Identification of secreted bacterial proteins by
noncanonical amino acid tagging
Alborz Mahdavi
a,b
, Janek Szychowski
a
, John T. Ngo
a
, Michael J. Sweredoski
c
, Robert L. J. Graham
c
, Sonja Hess
c
,
Olaf Schneewind
d
, Sarkis K. Mazmanian
e
, and David A. Tirrell
a,1
a
Division of Chemistry and Chemical Engineering,
b
Department of Bioengineering, and
c
Proteome Exploration Laboratory, Beckman Institute, California
Institute of Technology, Pasadena, CA 91125;
d
Department of Microbiology, University of Chicago, Chicago, IL 60637; and
e
Division of Biology and Biological
Engineering, California Institute of Technology, Pasadena, CA 91125
Edited by Ralph R. Isberg, Howard Hughes Medical Institute, Tufts University School of Medicine, Boston, MA, and approved November 25, 2013 (receive
dfor
review January 27, 2013)
Pathogenic microbes have evolved complex secretion systems to
deliver virulence factors into host cells. Identification of these fac-
tors is critical for understanding the infection process. We report
a powerful and versatile approach to the selective labeling and
identification of secreted pathogen proteins. Selective labeling
of microbial proteins is accomplished via translational incorpora-
tion of azidonorleucine (Anl), a methionine surrogate that requires
a mutant form of the methionyl-tRNA synthetase for activation.
Secreted pathogen proteins containing Anl can be tagged by
azide-alkyne cycloaddition and enriched by affinity purification.
Application of the method to analysis of the type III secretion
system of the human pathogen
Yersinia enterocolitica
enabled
efficient identification of secreted proteins, identification of dis-
tinct secretion profiles for intracellular and extracellular bacteria,
and determination of the order of substrate injection into host
cells. This approach should be widely useful for the identification
of virulence factors in microbial pathogens and the development
of potential new targets for antimicrobial therapy.
proteomics
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click chemistry
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BONCAT
|
Yop
M
any bacterial pathogens use elaborate secretion systems to
transfer effector proteins into target cells (1). The injected
proteins disrupt host cell functions, including cytoskeletal assembly
and cytokine production, to promote infection (2). An important
step in understanding virulence mechanisms is the identification
of injected and secreted bacterial proteins. Traditional methods
have included genetic screens and candidate protein approaches,
which can be laborious and noncomprehensive. Proteome-wide
labeling strategies offer the potential to rapidly identify secreted
pathogen proteins without bias and with limited previous knowl-
edge of host
–
pathogen interactions (3).
We have developed a method, termed bio-orthogonal nonca-
nonical amino acid tagging (BONCAT), for incorporating azide
functional groups into proteins as a general strategy for the en-
richment of newly synthesized cellular proteins, making it pos-
sible to elucidate the spatial and temporal character of proteomic
changes (4, 5). Our initial studies used the noncanonical amino
acid azidohomoalanine (Aha) (structure
2
, Fig. 1
A
), a methio-
nine (Met) surrogate, to label newly synthesized proteins (4, 5).
The azide side chain of Aha allows newly synthesized proteins to
be tagged with alkyne-functionalized affinity reagents and sepa-
rated from preexisting proteins by affinity chromatography. After
separation, proteins are identified by tandem MS. Enrichment of
newly synthesized proteins reduces the complexity of the sample
and facilitates identification of the proteins of interest.
We recently showed that introduction of a mutant form of
the methionyl-tRNA synthetase (designated NLL-MetRS) into
Escherichia coli
enables incorporation of the noncanonical amino
acid azidonorleucine (Anl) (structure
3
, Fig. 1
A
) into the bac-
terial proteome (6). Because Anl is not activated to any significant
extent by any of the WT synthetases (6), labeling is restricted to
cells in which NLL-MetRS is expressed (7). This approach has
prompted recent efforts to study proteomic changes in pathogens
during infection (8). At the same time, there has been considerable
interest in protein labeling strategies to study secreted pathogen
proteins, most notably through the use of stable-isotope labeling
of amino acids in cell culture (SILAC) (9, 10). Isotopic labeling
does not allow enrichment of secreted pathogen proteins, how-
ever, and enrichment is important for identification of virulence
factors that otherwise would go undetected among abundant host
proteins. Here, using a shotgun, bottom-up proteomics approach,
we show that noncanonical amino acid labeling enables enrich-
ment of secreted virulence factors and identification of injected
proteins from host cell lysates.
Pathogenic bacteria secrete proteins through various mecha-
nisms. Secretion via type III, type IV, and type VI systems occurs
by direct injection of proteins into host cells, whereas type II and
type V secretion systems use a two-step passage through the inner
and outer membranes of the pathogen. Secreted outer mem-
brane vesicles also mediate export of a complex array of proteins
(11). We focus here on the well-characterized type III secretion
system (T3SS) of
Yersinia enterocolitica
, a Gram-negative bac-
terium. In
Yersinia,
the majority of secreted proteins, designated
Yersinia
outer proteins (Yops), are encoded on the 70-kb viru-
lence plasmid pYV (2, 12). In addition to encoding Yops, the
plasmid encodes machinery consisting of needle-shaped struc-
tures that assemble on the bacterial surface and inject proteins
into the cytoplasm of host cells. The T3SS is activated by a tem-
perature shift from 26 °C to the host temperature (37 °C); injection
Significance
Microbial pathogens use complex secretion systems to deliver
virulence factors into host cells, where they disrupt host cell
function. Understanding these systems is essential to the de-
velopment of new treatments for infectious disease. A chal-
lenge in such studies arises from the abundance of host cell
proteins, which interfere with detection of microbial effectors.
Here we describe a metabolic labeling strategy that allows
selective enrichment of microbial proteins from the host cell
cytoplasm. The method enables efficient identification of mi-
crobial proteins that have been delivered to the host, identifies
distinct secretion profiles for intracellular and extracellular
bacteria, and allows for determination of the order of injection
of microbial proteins into host cells.
Author contributions: A.M., S.K.M., and D.A.T. designed research; A.M., S.K.M., and D.A.T.
performed research; A.M., J.S., J.T.N., M.J.S., R.L.J.G., S.H., O.S., S.K.M., and D.A.T. con-
tributed new reagents/analytic tools; A.M., J.S., M.J.S., R.L.J.G., S.H., O.S., S.K.M., and
D.A.T. analyzed data; and A.M., J.S., J.T.N., M.J.S., R.L.J.G., S.H., O.S., S.K.M., and D.A.T.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: tirrell@caltech.edu.
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1301740111/-/DCSupplemental
.
www.pnas.org/cgi/doi/10.1073/pnas.1301740111
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is initiated on surface contact with target cells (13, 14). The pYV
virulence plasmid also encodes a low calcium response (LCR)
that enables secretion of T3SS substrates into the medium in the
absence of host cells (15). As a control for type III secretion, we
used a YscU mutant strain (designated T3SS-Mut), which is unable
to secrete Yops (16). YscU is an inner membrane protein re-
quired for T3SS assembly and recruitment of substrates (17).
In this study, NLL-MetRS was introduced to both WT and
mutant
Yersinia
strains to enable selective Anl labeling of bac-
terial proteins (Fig. 1
B
). Because host cells do not express NLL-
MetRS, host cell proteins are not labeled with Anl. After Anl
was added to the infection medium, Anl-labeled proteins were
tagged by copper-catalyzed cycloaddition (18) (Fig. 1
C
) with
alkyne-functionalized dyes and detected by in-gel fluorescence or
by confocal fluorescence imaging of infected host cells. Similarly,
enrichment of Anl-labeled proteins was performed after at-
tachment of a cleavable affinity tag (structure
4
, Fig. 1
A
) that
permits binding of labeled proteins to immobilized streptavidin
resin and removal of unlabeled proteins. The small mass modi-
fication resulting from tagging of Anl residues is readily detected
by MS, thereby facilitating identification of enriched proteins (
SI
Appendix
, Figs. S1 and S2
).
In a HeLa cell infection model, we identified the
Yersinia
proteins that were secreted into the medium and injected into
HeLa cells. In addition to identifying known Yops, we identified
secreted proteins that may play important roles in
Yersinia
in-
fection. An extension of this approach allowed us to selectively
label proteins secreted by
Yersinia
that had invaded HeLa cells
and to reveal secretion of distinct subsets of virulence factors.
Pulse-labeling with Anl was used to investigate the order of in-
jection of type III substrates into HeLa cells, providing a simple
method to determine the hierarchy of injection of virulence
factors. The approach described here is not limited to the study
of T3SS substrates, but can be used to examine the many dif-
ferent secretion systems of microbial pathogens.
Results
Labeling of the
Yersinia
Proteome and T3SS Substrates.
E. coli
NLL-
MetRS was constitutively expressed in
Y. enterocolitica
under
control of its natural promoter to enable incorporation of Anl
into bacterial proteins (
SI Appendix
, Fig. S3
). Proteins secreted
under LCR conditions were tagged with an alkyne-functionalized
tetramethylrhodamine (TAMRA) dye (
SI Appendix
, Fig. S4
) and
detected by in-gel fluorescence imaging (Fig. 2
A
). Labeling was
observed only in samples treated with Anl (Fig. 2
A
, lane 3);
nonspecific labeling in the absence of Anl was negligible (Fig.
2
A
, lane 1). Lack of TAMRA labeling in the absence of Anl was
not related to the absence of secreted proteins, because these
proteins were detected by colloidal blue staining (Fig. 2
B
, lane
1). These results confirm the chemoselectivity of the copper-
catalyzed click reaction. As expected, the T3SS-Mut strain did
not secrete any labeled proteins (Fig. 2
B
, lanes 2 and 4). The
similarity of the protein secretion profiles in the Met- and Anl-
treated samples (Fig. 2
B
, lanes 1 and 3), along with the lack of
secretion by the mutant strain (lanes 2 and 4), indicate that Anl
incorporation does not interfere with type III secretion. Western
blot analysis with antibodies specific for YopD and YopE con-
firmed Yop secretion by the T3SS-Wt strain (Fig. 2
C
). Analysis
Fig. 1.
Incorporation of Anl into injected pathogen proteins and enrich-
ment of labeled proteins. (
A
) Structures of amino acids Met (
1
) and Met
analogs Aha (
2
) and Anl (
3
). The alkyne-functionalized biotin affinity probe
(
4
) contains an acid-cleavable silane linker. The probe can be appended to
the azide side chain of Anl. Cleavage with formic acid transfers a small mass
tag to each modified Anl residue. (
B
) NLL-MetRS charges the tRNA
Met
with
Anl; its expression in the pathogen allows cell-selective labeling of pathogen
proteins during infection. Injected pathogen proteins are enriched after
labeling with
4
and identified by tandem MS. (
C
) Copper-catalyzed azide-
alkyne cycloaddition yields a triazole linkage.
Fig. 2.
Labeling of secreted T3SS substrates. (
A
) Secretion competent (
+
)
and secretion mutant (
−
)
Y. enterocolitica
strains harboring the NLL-MetRS
were induced to secrete T3SS substrates under LCR conditions. Secreted
proteins were labeled with alkyne-TAMRA and detected by in-gel fluores-
cence. (
B
) Detection of all secreted proteins in
A
by colloidal blue staining.
(
C
) Western blot detection of YopD and YopE in samples from
A
.(
D
) In-gel
fluorescence detection of alkyne-TAMRA labeling of
Y. enterocolitica
lysates
from conditions corresponding to lanes 3 and 4 in
A
shows proteome-wide
incorporation of Anl. (
Inset
) Colloidal blue staining of the same samples.
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Mahdavi et al.
of
Yersinia
lysates showed proteome-wide incorporation of Anl
into
Yersinia
proteins (Fig. 2
D
).
Detection of Injected Proteins in Host Cells by Fluorescence Imaging.
We examined the injection of Yops into HeLa cells, a widely
used in vitro model for
Yersinia
pathogenesis (17). Yop injection
results in a characteristic rounded HeLa cell morphology that
can be used to track injection (
SI Appendix
, Fig. S5
) (19, 20).
Infections were performed with 1 mM Anl; incubation of
Yersinia
at 37 °C before infection increased the efficiency of injection (
SI
Appendix
, Fig. S6
). Digitonin was used to lyse HeLa cells selec-
tively without causing significant disruption of
Yersinia
mem-
branes (
SI Appendix
, Fig. S7
) (19, 21). Injected T3SS substrates
were labeled with alkyne-TAMRA and detected by in-gel fluo-
rescence imaging. We observed distinct bands of labeled proteins
corresponding to molecular weights of known YopD, YopE,
YopH, YopM, YopN, YopP, YopQ, and low calcium response
virulence (LcrV) protein (Fig. 3
A
, lane 1). These bands were not
observed in infections with the T3SS-Mut strain [although a low
level of background labeling was observed (Fig. 3
A
, lane 2)], and
proteins injected by
Yersinia
lacking NLL-MetRS were not labeled
(Fig. 3
A
, lane 3). Cell-specific proteome-wide incorporation of
Anl in
Yersinia
was confirmed in these cocultures (Fig. 3
A
,lanes4
and 5). Injected proteins were readily detected by this chemical
labeling approach despite the much greater abundance of host cell
proteins. Western blot analysis of the same samples with anti-
bodies for YopD, YopE, and YopH showed the presence of these
Yops in infected HeLa cells; antibody staining for
Yersinia
bac-
terial RNA polymerase A (RpoA) confirmed the absence of sig-
nificant
Yersinia
cell lysis (Fig. 3
B
). Transference of HeLa cell
lysates treated with alkyne-TAMRA to nitrocellulose membranes
and probing of the membranes with antibodies for YopD and
YopE revealed that the protein bands detected by these anti-
bodies were also labeled with the TAMRA dye (Fig. 3
C
).
To visualize injection, we used fluorescence confocal micros-
copy to detect Anl-labeled T3SS substrates in the HeLa cell
cytoplasm. Anl-labeled proteins were tagged with alkyne-Alexa
Fluor 488. We observed increased fluorescence in the cytoplasm
of HeLa cells infected with the T3SS-Wt strain compared with
infections with T3SS-Mut
Yersinia
(Fig. 3
D
and
E
), although
a low level of background labeling was observed in experiments
with the mutant strain (Fig. 3
E
). As expected, infections with
Yersinia
that lacked NLL-MetRS resulted in HeLa cell rounding,
but no evidence of labeled proteins in the cytoplasm (
SI Appendix
,
Fig. S8
). These results indicate that Anl labeling can be used to
detect injected virulence factors inside host cells.
Identification of Virulence Factors.
As a first step toward identifying
injected virulence factors, we performed a directed MS search
for Anl-labeled Yops secreted under LCR conditions. We were
able to detect incorporation of Anl at Met positions distributed
throughout the secreted proteins (
SI Appendix
, Figs. S9
–
S11
).
We next sought to enrich and identify injected T3SS substrates
from HeLa cells using a shotgun MS approach. HeLa cells were
infected with
Y. enterocolitica
in Anl-supplemented medium and
selectively lysed with digitonin after the infection. Cell lysates
were treated with probe
4
for affinity enrichment of Anl-labeled
proteins. Biotinylation was detectable by Western blot analysis
(
SI Appendix
,Fig.S12
), and labeled proteins were affinity-enriched
on streptavidin resin (
SI Appendix
, Fig. S13
). In-gel tryptic di-
gestion was performed on these samples, and the resulting peptide
mixtures were analyzed on a nanoliquid chromatography, linear
trap quadrupole, Fourier transform (nano-LC-LTQ-FT) mass
spectrometer (
SI Appendix
, Figs. S14 and S15
). Type III-specific
virulence factors were determined by comparing lysates of HeLa
cells infected by the T3SS-Wt and T3SS-Mut strains (Fig. 3
F
).
This analysis identified previously reported T3SS-specific sub-
strates, including YopD, YopE, YopH, YopM, YopN, YopP,
YopQ, and LcrV (17, 22). We did not detect YopT or YopO,
perhaps because these proteins are
associated with host membranes.
YopT was previously observed in th
e insoluble fraction of HeLa cell
lysates (23, 24), and YpkA
(the YopO counterpart in
Yersinia
pseudotuberculosis
) has been reported to associate with the plasma
membrane after injection (25). It is also possible that YopT and
YopO are made or secreted at levels below our limit of detection.
In addition to identifying proteins injected into HeLa cells, we
investigated proteins that were secreted into the medium during
infection (
SI Appendix
, Fig. S16
). This was done by precipitating
the proteins from the infection medium and enriching Anl-la-
beled proteins. Comparison of T3SS substrates that were injec-
ted into HeLa cells with those secreted into medium during
infection (Fig. 3
G
) showed that YopB, YscP, and YscH/YopR
Fig. 3.
Detection and identification of injected
Y. enterocolitica
virulence factors in HeLa cells. (
A
)
HeLa cells were infected with
Y. enterocolitica
in
media containing 1 mM Anl. Proteins were labeled
with alkyne-TAMRA and detected by in-gel fluores-
cence. (
Inset
) Colloidal blue staining of the same gels.
(
B
) Western blots of HeLa cell lysates with antibodies
specific to YopD, YopE, YopH, and RpoA. The legend
at the bottom of
A
applies to
B
as well. (
C
) Lysates of
infected HeLa cells were treated with alkyne-TAMRA
(click) and transferred to nitrocellulose membranes
after SDS/PAGE. The same membranes were probed
with antibodies specific to YopD and YopE. (
D
) De-
tection of injected
Yersinia
proteins in HeLa cells by
fluorescence confocal microscopy. Anl-tagged pro-
teins were labeled with alkyne-Alexa Fluor 488 (green).
An Alexa Fluor 633-WGA conjugate was used to label
membranes of HeLa cells (red). (
E
) The same analysis
was performed for infections with secretion mutant
Y. enterocolitica
.(
F
) Shotgun proteomic identifica-
tion of virulence factors injected into HeLa cells. Color
code indicates the number of independent experi-
ments in which each protein was detected. *YopQ
was detected in only one infection. (
G
) Venn dia-
gram showing Yops injected into HeLa cells or se-
creted into medium during infection.
Mahdavi et al.
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were found only in the medium and not inside infected HeLa
cells. This finding supports a previously proposed mechanism of
action of these Yops, in which secretion of YscP is followed by
secretion of YopR into the extracellular medium, resulting in
injection of YopN and other effector Yops into host cells (19).
Nineteen
Yersinia
proteins were found both in lysates pre-
pared from HeLa cells infected with the secretion-competent
strain and in lysates prepared from cells infected with the se-
cretion mutant strain (Fig. 3
F
). These proteins, which are not
T3SS substrates, fall into two classes: highly abundant bacterial
proteins and proteins associated with the bacterial cell surface.
The highly abundant proteins, defined as those ranked among
the top 10% of bacterial proteins in terms of abundance according
to the PaxDb database (26), are listed in italic type in Fig. 3
F
and
include Tuf1 (ranked as the first of 3,163 proteins in terms of
abundance), TufA (ranked fourth), GapA (ranked fifth), Eno
(ranked sixth), GroL (ranked seventh), and DnaK (ranked tenth).
We have also listed LivK in this group, as it is in the top 10.8% of
bacterial proteins in terms of abundance, as well as MetG (MetRS),
because it is overexpressed in both
Yersinia
strains. It seems
likely that these highly abundant proteins are found in the HeLa
cell lysate as a result of a low level of adventitious bacterial cell
lysis, although we cannot rule out other mechanisms of transfer.
Previous proteomic studies of factors secreted by
Yersinia
found
subsets of these proteins, including HtpG, OmpA, GroL, and
several elongation factors (27), as well as DnaK and Eno (28).
The second class of proteins found in experiments conducted
with the secretion mutant strain includes the membrane-associ-
ated proteins A1js30, Sif15, A1jpb8, OmpA, and Ail.
Yersinia
surface proteins invasin, Ail, and YadA mediate binding to host
cells (29), and it has been shown that Ail mediates the attach-
ment and uptake of bacterially secreted outer membrane vesicles
(11). Outer membrane protein A (OmpA), also identified in our
analysis, is known to be present in such vesicles and released by
Gram-negative bacteria (11, 30). OmpA has been detected in
monocyte cell lysates after infection with
Y. pestis
(27), is known
to bind scavenger receptors (31), and is considered a potent
Yersinia
virulence factor (30). Sif15, also known as systemic factor pro-
tein-a (Sfpa), is involved in systemic infection of
Y. enterocolitica
,
is induced at 37 °C, and is necessary for colonization of mesen-
teric lymph nodes in a mouse Peyer
’
s patch infection model (32).
We also found the putative exported protein A1jpb8 and the
outer membrane porin A1js30 in lysates prepared from HeLa
cells infected with the secretion-mutant strain. Transfer of these
proteins to the HeLa cell lysate could occur via various mechanisms,
including regulated release of outer membrane vesicles. Further
work is needed to establish the mode of transfer of each protein.
Identification of Virulence Factors Secreted by Internalized Bacterial
Cells.
Many pathogens, including
Yersinia
strains, invade host
cells during infection. Internalized pathogens may secrete viru-
lence factors that are distinct from those released by extracel-
lular bacteria (33). We used the gentamicin protection assay with
pulsed Anl labeling to compare the type III secretion profiles of
internalized and extracellular
Yersinia
cells. Two parallel infec-
tions were initiated with the T3SS-Wt strain. After 1 h, to allow
internalization of the pathogen by HeLa cells, gentamicin was
added to one of the samples, thereby inhibiting protein synthesis
in the extracellular bacteria in this sample. Thereafter, Anl was
introduced into both samples for identical labeling times of 3 h.
Confocal microscopy verified selective labeling of
Yersinia
pro-
teins inside infected HeLa cells (Fig. 4
A
). In the absence of gen-
tamicin, both extracellu
lar and intracellular
Yersinia
were labeled
(
SI Appendix
, Figs. S17 and S18
and
Movies S1
and
S2
).
Comparison of Anl-labeled proteins in HeLa cell lysates by in-
gel fluorescence detection revealed a distinct pattern of proteins
secreted by internalized
Yersinia
(Fig. 4
B
); internalized cells
appear to secrete a subset of virulence factors. MS analysis, after
enrichment of injected proteins, also indicated that a subset of
Yops is secreted by internalized
Yersinia
(
SI Appendix
, Fig. S19
);
YopM, YopP, and YopQ were not detected, whereas YopD,
YopN, LcrV, and effectors YopE and YopH were injected by the
intracellular subpopulation.
Yops Are Injected in a Temporally Distinct Manner.
The BONCAT
method is ideally suited to the study of time-dependent cellular
phenomena. To analyze the order in which Yops are injected
into host cells, we used an Anl pulse-labeling strategy in which
Anl was added to the medium at specified times after infection
(Fig. 4
C
). MS analysis revealed that injection of YopD, which is
part of the type III needle complex that inserts into the host cell
membrane, is followed by injection of effector YopE and YopH.
Identification of YopD as the earliest injected substrate is sup-
ported by previous reports indicating that its injection is required
to establish translocation of other Yops (34). YopD, YopE, and
YopH have previously been detected on the surface of bacteria
before contact with host cells, potentially allowing rapid injection
of these substrates to stop phagocytosis (35). YopE disrupts the
host cell cytoskeleton and can interfere with phagocytosis, and YopE
and YopH are thought to control inj
ection of effector Yops (36).
Our finding that YopN and YopM are injected after YopE is
supported by the fact that impassable YopE-DHFR fusion sub-
strates can be used to block injection of YopN and YopM (37).
YopP was first detected at 60
–
90 min after initiation of infection,
in agreement with previous findings that its cytotoxic effect is not
detected until 60 min postinfection and that its inhibition of NF-
κ
B signaling in dendritic cells is detected starting at 90 min after
infection (38). Detection of YscM and YopO may indicate that
Fig. 4.
Labeling of proteins injected into HeLa cells by internalized
Y. enterocolitica
and identification of the order of Yop injection into HeLa
cells. (
A
) Confocal fluorescence microscopy showed Anl incorporation into
the proteome of internalized
Y. enterocolitica
. HeLa cell membranes were
labeled with Alexa Fluor 633-WGA conjugate (red), and Anl residues were
labeled with alkyne-Alexa Fluor 488 (green). The arrow indicates labeled
Y. enterocolitica
inside HeLa cells. (
B
) Infected HeLa cells were selectively
lysed with digitonin and treated with alkyne-TAMRA to detect the proteins
injected by internalized
Y. enterocolitica
.(
Inset
) Colloidal blue staining of
the same gel. In the presence of gentamicin only, internalized
Yersinia
can
inject proteins into HeLa cells. (
C
) The order of injection of Yops was de-
termined by pulsed-Anl labeling and shotgun MS. Anl was added only dur-
ing the indicated times for each infection, and HeLa cells were lysed with
digitonin at the end of each time interval. (
D
) Western blot analysis detected
Yops in pulsed-Anl labeling experiments. RpoA served as a control for bac-
terial lysis; antibody for
α
-tubulin was used as a loading control for HeLa
lysates. BP, bacterial pellet.
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Mahdavi et al.
Anl pulse-labeling may be particularly good for identifying low-
abundance and transiently injected proteins that would other-
wise be undetected. Taken together, these results demonstrate
the capacity of the BONCAT method to elucidate the hierarchy
of secretion of virulence factors.
Discussion
Identification of effector proteins that are secreted or injected by
pathogenic bacteria offers new opportunities for understanding
mechanisms of pathogenesis and developing novel therapeutics.
Here we show that cell-selective, noncanonical amino acid tag-
ging enables labeling, enrichment, and identification of virulence
factors secreted by pathogenic bacteria. Cell-selective proteomic
labeling was achieved by outfitting
Yersinia
cells with
E. coli
NLL-MetRS, which charges
Yersinia
tRNA
Met
with the azide-
functionalized noncanonical amino acid Anl (Fig. 1
B
). Treatment
of Anl-labeled proteins with alkyne affinity reagents provided a
selective chemical tagging method and enabled enrichment of se-
creted virulence factors from abundant host proteins.
Yersinia
proteins isolated from HeLa cell lysates included eight T3SS
substrates and 19 proteins that were transferred via type III-
independent mechanisms (Fig. 3
F
). Because some (or perhaps all)
of the latter proteins may have been released via adventitious
bacterial cell lysis, the overall selectivity of the method might be
enhanced by further improvements in the removal of bacterially
shed proteins and better host cell lysis techniques.
For live cell applications, cyclooctyne-functionalized reagents
can be used to tag the azide side chain of Anl residues in a
copper-free manner (39, 40). Our chemical tagging strategy is
compatible with routine MS sample preparation methods such
as gel electro
phoresis liquid chromatogr
aphy-mass spectrometry
(GeLC-MS), filter-aided sample preparation (FASP) (41, 42), and
multidimensional protein identification technology (MudPIT)
(43), and is easily combined with SILAC, isobaric tag for rel-
ative and absolute quantification (iTRAQ), and multiple-reaction
monitoring (MRM) quantitative MS methods (44, 45). This ap-
proach can be complemented with candidate protein methods,
such as expression of tagged substrates, to verify the secretion and
identify the location of newly identified substrates inside host cells.
Pulsed Anl labeling was combined with a gentamicin pro-
tection assay to identify proteins injected into HeLa cells by in-
ternalized
Yersinia
(Fig. 4
A
and
B
). The results demonstrate that
the method can be used in different compartments of the host
and should be applicable to studies of functional redundancy, in
which multiple effectors perform similar functions (46). Pulsed
Anl labeling was used to study the hierarchy of Yop injection,
enabling direct elucidation of the order of injection of T3SS
substrates (Fig. 4
C
and
D
). As an alternative to pulsed Anl la-
beling, spatial and temporal resolution may be achieved in future
studies by placing NLL-MetRS under the control of specific
promoters that are stage-specific or spatiotemporally regulated
(47). This approach may allow for Anl labeling at different stages
of infection, particularly host cells, or subcellular compartments.
Straightforward extensions of the technology will enable in-
vestigation of both pathogen proteins and host proteins during
infections in animals. The simplicity of the approach makes it
suitable for the study of numerous host
–
microbe interactions.
Materials and Methods
Expression of NLL-MetRS in
Y. enterocolitica.
DH10B strains were used for
genetic manipulations.
E. coli
NLL-MetRS with mutations L13N, Y360L, and
H301L was isolated from pJTN1 (7) by Nhe1 digestion and inserted into
pQE80 (Qiagen). Kanamycin resista
nce was used for selection, because
Y. enterocolitica
is resistant to ampicillin (48) and nalidixic acid. The resulting
plasmid, which carries NLL-MetRS under control of the endogenous
E. coli
MetG promoter, is termed pAM1 and has been deposited in Addgene. The
plasmid was transformed into electrocompetent
Y. enterocolitica
,and
transformants were grown at 26 °C on agar plates or in LB medium, both
containing 50
μ
g/mL kanamycin.
Secretion of T3SS Substrates Under LCR Conditions.
Y. enterocolitica
W2273
was diluted 1:50 from an overnight LB culture into M9 medium at 26 °C with
agitation at 250 rpm. At OD
600
=
0.5, protein secretion was initiated by
a temperature shift to 37 °C. Labeling with Anl was performed in M9 me-
dium lacking calcium and containing 1 mM Anl. After 2.5 h, bacteria were
sedimented for 15 min at a relative centrifugal force of 15,000
×
g
at 4 °C.
The medium was passed through a 0.2-
μ
m filter, and proteins were pre-
cipitated with chloroform/methanol.
HeLa Cell Infection and Anl Labeling of T3SS Substrates.
HeLa cells (American
Type Culture Collection) were routinely cultured in DMEM supplemented
with 10% FBS, trypsinized (Gibco), and expanded every 72 h. Before infection,
cells were washed twice with PBS and resuspended in Opti-MEM medium
(Gibco).
Y. enterocolitica
was diluted 1:25 from overnight cultures in LB and
incubated at 26 °C with agitation at 250 rpm until an OD
600
=
0.5 was
reached.
Yersinia
were preincubated at 37 °C for 3 h before the start of
infection. The preincubation time was determined by tracking T3SS injection
of Anl-labeled proteins (
SI Appendix
, Fig. S6
). Labeling was performed at
a multiplicity of infection of 100, with 10
7
HeLa cells per condition, at 1 mM
Anl and 50
μ
g/mL kanamycin. Infections were carried out for 3.5 h. Infected
HeLa cells were lysed with digitonin as described below for analysis of
injected
Yersinia
proteins in HeLa cells (Fig. 3
F
).
Selective Lysis of HeLa Cells After Infection.
After infection, HeLa cells were
washed five times with PBS to remove surface-bound proteins. Cells were
incubated with 0.1% (wt/vol) digitonin in PBS for 20 min at room temperature
with agitation at 100 rpm. EDTA-free protease inhibitor (Roche) was added to
the lysis buffer. Bacterial cells were removed from the lysates by centrifu-
gation at 15,000
×
g
for 15 min at 4 °C and filtration through a 0.2-
μ
m filter.
Western blot analysis with an antibody for RpoA was used to confirm the
absence of
Yersinia
lysis.
Enrichment of Anl-Labeled Proteins.
Probe
4
was appended to Anl-labeled
proteins by copper-catalyzed azide-alkyne cycloaddition (
SI Appendix
, Fig.
S1
). Proteins were precipitated with acetone, dissolved in 250
μ
Lof4%SDS
in PBS, and diluted to 0.1% SDS by the addition of PBS supplemented with
EDTA-free protease inhibitor (Roche). Proteins were incubated with 400
μ
L
of Streptavidin Plus Ultralink resin (Pierce) for 1.5 h at room temperature.
Affinity purification was performed according to a previously published
protocol (49). Elution fractions were combined with Amicon Ultra 0.5 cen-
trifuge filters (3 kDa molecular weight cutoff; Millipore). Enrichment was
also performed with Click-iT alkyne-agarose resin (Invitrogen).
Detection of Proteins in Gels and Western Blots.
Bicinchoninic acid protein
quantification (Pierce) was used to equalize the amounts of proteins analyzed
under different conditions. After dye labeling via the copper-catalyzed click
reaction described above, proteins were washed with methanol to remove
unreacted dye and then electrophoresed on
a Novex 12% Bis-Tris polyacrylamide
gel (Invitrogen). Colloidal blue dye (Invitrogen) was used for nonspecific
protein detection. Antibodies were used at the following dilutions: YopD,
1:20,000; YopE, 1:40,000; YopH, 1:4,000; RpoA, 1:40,000,
α
-tubulin (Abcam)
and secondary antibody anti-rabbit IgG-Alexa Fluor 488 conjugate (Cell Signal
Technologies), 1:1,000. Fluorescence imaging of Western blots and gels was
performed with a Typhoon 9400 molecular imager (GE Healthcare).
Fluorescence Confocal Microscopy.
Adherent HeLa cells were infected as de-
scribed above and fixed with 3.7% for
maldehyde in PBS before labeling
with 10
μ
g/mL Alexa Fluor 633-wheat germ agglutinin (WGA) conjugate
(Invitrogen) in PBS for 30 min. Cell
s were permeabilized with ice-cold
methanol for 3 min. Labeling with alkyne-TAMRA (Invitrogen) was per-
formed as described above. Fluores
cence confocal images were obtained
on a Zeiss LSM 510 microscope.
Comparison of Yops Injected by Extracellular and Internalized
Y. enterocolitica.
Y. enterocolitica
(T3SS-Wt) was diluted 1:25 from overnight cultures in LB
and incubated at 26 °C with agitation until OD
600
=
0.5 was reached. Two
parallel infections of 5
×
10
7
HeLa cells each were initiated at a multiplicity
of infection of 100 in Opti-MEM without Phenol Red (Invitrogen). After 1 h
of infection, 80
μ
g/mL gentamicin was added to a sample of infected HeLa
cells; the other sample did not contain the antibiotic. After 1 h, the medium
was changed to Opti-MEM without gentamicin, and 1 mM Anl was added to
both samples. The infection that was initially treated with gentamicin was
supplemented with 4
μ
g/mL gentamicin to maintain inhibition of protein
synthesis by extracellular bacteria. After 3 h of labeling, HeLa cells in both
Mahdavi et al.
PNAS
|
January 7, 2014
|
vol. 111
|
no. 1
|
437
MICROBIOLOGY
samples were lysed with 0.1% digitonin for enrichment and MS analysis or
fixed with 3.7% formaldehyde for fluorescence confocal microscopy as
described above.
Determination of the Order of Yop Injection.
T3SS-Wt
Y. enterocolitica
was
diluted 1:25 from overnight cultures in LB and incubated at 26 °C with ag-
itation up to an OD
600
=
0.5. Cells were pelleted at 5,000
×
g
and washed
with PBS. Infection of four parallel samples, corresponding to the four time
windows of interest (Fig. 4
C
), was initiated as described above, with no
preincubation at 37 °C. Anl was added to the infection medium at 1 mM for
the indicated times (Fig. 4
C
), and HeLa cells were lysed with digitonin at the
end of each interval. HeLa cell lysates were treated with probe
4
as described
above for enrichment and identification of injected proteins by MS.
Mass Spectrometry.
AnalyseswereperformedwitheitherahybridLTQ-Orbitrap
or LTQ-FT Ultra (Thermo Fisher Scientific) equipped with a nanoelectrospray ion
sourceconnectedtoanEASY-nLCIIinstrument(ThermoFisherScientific).Details
of instrument setup and data analysis are provided in
SI Appendix
,TableS1
.
ACKNOWLEDGMENTS.
We thank Geoff Smith (Proteome Exploration Lab-
oratory, Beckman Institute, California Institute of Technology) for technical
assistance. This work was supported by the National Institutes of Health
(Grant R01 GM062523), the Institute for Collaborative Biotechnologies
(Grant W911NF-09-0001 from the US Army Research Office), the Burroughs
Wellcome Fund in the Pathogenesis of Infectious Disease, and the Gordon
and Betty Moore Foundation. A.M. is the recipient of a Natural Sciences and
Engineering Reasearch Council of Canada scholarship and a Donna and
Benjamin M. Rosen postgraduate scholarship.
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Mahdavi et al.