Burkholderia pseudomallei suppresses Caenorhabditis
elegans immunity by speciﬁc degradation of a GATA
, Rui-Rui Wong
, Chui-Yoke Chin
, Tian-Yeh Lim
, Su-Anne Eng
, Cin Kong
, Nur Aﬁfah Ijap
, Mei-Perng Lim
, Yunn-Hwen Gan
, Fang-Lian He
, Man-Wah Tan
, and Sheila Nathan
School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia;
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597; Departments of
Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5120; and
Malaysia Genome Institute, Jalan Bangi, 43000
Kajang, Selangor, Malaysia
Edited by Frederick M. Ausubel, Harvard Medical School and Massachusetts General Hospital, Boston, MA, and approved August 7, 2013 (received for review
June 24, 2013)
Burkholderia pseudomallei is a Gram-negative soil bacterium that
infects both humans and animals. Although cell culture studies
have revealed signiﬁcant insights into factors contributing to vir-
ulence and host defense, the interactions between this pathogen
and its intact host remain to be elucidated. To gain insights into
the host defense responses to B. pseudomallei infection within an
intact host, we analyzed the genome-wide transcriptome of infected
Caenorhabditis elegans and identiﬁed ∼6% of the nematode genes
that were signiﬁcantly altered over a 12-h course of infection. An
unexpected feature ofthe transcriptional response to B. pseudomal-
lei was a progressive increase in the proportion of down-regulated
genes, of which ELT-2 transcriptional targets were signiﬁcantly
enriched. ELT-2 is an intestinal GATA transcription factor with
a conserved role in immune responses. We demonstrate that B.
pseudomallei down-regulation of ELT-2 targets is associated with
degradation of ELT-2 protein by the host ubiquitin–proteasome
system. Degradation of ELT-2 requires the B. pseudomallei type
III secretion system. Together, our studies using an intact host
provide evidence for pathogen-mediated host immune suppres-
sion through the destruction of a host transcription factor.
Bacterial pathogens attack host cells to gain access to a privi-
leged niche within a host, whereas hosts respond by acti-
vating their immune system aimed at restricting and eliminating
infecting pathogens. Several conserved innate immune signaling
pathways have been revealed from studies of host–pathogen
interactions using vertebrate and invertebrate models. They in-
clude the Toll-like receptor signaling and a GATA transcription
factor with a conserved role in epithelial immunity in Caeno-
rhabditis elegans,Drosophila, and mammals (1, 2). The transcrip-
tion factor FOXO, which is regulated by the conserved insulin
signaling pathway, is another factor regulating the expression of
antimicrobial peptides in worms, ﬂy, and mammals (3–6). In the
coevolutionary arms race between host and pathogens, selec-
tive pressures imposed by the host drive pathogens to evolve
mechanisms to subvert or suppress host immune responses. For
example, Pseudomonas aeruginosa suppresses the expression of
a subset of FOXO-regulated immune defense genes in C. ele-
gans by activating the DAF-2/DAF-16 insulin-like signaling
Burkholderia pseudomallei is the causative agent of melioi-
dosis, a severe infectious disease endemic to Southeast Asia,
northern Australia, and other tropical areas (8). B. pseudomallei
infections are responsible for up to 40% of sepsis-related mor-
tality. No licensed vaccine is currently available for immuno-
prophylaxis of melioidosis. Despite much knowledge gained on
the pathogenic factors and immunogenic agents of B. pseudo-
mallei (8), molecular mechanisms underlying host susceptibility
and response to infection remain poorly understood.
Taking advantage of the tractability and simplicity of C. ele-
gans as a model, we investigated host responses to B. pseudo-
mallei infection in the context of a whole organism. Whole-
genome gene expression proﬁling in C. elegans identiﬁed a set of
ELT-2–regulated transcripts that are progressively down-regu-
lated over a time course of B. pseudomallei infection that is
concomitant with a progressive and speciﬁc loss of nuclear ELT-2
in the worm intestine. Loss of ELT-2 requires the host ubiq-
uitin–proteasome system (UPS) and is dependent on the
B. pseudomallei type III secretion system (T3SS). Given the
conserved role of GATA factors in epithelial immunity, this
ﬁnding may contribute new insights into B. pseudomallei in-
fection of humans.
Bacterial pathogens use multiple mechanisms to survive and
proliferate within an infected host, including blunting the host’s
ability to defend itself from pathogenic assaults. We identiﬁed
a new immune suppression mechanism by Burkholderia pseu-
domallei, the causative agent of melioidosis, which a life-
threatening disease in humans. Analyses of whole-genome
transcriptional responses of Caenorhabditis elegans to B. pseu-
domallei infection revealed that B. pseudomallei,throughits
type III secretion system, recruits the host ubiquitin–proteasome
system to speciﬁcally degrade a GATA transcription factor. This
GATA factor is critical for host immune defense; thus, its deg-
radation leads to suppression of the host’s ability to mount an
effective antimicrobial defense.
Author contributions: S.-H.L., C.-Y.C., M.-W.T., and S.N. designed research; S.-H.L., R.-R.W.,
C.-Y.C., T.-Y.L., S.-A.E., C.K., N.A.I., M.-S.L., M.-P.L., and F.-L.H. performedresearch; Y.-H.G.
and M.-W.T. contributed new reagents/analytic tools; S.-H.L., R.-R.W., C.-Y.C., T.-Y.L., S.-A.E.,
C.K., N.A.I., M.-W.T., and S.N. analyzed data; and S.-H.L., R.-R.W., M.-W.T., and S.N. wrote
The authors declare no conﬂict of interest.
This article is a PNAS Direct Submission.
Data deposition: The microarray data reported in this paper have been deposited in the
Stanford Microarray Database, http://smd.stanford.edu.
S.-H.L. and R.-R.W. contributed equally to this work.
Present address: Department of Integrative Biology, University of California, Berkeley,
Present address: Emory Vaccine Center, Emory University, Atlanta, GA 30329.
Present address: Department of Plant Biology, Carnegie Institution for Science, Stanford,
Present address: Department of Infectious Diseases, Genentech, Inc., South San Francisco,
To whom correspondence may be addressed. E-mail: email@example.com or sheila@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
September 10, 2013
Transcriptional Changes in Response to Pathogenic B. pseudomallei.
To assess temporal transcriptional changes in B. pseudomallei-
infected C. elegans, we performed a genome-wide transcriptome
analysis on age-matched adult worms infected with B. pseudo-
mallei strain R15 (henceforth referred to as BpR15), a clinical
isolate that is highly pathogenic on C. elegans (9). We compared
transcript levels in worms exposed to BpR15 for 2, 4, 8, and 12 h
to those exposed to Escherichia coli strain OP50—the standard
laboratory food source as uninfected control—over the same
time course. The initial time points precede the ﬁrst deaths,
whereas at 12 h postinfection (hpi), a mortality of less than 5% of
the population was observed (Fig. 1A). At each experimental time
point, microarray analysis compared gene expression between B.
pseudomallei-infected and -uninfected control animals relative to
a reference control, which comprised a mixed-stage population of
C. elegans. The application of a reference control to normalize C.
elegans gene expression enables an accurate comparison of the
average gene expression between any two time points (10).
B. pseudomallei provoked progressively greater transcriptional
changes in C. elegans over the course of infection. Between 2 and
8 hpi, an increasingly larger subset of genes was up-regulated in
response to BpR15 compared with E. coli (signiﬁcance analysis
of microarray analysis, at a false-discovery rate of ≤1%) (11).
However, at 12 hpi, fewer genes were up-regulated (454 genes)
and more were down-regulated (493 genes) (Table S1). Notably,
the proportion of down-regulated genes increased with time of
infection. Whereas only 16% of modulated genes were down-
regulated at 2 hpi, the proportion progressively increased to
∼30% at 4 and 8 hpi and to 53% at 12 hpi (Fig. S1), hinting at
the possibility that suppression of host defense genes expression
may be an important virulence mechanism used by B. pseudo-
mallei. Cluster analysis of time course data from 2 to 12 hpi
revealed that ∼6% (>1,200) C. elegans genes were differentially
expressed in response to B. pseudomallei, of which 694 genes
were up-regulated and 560 genes were down-regulated (Fig. 1B
and Dataset S1). Although a majority of induced genes after
8 hpi were involved in defense response (Dataset S1), some that
encode immune effectors, such as lys-7 and spp-1, were signiﬁ-
cantly repressed; the signiﬁcance of this observation will be
addressed below. Quantitative real-time PCR (qRT-PCR) mea-
surements conﬁrmed microarray results for 30 genes identiﬁed
as infection and stress response genes with altered expression at
12 hpi (Table S2) (7). Functional category analysis revealed that
defense-speciﬁc genes, including those with putative roles in
pathogen detection, activation of immune-related signaling path-
ways, and candidate immune effector genes, were enriched upon
B. pseudomallei infection, which is in concordance with responses
to other bacterial pathogens (1, 12, 13). Numerous cellular pro-
cesses were transcriptionally altered over the course of B.
pseudomallei infection: genes involved in metabolic processes,
longevity, and stress responses were enriched and host lipid me-
tabolism genes were among the most signiﬁcantly repressed.
Comparing our infection-response gene list with published
regulated gene lists for the p38 MAPK (12, 14), DAF-2 insulin-like
(3, 15), and SMA/TGF-βpathways (16, 17) revealed signiﬁcant
enrichment of genes regulated by these factors (Fig. S2A) (hyper-
geometric probability, P<1×10
). Speciﬁcally, expression of 42
of 92 PMK-1–regulated genes was signiﬁcantly altered during in-
fection [representation factor (Rf) =6.51]. Of the 440 (class 1 and
2) DAF-16–regulated genes (3), 149 genes were differentially
expressed in infected worms (Rf =4.83). Among the 1,122 genes
identiﬁed as SMA-6 regulated (17), 144 of these overlapped with
the B. pseudomallei infection response (Rf =1.83). The GATA
transcription factor ELT-2 is a positive regulator of intestinal im-
mune responses (1, 18). A signiﬁcant enrichment (Rf =8.43,
hypergeometric probability P<1×10
) was observed between
ELT-2–regulated genes (19) and B. pseudomallei infection re-
sponse genes, suggesting that ELT-2 might also contribute to the
infection response of B. pseudomallei (Fig. S2A). By contrast, the
noncanonical unfolding protein response (ncUPR), an important
defense mechanism against bacterial pathogens (20) and repre-
sented by the pqn/abu genes, was not signiﬁcantly induced (Fig.
S2B), suggesting that B. pseudomallei infection did not elicit the
B. pseudomallei Down-Regulates ELT-2 Transcriptional Targets. ELT-
2 transcriptional targets are induced by P. aeruginosa PA14 (1).
Unexpectedly, of the 42 ELT-2–regulated genes that were dif-
ferentially expressed following B. pseudomallei infection, 83%
were repressed over the course of infection (Fig. S3). They in-
clude genes predicted to encode for proteins involved in bacte-
rial lysis, luminal degradation of macromolecules, detoxiﬁcation,
and stress response (Table S3) (19). When we compared the
expression of a subset of ELT-2 transcriptional targets (elo-6,
F55G11.2, F57F5.1, K10C2.3, K12H4.7, and spp-8) in worms
infected by BpR15 and PA14 using qRT-PCR, an opposite
pattern of gene expression was observed (Fig. 2A). ELT-2–reg-
ulated genes that were induced by PA14 were consistently sup-
pressed by BpR15. Suppression of ELT-2 transcriptional targets
by BpR15 does not appear to be a consequence of a general
shutdown in transcription because ELT-2–independent tran-
scripts (pgp-5,ugt-29,thn-1,pqm-1, and skr-3) were similarly in-
duced by PA14 and BpR15 (Fig. 2B). To conﬁrm that the
repression of ELT-2 transcriptional targets by B. pseudomallei is
associated with inactivation of elt-2, we measured the transcript
levels of ELT-2 targets in adult worms following elt-2 knockdown
by RNAi and upon BpR15 infection. We observed a more pro-
nounced reduction of ELT-2 transcriptional targets (asp-3,mtl-1,
mtl-2,elo-6, F55G11.2) expression in elt-2 RNAi-treated adult
animals after BpR15 infection (Fig. 2C).
To determine whether ELT-2 is required to protect against
B. pseudomallei infection, we compared survival of elt-2 RNAi-
treated and vector control adult animals following BpR15 in-
fection. Exposure to elt-2 RNAi resulted in a signiﬁcant re-
duction in worm survival on BpR15 (Fig. S4A) but did not
signiﬁcantly alter normal life span (Fig. S4B). Thus, elt-2 is re-
quired to protect C. elegans from B. pseudomallei infection.
B. pseudomallei Affects ELT-2 at the Posttranscriptional Level. The
juxtaposition between the requirement of ELT-2 for defense
against B. pseudomallei and down-regulation of ELT-2 transcrip-
tional targets in BpR15-infected worms led us to hypothesize that
this pathogen could target ELT-2 for immune suppression either
at the transcriptional or posttranscriptional level. We ruled out the
former by showing that elt-2 transcripts in BpR15-infected worms
Fig. 1. B. pseudomallei induces a rapid transcriptional response. (A) Kinetics
of C. elegans killing by BpR15. The dotted arrows indicate time points chosen
for gene expression analyses. (B) Hierarchically clustered expression proﬁle
(rows) for 1,254 genes differentially expressed upon exposure to BpR15
compared with OP50 at 2, 4, 8, and 12 h after exposure. Data from three
independent experiments (columns) are shown for each time point. The
vertical bars mark clusters of genes induced (Upper) or repressed (Lower).
www.pnas.org/cgi/doi/10.1073/pnas.1311725110 Lee et al.
were not signiﬁcantly reduced at 4 and 12 hpi compared with
uninfected (Fig. 3A). We next determined the effects of infection
on ELT-2 protein levels in intact worms using a nuclear ELT-2::
GFP quantitation assay. First, we infected JM90 transgenic worms
that express an ELT-2::GFP fusion protein under the control of its
native promoter (21). Over the course of infection, we compared
the intensity and distribution of ELT-2::GFP ﬂuorescence (Fig. 3
B–D)andenumeratedthenumberofﬂuorescent nuclei in the
worm intestine (Fig. 3E). ELT-2::GFP ﬂuorescence remain lo-
calized to intestinal nuclei of worms exposed to E. coli OP50 (Fig.
3B) and PA14 (Fig. 3C). In stark contrast, only faint ELT-2::GFP
ﬂuorescence could be detected in the intestinal nuclei of BpR15-
infected animals (Fig. 3D). Whereas the worms exposed to E. coli
and PA14 retained the ELT-2::GFP fusion protein in the nucleus
throughout infection (Fig. 3E), worms exposed to BpR15 showed
asigniﬁcant reduction in the number of nuclear GFP from 12 to
24 hpi. To demonstrate that the effect does not simply represent
changes due to intestinal damage or the dying process due to the
more rapid killing by BpR15, the number of nuclear localized
GFP was enumerated in worms exposed to PA14 up to the point
where 50% of the worm population was killed (Fig. S5A). These
worms retained a similar number of nuclear GFP as worms ex-
posed to E. coli over the course of infection (Fig. S5B). Thus, the
loss of ELT-2 is speciﬁctoB. pseudomallei infection and not
a consequence of intestinal damage. To further conﬁrm that the
level of ELT-2 protein had diminished over the course of BpR15
infection, we immunoblotted lysates obtained from age-matched
JM90 adults exposed to E. coli, PA14, or BpR15, using a
monoclonal antibody speciﬁc to either GFP or ELT-2 (21).
Consistent with the nuclear enumeration assay, worms infected
with PA14 for 12 and 18 h had comparable levels of ELT-2::GFP
and ELT-2 as that observed in worms exposed to E. coli (Fig.
3F). In contrast, much lower levels of ELT-2::GFP and ELT-2
protein were observed in worms infected with BpR15 after 12
and 18 h (Fig. 3F). A similar observation was obtained in worms
infected with BpR15 up to 24 h (Fig. S5C).
We also enumerated nuclear ELT-2::GFP in worms infected
with other Gram-negative (e.g., Salmonella typhimurium)and
Gram-positive (e.g., Enterococcus faecalis and Staphylococcus au-
reus) bacteria known to cause a comparable killing rate (Fig. 4A).
None of these pathogens induced signiﬁcant loss of ELT-2::GFP
over the course of infection (Fig. 4B). Infections by other Bur-
kholderia species—B. thailandensis,B. cepacia,andB. vietnamiensis—
also did not trigger the loss of ELT-2::GFP protein (Fig. 4 Cand
D). By contrast, all other B. pseudomallei isolates tested (Orang
Utan 320, K96243, Argus4533, and Goat 2124) were capable of
causing loss of ELT-2::GFP (Fig. 4 Eand F). Additionally, the
extent of ELT-2::GFP loss correlated with levels of virulence,
with the most marked ELT-2 loss seen in worms infected by the
most virulent B. pseudomallei isolate. These results suggest that
repression of ELT-2 transcriptional targets is associated with
reduction of ELT-2 protein and that targeting ELT-2 is a unique
pathogenic strategy of B. pseudomallei.
Fig. 2. Down-regulation of ELT-2 transcriptional targets by
B. pseudomallei.(Aand B) Gene expression changes during
infection by BpR15 (open columns) and PA14 (ﬁlled columns).
Shown are fold relative to uninfected animals. Columns rep-
resent mean ±SD; n=2. (A) ELT-2 and (B) non–ELT-2 tran-
scriptional targets. (C) Effects of elt-2 RNAi knockdown on
BpR15-infected worms measured by qRT-PCR. Shown are nor-
malized fractions of speciﬁc RNA levels in BpR15-infected ani-
mals relative to uninfected animals in elt-2 RNAi animals (ﬁlled
column) and animals fed on empty vector (open columns). The
columns represent mean ±SEM; n=2.
Fig. 3. B. pseudomallei causes the loss of nuclear ELT-2 in the intestine of C.
elegans.(A)elt-2 transcripts at 4 and 12 hpi with BpR15 measured by qRT-
PCR. Shown are normalized fractions of elt-2 RNA levels in infected relative
to OP50-exposed animals. Shown are mean ±SD; n=3. (B–D) Representative
ﬂuorescence micrographs of ELT-2::GFP-expressing adult worms with nuclear
localized ELT-2::GFP fusion protein after 24-h exposure to (B) OP50, (C) PA14,
and (D) BpR15. (E) Mean number of nuclei with detectable ELT-2::GFP signal
after 4-, 12-, 18-, and 24-h exposure to OP50 (black columns), PA14 (white
columns), and BpR15 (gray columns). *ttest, P<0.001 relative to OP50 at the
corresponding time point. The columns represent mean ±SEM; n=3. (F)
Representative immunoblots of ELT-2::GFP protein in OP50-, PA14-, and
BpR15-infected worms at 12 and 18 hpi using an ELT-2–speciﬁc mAb and
anti-GFP antibody. Anti-actin antibody was used as loading control.
Lee et al. PNAS
September 10, 2013
The Loss of Nuclear Protein in the Intestine Is Speciﬁc to ELT-2. To
test whether the loss of ELT-2 is mediated by B. pseudomallei
infection rather than an indirect consequence of general stress,
we exposed JM90 to cadmium, a toxic heavy metal known to
induce stress responses in C. elegans (22). No loss of nuclear
GFP was observed even with a high concentration of cadmium
(50 mM) (Fig. S6A), indicating that the loss of nuclear ELT-2::
GFP is a speciﬁc response to B. pseudomallei infection and not to
general stress. To test whether B. pseudomallei speciﬁcally tar-
gets nuclear-localized ELT-2, we compared the effect of BpR15
infection on ELT-2::GFP to other nuclear-localized transcription
factors: (i) PHA-4, using a transgenic strain AD84 that expresses
functional PHA-4::GFP fusion protein (23); and (ii) DAF-16,
using a transgenic strain TJ356 that expresses functional DAF-16::
GFP fusion protein (24). The number and intensity of nuclear
localized PHA-4::GFP (Fig. S6B) remained constant throughout
the BpR15 infection. For DAF-16, which is distributed pre-
dominantly in the cytoplasm under normal growth conditions, we
ﬁrst localized DAF-16::GFP fusion protein to the nuclei by heat
shock before exposing the worms to either BpR15 or E. coli.In
contrast to ELT-2::GFP ﬂuorescence, DAF-16::GFP ﬂuores-
cence remained nuclear localized and at a constant intensity over
the course of a 24-h infection (Fig. S6C). Heat treatment did
not signiﬁcantly affect the quantitation of ELT-2::GFP (Fig.
S6D). We conclude that ELT-2::GFP loss was not due to a
generalized translational shutdown nor a general loss of GFP-
fused proteins. Instead, it was speciﬁctoELT-2andspeciﬁcto
B. pseudomallei infection.
Host UPS Mediates ELT-2 Degradation During B. pseudomallei
Infection. The UPS plays a critical role in protein degradation
and is exploited by numerous pathogens to facilitate infection (25).
Protein degradation via UPS involves two processes: (i) covalent
attachment of ubiquitin (Ub) to target protein and (ii) degradation
of ubiquitin-tagged protein by the 26S proteasome. Covalent at-
tachment is a multistep process that involves Ub-activating (E1),
Ub-conjugating (E2), and Ub-ligating (E3) enzymes, which selec-
tively interacts with Ub-loaded E2, recruits and binds speciﬁc
substrates, thus transferring Ub to speciﬁc target proteins. Ub-
tagged proteins are then degraded in the proteasome (Fig. S7).
To investigate whether loss of ELT-2 during a B. pseudomallei
infection is mediated by the host UPS, we enumerated nuclear
ELT-2::GFP in RNAi-mediated knockdown of C. elegans ubq-1,
ubq-2,orrpt-2 genes. ubq-1 and ubq-2 encode for C. elegans
ubiquitin. RPT-2 is a regulatory proteasome particle and RNAi
of rpt-2 has been shown to inhibit proteasome function in live
C. elegans (26). As proteasome inactivation is lethal to developing
worms, we initiated RNAi only in young adults. As expected, in
adult animals exposed to E. coli OP50 and PA14, the number of
nuclear ELT-2::GFP retained was indistinguishable between ani-
mals treated with ubq-1 or ubq-2 RNAi or control vector (Fig. 5A).
By contrast, in BpR15-infected worms, although loss of ELT-2::
GFP was observed in worms treated with control vector, the
number of ELT-2::GFP in ubq-1 or ubq-2 RNAi-treated worms
was indistinguishable from those exposed to E. coli. The relative
importance of ubq-1 vs. ubq-2 is not known as RNAi is expected to
inactivate both genes due to their extensive homology. Similar
results were obtained with rpt-2 RNAi-treated worms (Fig. 5B).
Together, they indicate that UPS is required for the loss of ELT-2
and further supports the idea that ELT-2 is actively degraded
rather than translationally inhibited.
Speciﬁc E3 ligases coordinate ubiquitylation and subsequent
degradation of targeted proteins. Given that only ELT-2 is spe-
ciﬁcally degraded, we hypothesized that this process may involve
speciﬁc E3 ligase(s) that transfer Ub to ELT-2 and subsequently
signal the ELT-2 for degradation by proteasomes. Alternatively,
but not mutually exclusive, degradation of ELT-2 could be me-
diated by B. pseudomallei effectors that mimic host E3 ligase and
subvert the normal ubiquitylation events as a pathogenic strategy
(27). The C. elegans genome contains several hundred genes
predicted to encode for E3 ligase. We explored the possible in-
volvement of host E3 ligases in ubiquitylation and degradation of
ELT-2 by looking for E3 ligase encoding genes in the microarray
dataset that were induced by BpR15. Only two genes, F54B11.5
and ZK637.14, were signiﬁcantly up-regulated by BpR15 but not
by a nonpathogenic B. pseudomallei isolate (Table S4). We
knocked down the expression of these genes individually by
RNAi and tested for the loss of ELT-2 upon BpR15 infection.
Knockdown of each of these genes was sufﬁcient to rescue the
loss of ELT-2::GFP in BpR15-infected animals with the effect
being more pronounced for F54B11.5 (Fig. 5C). Knocking down
another E3 ligase R1010A.2, of a similar class but whose ex-
pression was not signiﬁcantly altered during BpR15 infection did
not rescue the loss of ELT-2::GFP in BpR15-infected animals
(Fig. 5C). The induction of F54B11.5 and ZK637.14 E3 ligases
by BpR15 suggests a strategy used by B. pseudomallei to promote
active degradation of ELT-2 as a means to counter the worm
immune response. These experiments do not, however, rule out
the possibility that bacterial effectors could also contribute to
ELT-2 degradation by mimicking an E3 ligase.
Loss of ELT-2 Requires B. pseudomallei T3SS. T3SS is important for
virulence in many Gram-negative bacteria (28) and T3SS effec-
tor proteins have been implicated in hijacking the eukaryotic
UPS (29). To determine whether T3SS is required for the loss of
ELT-2 during B. pseudomallei infection, we enumerated nuclear
ELT-2::GFP in JM90 worms infected with wild-type B. pseudo-
mallei strains KHW or K96243 and their respective isogenic
T3SS mutants in a KHW (bsaM and bspR) (30) or K96243 (bipB)
(31) background that completely lack the T3SS. B. pseudomallei
Fig. 4. Loss of ELT-2 in the intestine is B. pseudomallei speciﬁc. (A, C, and E)
Kinetics of C. elegans killing by (A) other known pathogens, (C) different
Burkholderia species, and (E) different B. pseudomallei isolates. (B, D, and F)
Mean number of nuclei with detectable ELT-2::GFP signal on worms exposed
to (B) other known pathogens for up to 6 d, (D) different Burkholderia
species for up to 72 hpi, and (F) different B. pseudomallei isolates at 24 hpi.
Shown are mean ±SD; n=3.
www.pnas.org/cgi/doi/10.1073/pnas.1311725110 Lee et al.
BsaM is homologous to Salmonella PrgH, which together with
InvG and PrgK/EscJ forms the multiring base of the needle
complex. BspR is the TetR family transcription regulator re-
quired for the expression of structural and secretion compo-
nents of T3SS, whereas BipB is a translocon in the T3SS
apparatus. Similar to BpR15, the number of nuclear GFP was
signiﬁcantly reduced in worms exposed to B. pseudomallei strain
KHW (Fig. 6A) and K96243 (Fig. 6B) at 24 hpi compared with
worms fed with E. coli OP50. In contrast, the number of nuclear
GFP retained in worms exposed to mutants bsaM,bspR (Fig.
6A), and bipB was similar to the control (P<0.0001, Student t
test) (Fig. 6B). This indicated a role for the T3SS in the loss of
ELT-2 protein during B. pseudomallei infection.
Whole-genome transcriptional analysis revealed that B. pseudo-
mallei-infected C. elegans mount a complex transcriptional re-
sponse that includes expression of a wide array of defense-
speciﬁc as well as non–immune-related genes. A unique feature
revealed from these studies is the repression of ELT-2–regulated
transcripts and the concomitant loss of ELT-2 protein in infected
worms. The loss of nuclear protein appears to be speciﬁcto
ELT-2, requires the host UPS and an intact T3SS of B. pseu-
domallei. Together, they suggest that B. pseudomallei suppresses
host immunity by degrading the ELT-2 protein.
Several conserved transcription factors including GATA/ELT-2
(1), FOXO/DAF-16 (15), and bZIP/ZIP-2 (32) regulate the ex-
pression of immunity-related genes and protect C. elegans from
lethal infection by P. aeruginosa (1, 32). We found that disruption
of ELT-2 rendered worms hypersusceptible to B. pseudomallei
infection, indicating that ELT-2 also protects C. elegans from
a lethal B. pseudomallei infection. Down-regulation of ELT-2–
regulated immunity genes, such as spp-8 and F55G11.2, during
B. pseudomallei infection suggests that this pathogen is able to
repress host defenses by subverting ELT-2. B. pseudomallei
suppresses ELT-2 transcriptional targets by affecting ELT-2
protein stability, as evidenced by loss of nuclear-localized ELT-
2::GFP in the intestine and decrease in ELT-2 protein in
B. pseudomallei-infected worm extracts. The progressive loss of
ELT-2 protein from the intestinal nuclei is speciﬁc to ELT-2 as
the PHA-4::GFP and DAF-16::GFP remained stably retained in
the intestinal nuclei. The progressive loss of ELT-2 protein also
appears to be speciﬁctoB. pseudomallei infection because ELT-2
protein levels remained stable over the course of infection by
several other bacteria as well as other Burkholderia species.
How do pathogens target host proteins? Pathogenic bacteria
could suppress host immune response by inhibiting host trans-
lation (33, 34), interfering with ubiquitination of signaling
intermediates (35), or disrupting signaling complexes (36). For
example, P. aeruginosa infection inhibits mRNA translation in
the intestine via the endocytosed translation inhibitor Exotoxin
A (33, 34). Other bacterial pathogens interfere with the NF-κB
pathway by deubiquitination (37). In this study, we demonstrate
that B. pseudomallei subverts the host UPS to speciﬁcally de-
grade the intestinal transcription factor ELT-2, which has a sig-
niﬁcant role in protecting worms from infection. We showed that
disruption of important components in the UPS in worms such as
ubq-1,ubq-2, and rpt-2 limited the loss of ELT-2 seen in B.
pseudomallei-infected worms. This is in concordance with nu-
merous ﬁndings demonstrating that subversion of the UPS is one
of the mechanisms used by bacterial pathogens to manipulate
host cells (25). Similarly, a recent report also demonstrated that
B. pseudomallei actively inhibits NF-қB and type I IFN-related
pathway activation by interfering with the ubiquitination of
critical signaling intermediates including TNFR-associated fac-
tor-3 and factor-6, and IқBαthrough TssM (a broad-base
deubiquitinase) (38). Many bacterial effector proteins act as E3
Ub ligase mimics and can interact with a host Ub-bound E2
enzyme and facilitate ubiquitination of host target. Our study,
however, suggests that host E3 ligases are responsible for the
observed ubiquitination event. We identiﬁed two C. elegans E3
ligases that were up-regulated upon B. pseudomallei infection
and knockdown of these E3 ligases limited ELT-2 loss in infected
worms. Loss of ELT-2 required an intact B. pseudomallei T3SS.
Previous ﬁndings indicate that T3SS and T4SS effector proteins
are important in facilitating ubiquitination of target host proteins
(29). We used a transgenic strain JM90 that overexpresses ELT-
2::GFP to directly observe degradation of ELT-2 within an intact
host. Although this implies that the conclusions are based on
overexpression studies, other data, including the down-regula-
tion of ELT-2–regulated transcripts in wild-type worms are
consistent with B. pseudomallei using the T3SS to induce speciﬁc
host E3 ligases to target ELT-2 for destruction by UPS, resulting
ultimately in the down-regulation of ELT-2–regulated transcripts
and increased host susceptibility. Further work is needed to
unravel the identity of B. pseudomallei type 3 effectors and the
mechanism by which T3SS functions to subvert the host UPS to
Our study also identiﬁed genes with putative roles in activation
of immunity-related signal transduction pathways, such as the
p38 MAPK, DAF-2/DAF-16, and Sma/TGF-βpathways as well
as their respective immune effectors. For example, up-regulation
of tir-1 and vhp-1, which encode defense proteins TIR-1 (39) and
VHP-1, a regulator of the c-Jun N-terminal kinase (JNK) and
Fig. 5. B. pseudomallei infection triggers ELT-2 degradation by the host
ubiquitin–proteosome system. (A–C) Mean number of nuclei with detectable
ELT-2::GFP signal of empty vector-treated worms and (A)ubq-1 and ubq-2,
(B)rpt-2, and (C) E3 ligases knockdown worms after 24-h exposure to OP50
and BpR15. **ttest, P<0.005; ***ttest, P<0.0001. n=3.
Fig. 6. B. pseudomallei T3SS is required for the loss of ELT-2. (Aand B)
Mean number of nuclei with detectable ELT-2::GFP signal after 24-h expo-
sure to B. pseudomallei.(A) KHW and mutants bsaM and bspR;(B) K96243
and mutant bipB. ***ttest, P<0.0001. The columns represent mean ±SEM;
n=3. Each experiment included 17–20 animals per group.
Lee et al. PNAS
September 10, 2013
p38 MAPKs (40), respectively, implicates the involvement of the
p38 MAPK signaling pathway during B. pseudomallei infection.
Interestingly, several known DAF-2/DAF-16–regulated immune
effectors such as lys-7,spp-1, and thn-2 are signiﬁcantly repressed
upon B. pseudomallei infection similar to that previously seen in a
P. aeruginosa infection (7). P. aeruginosa accomplished this by
inducing INS-7, a ligand of the DAF-2/DAF-16 pathway that
through a series of phosphorylation events ultimately leads to
ejection of DAF-16 from the intestinal nuclei and down-regu-
lation of immune effectors (4, 7). By contrast, B. pseudomallei
retains DAF-16 in the nuclei of intestinal cells over the course of
infection, indicating an immune suppression mechanism that is
distinct from P. aeruginosa (Fig. S6C). Thus, gene expression and
protein localization studies showed that B. pseudomallei and
P. aeruginosa use distinct immune suppression mechanisms.
Further work is in progress to decipher the underlying mecha-
nism that contributes to the down-regulation of DAF-16 tran-
scriptional targets while retaining nuclear localized DAF-16.
In summary, we have identiﬁed a mechanism by which
B. pseudomallei suppresses host immunity. B. pseudomallei targets
ELT-2, a component of a multipathogen defense pathway (1), by
actively degrading the ELT-2 protein leading to down-regulation
of ELT-2 transcriptional targets and suppression of host immu-
nity. This process is mediated by host UPS and requires the
bacterial T3SS. The conserved role for GATA factors in
epithelial immunity may lead to insights that are generalizable to
other hosts of B. pseudomallei, including humans.
Materials and Methods
All experimental procedures are detailed in SI Materials and Methods. RNA
extraction, microarray experiments, and qRT-PCR were performed as de-
scribed (1). C. elegans RNAi knockdown and survival assays were performed
as previously described (41). Nuclear localized ELT-2 was assayed by enu-
merating total visible ELT-2::GFP nuclei from 1-d-old individual worm strain
(JM90) exposed to various pathogenic bacteria, bacterial mutants (bsaM,
bspR,andbipB), or heavy metal cadmium under 400×total magniﬁcation
using an upright ﬂuorescent microscope. For immunoblot analyses, ELT-2–
speciﬁc mouse monoclonal antibody (a gift from James McGhee at the
University of Calgary, Calgary, AB, Canada), anti-GFP antibody (Calbiochem,
EMD Biosciences), and anti-actin antibody (Sigma-Aldrich) were used.
ACKNOWLEDGMENTS. We thank Yee-Chin Wong for her technical assis-
tance and the Institute for Medical Research, Kuala Lumpur, for provision of
the B. pseudomallei isolates. We also thank Prof. James McGhee (University
of Calgary) for providing anti–ELT-2 antibodies, Prof. Andrew Dillin (Univer-
sity of California, Berkeley) for providing PHA-4::GFP strain, and Prof. Sunee
Korbsrisate (Mahidol University) for providing the bipB mutant. This study
was supported by the Malaysia Genome Institute–Stanford University Inter-
national Research Grant awarded to S.N. and M.-W.T. by the Government of
Malaysia. S.-H.L. was supported by a National Science Fellowship provided by
the Ministry of Science, Technology and Innovation, Malaysia.
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