Orthogonal alkynyl amino acid reporter for selective labeling of bacterial proteomes during infection

Article abstract of DOI:10.1002/anie.201002050

(figure Presented) Bacterial proteins in the haystack: The alkynyl amino acid reporter AOA (see scheme) can be used to visualize and identify newly synthesized bacterial proteins during Salmonella infection of mammalian cells by the copper(I)-catalyzed az

Full text of DOI:10.1002/anie.201002050

Communications
DOI: 10.1002/anie.201002050
Protein Identification
Orthogonal Alkynyl Amino Acid Reporter for Selective Labeling of
Bacterial Proteomes during Infection**
Markus Grammel, Mingzi M. Zhang, and Howard C. Hang*
Bacterial pathogens have evolved sophisticated mechanisms
to evade host defenses and cause disease.[1] The emergence of
new and antibiotic-resistant bacterial pathogens demands a
better understanding of virulence mechanisms for antibacte-
rial-drug discovery. Although the discovery of bacterial
toxins, quorum sensing, and protein-secretion pathways has
revealed some key virulence mechanisms, the precise mech-
anisms by which intracellular bacterial pathogens subvert host
immune responses are still unclear.[1] The analysis of individ-
ual virulence factors has demonstrated that bacterial patho-
gens alter their protein expression to infect and replicate in
host tissues.[2] However, the system-wide identification and
analysis of bacterial proteins that are uniquely expressed or
secreted during infection is paramount for understanding
mechanisms of bacterial pathogenesis.[2,3] Comparative
genomics and mutagenesis studies have revealed bacterial
genes that are important for infection, but their precise
biochemical mechanisms and temporal expression patterns
can be elusive as a result of posttranscriptional regulation.[2,3]
Direct biochemical analysis of bacterial proteomes during
infection is needed.[3] The large excess of host proteins in
mixed pathogen–host lysates presents a significant challenge
for proteomic analysis of bacterial proteins during infec-
tions,[3] and even after the physical isolation of intact bacteria,
significant amounts of host proteins still remain.[4,5] This
factor is particularly important since many bacterial virulence
factors are often expressed at low levels.[2] New strategies are
therefore required to selectively enrich bacterial proteins
from host proteomes for their analysis during infection.
The incorporation of nonnatural amino acids in bacteria
has provided new methods to differentiate bacterial proteins
from host proteomes. For example, the incorporation of
phenylalanine analogues in mycobacteria by amber-stop-
codon-suppression technology has enabled the selective
labeling of green fluorescent protein expressed in Mycobac-
terium tuberculosis during the intracellular infection of
macrophages.[6] Alternatively, alkyne- or azide-functionalized
methionine (Met) surrogates can be incorporated by the
endogenous methionyl-tRNA synthetase (MetRS) into bac-
terial proteomes.[7,8] These amino acid chemical reporters
enable the metabolic labeling and detection of newly
synthesized proteins using bioorthogonal ligation methods,
ssuch as the copper(I)-catalyzed azide–alkyne cycloaddi-
tion.[10] Additionally, MetRS mutants have been identified
that can incorporate azidonorleucine (ANL, Figure 1a),[11,12]
a Met surrogate that is not efficiently activated by the wild-
type (wt) MetRS or other endogenous aminoacyl-tRNA
synthetases.
ANL can therefore be used as an orthogonal amino acid
reporter to selectively label proteins in nonpathogenic
Escherichia coli by the bacterial expression of MetRS mutants
in the presence of mammalian cells.[13] Although these studies
have demonstrated the selective targeting of bacterial pro-
teomes in the presence of host cells,[6,13] the use of nonnatural
amino acid reporters for the analysis of endogenously
expressed bacterial proteins during infection has not been
reported. Herein, we report a new orthogonal alkynyl amino
acid reporter for the specific imaging and enrichment of
bacterial proteomes during the infection of mammalian cells
with the Gram-negative intracellular bacterial pathogen
Salmonella typhimurium (Figure 1b).
On the basis of the reported superior selectivity of azide-
over alkyne-functionalized secondary CuAAC reagents[14]
and our own experience with fatty-acid chemical reporters,
we evaluated whether an alkynyl isostere of ANL, 2-amino-
octynoic acid (AOA, Figure 1a), could be accepted by
previously reported MetRS mutants in S. typhimurium.[13,15,16]
MetRS mutants were generated from the E. coli metG gene
(the S. typhimurium metG gene is 95% identical to E. coli
metG) by site-directed mutagenesis and ligated into the low-
copy-number plasmid pWSK29 under the expression control
of the lac promoter, which provides constitutive expression in
S. typhimurium. All plasmids were transformed into the
S. typhimurium strain IR715. AOA was synthesized by the
alkylation of diethyl acetamidomalonate with hex-5-ynyl-4-
methylbenzenesulfonate and sequentially deprotected to
yield the racemic product (see Scheme 1 in the Supporting
Information). For the in vitro analysis of the S. typhimurium
strains expressing MetRS mutants, bacteria were grown in full
Luria–Bertani (LB) medium to the stationary phase and
diluted into minimal medium containing Met or AOA.
Protein lysates were reacted with the azido-rhodamine (az-
Rho)[15] detection tag by CuAAC and analyzed by SDS-
PAGE and in-gel fluorescence scanning. AOA selectively and
[*] M. Grammel, M. M. Zhang, Dr. H. C. Hang
Laboratory of Chemical Biology and Microbial Pathogenesis
The Rockefeller University
1230 York Avenue, New York, NY 10065 (USA)
Fax: (+1)212-327-7276
E-mail: hhang@rockefeller.edu
[**] We thank The Rockefeller University Proteomics Resource Center for
mass spectrometry analysis, James Flexner for help with the
azidonorleucine synthesis, and members of the Hang Laboratory for
providing secondary detection reagents. M.M.Z. is supported by
A*STAR, Singapore. H.C.H. acknowledges support from The Rock-
efeller University, the Irma T. Hirschl/Monique Weill-Caulier Trust,
the Lerner Trust, and the Northeastern Biodefense Center NIH/
NIAID (2 U54 AI057158-06).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201002050.
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for subsequent experiments.
These results demonstrate that
AOA can selectively label newly
synthesized Met-containing pro-
teins in Gram-negative bacterial
pathogens, such as MetRS-NLL
S. typhimurium.
To determine the most effi-
cient combination of MetRS
mutant and orthogonal amino
acid reporter for the selective
labeling of bacterial proteomes
during the infection of host cells,
we evaluated other reported
MetRS mutants with ANL and
AOA in Salmonella. We gener-
ated three previously reported
MetRS mutants with high acti-
vation efficiency for ANL to
evaluate their utility for the
metabolic labeling of S. typhimu-
rium proteins with orthogonal
amino acid reporters: L13G
(MetRS-L13G), L13N-Y260L-
H301L (MetRS-NLL), and
L13P-Y260L-H301L (MetRS-
PLL).[11] All three MetRS
mutants permit the incorpora-
tion of ANL and AOA into
Salmonella proteomes (Fig-
ure 2a). Neither ANL nor
AOA were greatly incorporated
into Salmonella transformed
with control plasmid or over-
expressing wt MetG. However,
AOA displayed some back-
ground incorporation by the
endogenous protein translation
machinery (Figure 2a). The
Figure 1. Selective labeling of bacterial proteomes with orthogonal amino acid reporters during infection.
a) Structures of methionine (Met), azidonorleucine (ANL), and 2-aminooctynoic acid (AOA). b) Infection
of host cells with bacterial pathogens expressing a mutant methionyl-tRNA synthetase (MetRS), followed
by pulse labeling with the orthogonal amino acid reporter AOA, enables the specific imaging and
proteomic analysis of bacterial proteomes by using CuAAC or “click chemistry”. c) MetRS-NLL
S. typhimurium was treated with Met (1 mm) or AOA (2 mm), in the presence or absence of tetracycline,
for 30 min. The cell lysates were subjected to CuAAC with az-Rho and analyzed by in-gel fluorescence.
d) Competition experiment with Met for labeling with AOA (2 mm). e) Comparison of the labeling
efficiency of d/l-AOA and l-AOA. CM=Coomassie; “fluor.” indicates in-gel fluorescence scanning.
efficiently labeled proteins in S. typhimurium expressing the
MetRS-NLL mutant (L13N-Y260L-H301L; Figure 1c).
To confirm the selectivity of AOA for newly synthesized
Met-containing proteins, we conducted protein synthesis
inhibition and Met-competition experiments. The pre- and
coincubation of MetRS-NLL S. typhimurium with the protein
synthesis inhibitor tetracycline (Tet) effectively abolished
AOA labeling (Figure 1c), and Met competed efficiently
against AOA incorporation in a dose-dependent manner
(Figure 1d).
Since the synthetic route to AOA results in a racemic
mixture, we investigated whether the presence of the d-AOA
isomer in the racemate impairs S. typhimurium labeling. Pig
acylase I was used for the kinetic resolution of the racemic
mixture of AOA[17] to afford enantiomerically pure l-AOA
(see Figure 1 in the Supporting Information). The activity of
racemic AOA at a 2 mm concentration was compared to that
of 1 mm l-AOA. Since both preparations showed identical
labeling efficiencies at comparable effective concentrations
(Figure 1e), we decided to use the racemic AOA preparation
expression levels of all MetRS mutants were comparable, as
judged by S-tag western blot analysis of the epitope tag fused
to all MetRS constructs (see Figure 2 in the Supporting
Information). Both triple mutants (NLL, PLL) enabled more
efficient incorporation of ANL and AOA over the single
mutant (L13G). Furthermore, AOA showed superior signal-
to-noise ratios relative to labeling with ANL for all three
MetRS mutants (see Figure 3a in the Supporting Informa-
tion). These results demonstrate that both orthogonal amino
acid reporters function in Salmonella expressing MetRS
mutants, but MetRS-NLL in combination with AOA affords
the optimal orthogonal enzyme–substrate pair for the meta-
bolic labeling of Salmonella proteins.
We then evaluated the efficiency of the ANL and AOA
labeling of S. typhimurium proteomes during the intracellular
infection of mammalian cells. To analyze the selective
incorporation of orthogonal amino acid reporters in intra-
cellular bacterial pathogens, we infected Raw264.7 murine
macrophages with MetRS-NLL S. typhimurium at a multi-
plicity of infection (MOI) of 100 for 30 min. Following
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controls for samples treated
with AOA or ANL demon-
strates the considerably higher
nonspecific background signal
of the alkyne-functionalized
detection tag (Figure 2b), as
observed earlier in vitro (Fig-
ure 2a). Close inspection of
AOA-treated uninfected mac-
rophages revealed marginal
concentration-dependent label-
ing of mammalian proteomes
that was not observed with
ANL (Figure 2b). Given the
higher reactivity of alkyne
detection
reagents
(Fig-
ure 2a,b), low levels of ANL
labeling in mammalian cells are
probably undetectable above
the background. Quantification
of the relative fluorescence
intensities of ANL and AOA
suggests that AOA, at a con-
centration of 2 mm, affords the
most sensitive labeling of Sal-
monella proteins during infec-
tion (see Figure 3b in the Sup-
porting Information). These
results show that ANL and
AOA can be selectively incor-
porated into S. typhimurium
during intracellular infection
by the bacterial expression of
MetRS-NLL. We found that
AOA, together with azide
detection reagents, displayed
superior labeling sensitivity at
all tested concentrations (see
Figure 3b in the Supporting
Information).
Figure 2. Comparative analysis of orthogonal amino acid reporters and MetRS mutants in S. typhimurium.
a) S. typhimurium expressing different MetRS mutants was labeled with AOA (2 mm) or ANL (1 mm) in
minimal medium. Cell lysates were analyzed by CuAAC with az- or alk-Rho and in-gel fluorescence
scanning. b) Raw264.7 cells infected with MetRS-NLL S. typhimurium, or uninfected, were labeled with
ANL or AOA at different concentrations 2h post infection. The total cell lysates were analyzed by CuAAC
with az- or alk-Rho and in-gel fluorescence scanning. Western blot analysis was performed for mutant
MetRS expression and levels of the host protein b-actin. CM=Coomassie; “fluor.” indicates in-gel
fluorescence scanning.
To confirm the selective
labeling of Salmonella with
AOA inside mammalian cells,
we performed fluorescence-
imaging studies of infected
cells. HeLa cells were infected
infection, extracellular bacteria were killed by the addition of
gentamicin, a cell-impermeable antibiotic, to the cell-culture
medium. After 2 h, macrophages infected with MetRS-NLL
S. typhimurium were labeled with different concentrations of
Met, ANL, or AOA in the growth medium for 3 h. Cell pellets
containing bacteria and mammalian cells were lysed with 4%
SDS lysis buffer, treated with alk-Rho (alkyne-rhodamine)[15]
or az-Rho, and analyzed by in-gel fluorescence.
Samples infected with MetRS-NLL S. typhimurium, as
validated by S-tag Western blot analysis, and labeled with
ANL or AOA displayed a strong fluorescence signal above
background in contrast to the uninfected samples labeled with
ANL or AOA (Figure 2b). Comparison of the respective Met
with MetRS-NLL S. typhimurium (MOI = 100), 16h post-
infection cells were pulse-labeled with AOA or Met for 1 h,
fixed, permeabilized, reacted with az-Rho, and stained with
anti-Salmonella serum. Imaging of rhodamine fluorescence
demonstrated robust and selective labeling of intracellular
bacteria with AOA (Figure 3a). No rhodamine fluorescence
was observed in host cells or MetRS-NLL S. typhimurium
infected cells treated with Met under these conditions (Fig-
ure 3a). Colocalization of AOA-labeled S. typhimurium with
anti-Salmonella serum (Figure 3a) validated the selective
incorporation of AOA into the bacterial proteome and
exclusion from host proteins. We also analyzed S. typhimu-
rium infected Raw264.7 macrophages by staining with a
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Figure 3. Fluorescence microscopy of AOA-labeled MetRS-NLL S. typhimurium infected mammalian cells (scale bar: 10 mm). a) HeLa cells were
infected with MetRS-NLL S. typhimurium and were labeled 16 h postinfection with AOA (2 mm) or Met (1 mm). Fixed cells were stained for
S. typhimurium (a-STM alexa 488, green) and DNA (TO-PRO-3, blue) after CuAAC with az-Rho (rhodamine, red). b) Raw264.7 cells were infected
with MetRS-NLL S. typhimurium and were labeled 16 h postinfection with AOA (2 mm). Fixed cells were stained for LAMP-1 (a-LAMP-1 alexa 488,
green) and DNA (TO-PRO-3, blue) after CuAAC with az-Rho (rhodamine, red).
fluorescently labeled LAMP-1 antibody. LAMP-1 serves as a
marker for Salmonella-containing vacuoles of infected host
cells.[2] AOA-labeled MetRS-NLL S. typhimurium inside
Raw264.7 macrophages were enclosed in LAMP-1-positive
compartments (Figure 3b), which suggests that AOA labeling
does not significantly disturb intracellular trafficking of
Salmonella in host cells. These results further validate the
utility of AOA for the selective metabolic labeling of
Salmonella proteins inside mammalian cells.
After the selectivity and efficiency of AOA labeling had
been established, we compared Salmonella proteins that are
metabolically labeled by AOA during growth in a liquid
culture with Salmonella proteins that are labeled during the
infection of macrophages. For this comparative analysis,
MetRS-NLL S. typhimurium grown in a liquid culture (in
vitro) and Raw264.7 macrophages infected with MetRS-NLL
S. typhimurium (16 h postinfection) were pulse-labeled with
AOA or Met for 1 h. Cell lysates were then treated with
azido-diazo-biotin,[18] affinity-purified with streptavidin, and
eluted from beads for protein identification by gel-based
proteomics (see Figure 4 in the Supporting Information).
The generated raw data was searched against a concaten-
ated mouse–Salmonella database for protein identification
(see the Supporting Information). Only proteins that con-
tained two unique peptides and were not detected in the Met
control were considered for this study. The analysis of
infected Raw264.7 macrophages identified a total of 218
proteins that met our filter criteria (see Table 1 in the
Supporting Information). Of these proteins, 185 were
assigned to the Salmonella proteome (85%), and the
remainder were mouse proteins (Figure 4a). We also identi-
fied 472 proteins from MetRS-NLL S. typhimurium pulse-
labeled with AOA in minimal medium (see Table 2 in the
Supporting Information). Between the in vitro and intra-
cellular Salmonella datasets, we could identify 96 common
proteins, whereas 89 proteins (48%) were observed exclu-
sively in the sample derived from infected macrophages
(Figure 4b). Interestingly, at least five Salmonella proteins
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mammalian cells as well as the enrichment and proteomic
analysis of endogenously expressed Salmonella proteins from
infected mammalian cells. The application of orthogonal
amino acid reporters should open new and exciting oppor-
tunities for the imaging and proteomic investigation of
different bacterial pathogens during infection.
Received: April 6, 2010
Published online: July 14, 2010
Figure 4. Proteomic analysis of AOA-labeled MetRS-NLL S. typhimu-
Keywords: bacterial pathogenesis · click chemistry ·
nonnatural amino acids · protein labeling · proteomics
rium infected mammalian cells. a) A total of 218 S. typhimurium
proteins were identified from infected Raw264.7 cells. Of these
proteins, 185 (85%) were Salmonella proteins and 33 were mouse
proteins. b) Comparison of Salmonella proteins identified from infected
macrophages and Salmonella grown in minimal liquid culture: 376
proteins were identified in liquid culture only, 89 proteins were
identified in infected macrophages only, and 96 were identified in both
samples. c) Comparison of proteins identified from AOA-pulse-labeled
infected Raw264.7 cells (amino acid reporter) and a previously
published dataset of Salmonella proteins identified from infected
Raw246.7 cells under comparable conditions without AOA labeling (no
amino acid reporter):[4] 100 proteins were exclusively identified in our
dataset, 85 proteins were identified in both datasets, and 49 proteins
were only identified in the previously published dataset.[4]
.
[1] A. P. Bhavsar, J. A. Guttman, B. B. Finlay, Nature 2007, 449, 827.
[2] A. Haraga, M. B. Ohlson, S. I. Miller, Nat. Rev. Microbiol. 2008,
6, 53.
[3] K. D. Rodland, J. N. Adkins, C. Ansong, S. Chowdhury, N. P.
Manes, L. Shi, H. Yoon, R. D. Smith, F. Heffron, Future
Microbiol. 2008, 3, 625.
[4] L. Shi, J. N. Adkins, J. R. Coleman, A. A. Schepmoes, A.
Dohnkova, H. M. Mottaz, A. D. Norbeck, S. O. Purvine, N. P.
Manes, H. S. Smallwood, H. Wang, J. Forbes, P. Gros, S. Uzzau,
K. D. Rodland, F. Heffron, R. D. Smith, T. C. Squier, J. Biol.
Chem. 2006, 281, 29131.
[5] D. Becker, M. Selbach, C. Rollenhagen, M. Ballmaier, T. F.
Meyer, M. Mann, D. Bumann, Nature 2006, 440, 303.
[6] F. Wang, S. Robbins, J. Guo, W. Shen, P. G. Schultz, PLoS ONE
2010, 5, e9354.
[7] K. E. Beatty, J. C. Liu, F. Xie, D. C. Dieterich, E. M. Schuman,
Q. Wang, D. A. Tirrell, Angew. Chem. 2006, 118, 7524; Angew.
Chem. Int. Ed. 2006, 45, 7364.
(SodM, SsrB, SseA, PipB2, and PhoP) from infected
Raw264.7 cells were previously described as virulence
factors.[19]
When compared with other proteomic datasets generated
from macrophages infected with S. typhimurium,[4] our results
demonstrate that AOA pulse labeling in conjunction with
bioorthogonal ligation enables greater enrichment of bacte-
rial proteins (identification of 85% of Salmonella proteins
with AOA labeling in comparison to approximately 45%
under comparable conditions without AOA)[4] and reveals
additional bacterial proteins that may be expressed prefer-
entially during infection (Figure 4c). Although the use of
AOA and MetRS-NLL only targets Met-containing proteins,
96% of the S. typhimurium LT2 proteome contains one or
more Met residues in addition to the N-terminal initiator Met
residue. Hence, the majority of the Salmonella proteome is
amenable to labeling with AOA (see Figure 5 in the
Supporting Information). Our preliminary proteomic studies
demonstrate that AOA pulse labeling in combination with
CuAAC enables the efficient enrichment and identification of
endogenously expressed bacterial proteins that are synthe-
sized differentially by Salmonella during the infection of host
cells.
[8] K. L. Kiick, E. Saxon, D. A. Tirrell, C. R. Bertozzi, Proc. Natl.
Acad. Sci. USA 2002, 99, 19.
[9] M. Meldal, C. W. Tornøe, Chem. Rev. 2008, 108, 2952.
[10] D. C. Dieterich, A. J. Link, J. Graumann, D. A. Tirrell, E. M.
Schuman, Proc. Natl. Acad. Sci. USA 2006, 103, 9482.
[11] I. C. Tanrikulu, E. Schmitt, Y. Mechulam, W. A. Goddard III,
D. A. Tirrell, Proc. Natl. Acad. Sci. USA 2009, 106, 15285.
[12] A. J. Link, M. K. Vink, N. J. Agard, J. A. Prescher, C. R.
Bertozzi, D. A. Tirrell, Proc. Natl. Acad. Sci. USA 2006, 103,
10180.
[13] J. T. Ngo, J. A. Champion, A. Mahdavi, I. C. Tanrikulu, K. E.
Beatty, R. E. Connor, T. H. Yoo, D. C. Dieterich, E. M. Schu-
man, D. A. Tirrell, Nat. Chem. Biol. 2009, 5, 715.
[14] A. E. Speers, B. F. Cravatt, Chem. Biol. 2004, 11, 535.
[15] G. Charron, M. M. Zhang, J. S. Yount, J. Wilson, A. S. Raghavan,
E. Shamir, H. C. Hang, J. Am. Chem. Soc. 2009, 131, 4967.
[16] K. E. Beatty, D. A. Tirrell, Bioorg. Med. Chem. Lett. 2008, 18,
5995.
[17] H. K. Chenault, J. Dahmer, G. M. Whitesides, J. Am. Chem. Soc.
1989, 111, 6354.
[18] Y. Y. Yang, J. M. Ascano, H. C. Hang, J. Am. Chem. Soc. 2010,
132, 3640.
[19] J. Yang, L. Chen, L. Sun, J. Yu, Q. Jin, Nucleic Acids Res. 2008,
36, D539.
In summary, we report a new and efficient orthogonal
amino acid reporter for the selective labeling of bacterial
proteomes during infection. The combination of CuAAC with
AOA pulse labeling enables the imaging of bacteria within
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