degree. C.
Samples were again pelleted and resuspended in 1 ml water and
boiled in SDS (1% w/v) with stirring for 30 min. The sacculus
preparations were washed a final time and resuspended in a minimal
amount of water. When needed, sacculi were further stained with
Wheat Germ Agglutinin, Alexa Fluor.RTM. 594 Conjugate (WGA-594, 15
.mu.g/ml).
[0128] HPLC Analysis of PG and Muropeptide Identification
[0129] PG from FDAA labeled cells was purified by the boiling SDS
extraction method and muramidase digestion treatment (Cellosyl) as
previously described in Brown et al. (2012) Proc. Natl. Acad. Sci.
USA 109:1697-1701. Solubilized muropeptide mixtures were then
either directly injected into the H PLC system (native or
non-reduced samples) or subjected to BH.sub.4Na reduction as
described in Brown et al. (2012), supra. Muropeptides were analyzed
using a binary-pump Waters*) HPLC System (Waters Corporation)
fitted with a reverse phase RP18 Aeris.RTM. Peptide Column (250
mm.times.4.6 mm; 3.6 .mu.m particle size) (Phenomenex) and a dual
wavelength absorbance detector. Elution conditions were: flow rate
1 ml/min; temperature 35.degree. C.; 3 minutes isocratic elution in
50 mM sodium phosphate, pH 4.35 followed by a 57 minute linear
gradient to 75 mM, sodium phosphate, pH 4.95 in 15% (v/v) methanol
(90 mM sodium phosphate, pH 5.2 in 30%(v/v) methanol for B.
subtilis analyses), and 10 minute isocratic elution under the
gradient final conditions. Elution was monitored setting one
channel to 204 nm and the second to a wavelength appropriate for
detection of corresponding FDAA. Muropeptides of interest were
collected following HPLC separation; vacuum dried, and subjected to
MALDI-mass spectrometry and electrospray ionization MS/MS as
described in Brown et al. (2012). supra.
[0130] Results
[0131] Growth of the phylogenetically diverse model species E.
coli, A. tumefaciens and B. subtilis in the presence of FDAAs for
as little as one generation resulted in strong peripheral and
septal labeling of entire cell populations (FIG. 8) without
affecting growth rate. Neither of the FLAAs prepared from
3-amino-L-alanine resulted in significant labeling, indicating that
labeling is specific to the D-enantiomers. The labeling was
exclusive to viable cells treated with the FDAAs and was not the
result of non-specific interaction of FDAAs with the PG.
Additionally, incorporation did not occur into teichoic acids for
B. subtilis as indicated by identical labeling of wild-type and a
.DELTA.dltA mutant that does not D-alanylate its teichoic
acids.
[0132] Retained fluorescence on the purified sacculi (FIG. 8)
demonstrated that the labeling of PG by the FDAAs was covalent.
HPLC analyses of muropeptides isolated from labeled cells (FIGS.
9B-C) revealed that 0.2%-2.8% of total muropeptides were modified
(FIG. 9C), which is sufficient for detection in various experiments
while avoiding possible toxicity issues that could result from
abundant incorporation. Significantly, the FDAA-specific peaks,
which were absent in samples treated with FLAAs, could be
distinguished from unlabeled muropeptides at FDAA-specific
absorption wavelengths (FIG. 9B). MS/MS analyses of FDAA-modified
muropeptides in B. subtilis indicated that FDAAs were exclusively
incorporated in the fifth position of the stem peptide (FIG. 9D).
Interestingly, in a .DELTA.dacA mutant of B. subtilis, the fraction
of labeled muropeptides and the fluorescent signal were
substantially higher than in wild-type B. subtilis, which is likely
due to the D,D-carboxypeptidase activity of DacA. In contrast, the
detectable incorporation was solely at the fourth position in E.
coli and A tumefaciens (FIG. 9D). These results are in agreement
with the known sites of incorporation of various natural DAAs in
these species and suggest that, similar to DAAs, FDAAs incorporate
mainly through periplasmic exchange reactions with the muropeptides
catalyzed either by D,D-transpeptidases (e.g., in B. subtilis) or
by L,D-transpeptidases (e.g., in E. coli and A. tumefaciens). This
DAA-like behavior together with the ease of fluorescent detection
make FDAAs a novel alternative to radioactive probes for studying
in vitro and in vivo activities of PG synthesis enzymes.
[0133] Pulse-chase experiments with HADA allowed the real-time
tracking of new PG incorporation during growth via time-lapse
microscopy. In E. coli and B. subtilis .DELTA.dacA (FIG. 10A), the
polar caps retained the HADA signal, but the signal from the
lateral walls dispersed as the cells grew, in agreement with
previous reports of cell wall growth along the length of the
lateral walls.
[0134] Strikingly, short labeling times (2%-8% of doubling time)
using E. coli and B. subtilis .DELTA.dacA with HADA resulted in
preferential localization of the signal at the septal plane of
predivisional cells and in punctate patterns on the lateral walls
of elongating cells (FIG. 11). Super-resolution microscopy of E.
coli revealed reticulated hoop-like patterns of HADA labeling
around the lateral wall (FIG. 10B), supportive of bursts of PG
incorporation in the side-walls. This ability of FDAAs to resolve
insertion of new PG provides the first direct detection of zones of
PG synthesis in a structured rather than a random pattern in E.
coli, consistent with recent results following the movement of the
cell wall elongation machinery. Short labeling times with A.
tumefaciens, whose growth occurs predominantly from a single pole
and the site of cell division while the mother cell remains inert,
resulted in polar and septal labeling. Super-resolution
fluorescence microscopy of labeled cells further enhanced the
spatial resolution of the site of active PG synthesis (FIG.
10C).
[0135] PG labeling in three evolutionarily distant species
suggested that FDAAs could specifically label the active site of PG
synthesis across the entire bacterial domain. When species
representing diverse phyla and modes of growth were briefly
incubated with FDAAs, we observed strong labeling at the sites of
cell division in actively dividing cells (FIG. 11). This septal
probe incorporation was the sole mode in Synechocystis sp. PCC
6803, L. lactis and S. aureus. Super-resolution microscopy of S.
aureus further highlighted the different stages of these
constricting septal rings (FIG. 10D). Labeling of S. pneumoniae
occurred in single or split equatorial rings depending on the
length of the cell, with peripheral labeling between the split
rings (FIG. 11). Labeling of S. venezuelae was predominantly
apical, with some weak labeling of vegetative septa and lateral
walls suggestive of a low but continuous lateral PG synthesis (FIG.
11). In C. crescentus, labeling occurred at the sites of septal
elongation, lateral elongation, and stalk synthesis (FIG. 11). B.
phytofirmans exhibited polar and mid-cell PG synthesis, B.
conglomeratum exhibited prominent peripheral PG synthesis in
addition to seemingly alternating perpendicular division planes,
and V. spinosum exhibited strong peripheral PG synthesis and
asymmetric septal labeling (FIG. 11).
[0136] The efficient label incorporation in all the bacteria
indicates that FDAAs, therefore DAAs, incorporation is common to
the bacterial domain and that FDAAs can thus be used to analyze
natural bacterial populations, providing a convenient and quick
standard to measure bacterial activity and to probe the diversity
of growth modes in complex microbiomes. Indeed, labeling times with
FDAAs as short as 2 hours revealed diverse modes of growth in
saliva and freshwater samples in situ, but did not label dead cells
as suggested by the strong correlation with Live-Dead staining.
[0137] Encouraged by the efficiency of FDAAs, additional and
differently functionalized unnatural DAAs were prepared and used.
Following a similar approach, a brighter and more versatile core
fluorophore, fluorescein (emission maximum .about.515 nm, green),
and its analogue, carboxytetramethylrhodamine (TAMRA, emission
maximum .about.565 nm, red) were derivatized and linked with D-Lys
to separate the bulky fluorophore from the DAA backbone, generating
FDL and TDL (see, for example, structures in FIG. 7). Incubation of
both E. coli and B. subtilis with FDL showed patterns similar to
NADA, although labeling of B. subtilis was stronger than E. coli.
In contrast, the larger TDL did not label E. coli cells, but
labeling of B. subtilis was prominent and showed patterns similar
to other FDAAs.
[0138] CDAAs, namely ethynyl-D-alanine (EDA) or azido-D-alanine
(ADA) (FIG. 7), that can be specifically captured by any molecule
carrying the conjugate functional group via click-chemistry also
were used. Similar to FDAAs, these bioorthogonal DAAs, but not the
L-enantiomer control ELA, labeled both E. coli and B. subtilis when
captured by commercially available azido/alkyne fluorophores.
[0139] Furthermore, custom DAAs containing different colored
fluorophores can be used sequentially to enable "virtual time-lapse
microscopy." Since addition of each new probe indicates the
location and extent of PG synthetic activity during the respective
labeling periods, this approach provides a chronological account of
shifts in PG synthesis of individual cells over time. Examples of
such serial labeling, including a combination with click chemistry,
were performed in Gram-negative A. tumefaciens (FIG. 10E) and in
Gram-positive S. venezuelae (FIG. 10F).
[0140] In view of the foregoing, disclosed herein are compositions
for and methods of covalently labeling PG in live bacterial cells.
This method works very efficiently in both Gram-positive and
Gram-negative organisms (Gram-negative organisms represent a
liability for approaches using fluorescently modified
vancomycin/ramoplanin), and the probe substrates do not appear to
be toxic to cells and show no adverse effects on cell morphology,
even at concentrations as high as 1 mM. The probes rapidly label
sites of active peptidoglycan biosynthesis and can be used in
time-lapse and dual labeling experiments.
[0141] Thus, compositions and methods have been described herein
for in situ labeling/probing of PG synthesis in bacteria with
fluorescent D-amino acids (FDAAs), as well as for screening for
bacterial cell wall-acting and/or cell wall-disrupting agents. The
FDAAs are based upon D-amino acids (DAAs) derivatized to covalently
include a small fluorophore. As such, the FDAAs can be directly
incorporated into bacterial cell walls during PG biosynthesis, as
occurs at sites of cell division in actively dividing cells.
[0142] The compositions include FDAAs. The FDAAs have a DAA
covalently attached to a fluorophore such as 7-hydroxycoumarin
3-carboxylic acid (HCC--OH), 7-nitrobenzofurazan (NBD),
4-chloro-7-nitrobenzofurazan (NBD-CI), fluorescein (F) or
carboxytetramethylrhodamine (T). The DAA can be any of the twenty
known, standard amino acids, such as D-Ala, D-Asp, D-Cys, D-Glu or
D-Lys.
[0143] The compositions also include clickable DAAs (CDAAs). The
CDAAs have a DAA backbone including an alkyne or azide functional
group that can be captured by any labeled detecting agent carrying
a conjugate functional group via click-chemistry, where the label
can be a fluorescent molecule.
[0144] The compositions also include fluorescent
muramylpentapeptide precursor units (FMPUs). The FMPUs have an
N-acetyl muramic acid (NAM) moiety with a stem peptide of three to
five amino acids in which one or more of the amino acids in the
stem peptides are FDAAs and/or CDAAs as described herein.
[0145] The compositions also include fluorescent PG units (FPGUs).
The FPGUs have a FMPU as described herein linked to an N-acetyl
glucosamine (NAG) moiety.
[0146] The compositions also include live bacteria having one or
more FDAA, CDAA, FMPU and/or FPGU as described herein incorporated
into PG in a cell wall.
[0147] The compositions also include kits having one or more FDAA,
CDAA, FMPU and/or FPGU as described herein and optionally one or
more labeled, detecting agents for use in in situ labeling/probing
of PG synthesis, as well as for screening for bacterial cell
wall-acting and/or cell wall-disrupting agents. The kits also can
include additional reagents such as unlabeled DAAs, unlabeled
L-amino acids (LAAs), fluorescent LAAs (FLAAs) and/or clickable
LAAs (CLAAs). The kits also can include positive and/or negative
bacterial controls, where the bacterial controls have unlabeled
DAAs, CDAAs, LAAs and CLAAs and/or labeled DAAs, CDAAs, LAAs and
CLAAs incorporated into PG in a cell wall.
[0148] In view of the foregoing, the methods have been disclosed
herein that include assessing bacterial cell wall synthesis in real
time by providing live bacteria with one or more FDAA, CDAA, FMPU
and/or FPGU as described herein, where the bacteria covalently
incorporate the one or more FDAA, CDAA, FMPU and/or FPGU into PG of
a bacterial cell wall. The one or more FDAA, CDAA, FMPU and/or FPGU
can be provided to live bacteria together or sequentially during
cell wall synthesis.
[0149] The methods also include screening for putative cell
wall-acting or cell wall-disrupting agents by contacting bacteria
with a putative cell wall-acting agent or putative cell
wall-disrupting agent, where the agent is cell wall-acting if the
agent interferes with ongoing PG biosynthesis in a bacterial cell
wall or is cell wall-disrupting if the agent weakens integrity of
PG in an existing bacterial cell wall. When screening for putative
cell wall-acting agents, the bacteria can be provided with one or
more FDAA, CDAA, FMPU and/or FPGU as described herein
simultaneously with the putative agent. When screening for putative
cell wall-disrupting agents, the bacteria can have one or more
FDAA, CDAA, FMPU and/or FPGU as described herein covalently
incorporated into PG of the cell wall prior to being contacted with
the putative agent.
[0150] The methods also include identifying a bacteria by providing
live, unknown bacteria with one or more FDAA, CDAA, FMPU and/or
FPGU as described herein under conditions sufficient for bacterial
cell wall synthesis, where the bacteria covalently incorporate one
or more FDAA, CDAA, FMPU and/or FPGU into a cell wall, and where
each of the one or more FDAA, CDAA, FMPU and/or FPGU includes a
distinct fluorophore. The methods also include observing an
incorporation pattern of the one or more FDAA, CDAA, FMPU and/or
FPGU, where the incorporation pattern identifies the bacteria. Such
methods are amenable for use in screening platforms to identify
novel compounds having bacteriostatic or bacteriotoxic
properties.
[0151] In the methods, the bacteria can be Gram-positive or
Gram-negative bacteria.
[0152] The methods also include detecting one or more FDAA, CDAA,
FMPU and/or FPGU as described herein that have been incorporated in
the bacterial cell wall or that have been disrupted from the
bacterial cell wall by, for example, fluorescence microscopy and
other methods, depending upon the label used.
[0153] The methods also can include comparing the results of the
putative agent with a known cell wall-acting agent or with a known
cell wall-disrupting agent.
[0154] The compositions and methods described herein therefore find
use in the study of bacterial cell wall biosynthesis and in the
discovery of bacterial cell wall-acting and/or cell wall-disrupting
agents. Advantageously, the FDAAs, CDAAs, FMPUs and/or FPGUs as
described herein simultaneously are non-toxic and can be tunable to
label sites of active PG biosynthesis, enabling fine spatiotemporal
tracking of cell wall dynamics in phylogenetically and
morphologically diverse bacteria.
[0155] All of the patents, patent applications, patent application
publications and other publications recited herein are hereby
incorporated by reference as if set forth in their entirety.
[0156] The present invention has been described in connection with
what are presently considered to be the most practical and
preferred embodiments. However, the invention has been presented by
way of illustration and is not intended to be limited to the
disclosed embodiments. Accordingly, one of skill in the art will
realize that the invention is intended to encompass all
modifications and alternative arrangements within the spirit and
scope of the invention as set forth in the appended claims.
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