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Burkholderia pseudomallei suppresses Caenorhabditis elegans immunity by specific degradation of a GATA transcription factor


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Significance 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 pseudomallei , the causative agent of melioidosis, which a life-threatening disease in humans. Analyses of whole-genome transcriptional responses of Caenorhabditis elegans to B. pseudomallei infection revealed that B. pseudomallei , through its 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 degradation leads to suppression of the host’s ability to mount an effective antimicrobial defense.
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Burkholderia pseudomallei suppresses Caenorhabditis
elegans immunity by specic degradation of a GATA
transcription factor
Song-Hua Lee
, Rui-Rui Wong
, Chui-Yoke Chin
, Tian-Yeh Lim
, Su-Anne Eng
, Cin Kong
, Nur Afah Ijap
Ming-Seong Lau
, 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
Genetics and
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 signicant 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 identied 6% of the nematode genes
that were signicantly 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 signicantly
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 ubiquitinproteasome
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.
innate immunity
ubiquitinproteosomal system
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 hostpathogen
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, y, and mammals (36). 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
pathway (7).
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 proling in C. elegans identied a set of
ELT-2regulated transcripts that are progressively down-regu-
lated over a time course of B. pseudomallei infection that is
concomitant with a progressive and specic loss of nuclear ELT-2
in the worm intestine. Loss of ELT-2 requires the host ubiq-
uitinproteasome 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
nding 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 hosts
ability to defend itself from pathogenic assaults. We identied
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 ubiquitinproteasome
system to specically degrade a GATA transcription factor. This
GATA factor is critical for host immune defense; thus, its deg-
radation leads to suppression of the hosts 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 paper.
The authors declare no conict 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,
S.-H.L. and R.-R.W. contributed equally to this work.
Present address: Department of Integrative Biology, University of California, Berkeley,
CA 94720-3102.
Present address: Emory Vaccine Center, Emory University, Atlanta, GA 30329.
Present address: Department of Plant Biology, Carnegie Institution for Science, Stanford,
CA 94305-4101.
Present address: Department of Infectious Diseases, Genentech, Inc., South San Francisco,
CA 94080.
To whom correspondence may be addressed. E-mail: or sheila@
This article contains supporting information online at
1073/pnas.1311725110/-/DCSupplemental. PNAS
September 10, 2013
vol. 110
no. 37
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 OP50the standard
laboratory food source as uninfected controlover the same
time course. The initial time points precede the rst 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 (signicance 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 signi-
cantly repressed; the signicance of this observation will be
addressed below. Quantitative real-time PCR (qRT-PCR) mea-
surements conrmed microarray results for 30 genes identied
as infection and stress response genes with altered expression at
12 hpi (Table S2) (7). Functional category analysis revealed that
defense-specic 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 signicantly 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 signicant
enrichment of genes regulated by these factors (Fig. S2A) (hyper-
geometric probability, P<1×10
). Specically, expression of 42
of 92 PMK-1regulated genes was signicantly altered during in-
fection [representation factor (Rf) =6.51]. Of the 440 (class 1 and
2) DAF-16regulated genes (3), 149 genes were differentially
expressed in infected worms (Rf =4.83). Among the 1,122 genes
identied 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 signicant enrichment (Rf =8.43,
hypergeometric probability P<1×10
) was observed between
ELT-2regulated 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 signicantly induced (Fig.
S2B), suggesting that B. pseudomallei infection did not elicit the
ncUPR response.
B. pseudomallei Down-Regulates ELT-2 Transcriptional Targets. ELT-
2 transcriptional targets are induced by P. aeruginosa PA14 (1).
Unexpectedly, of the 42 ELT-2regulated 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, detoxication,
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-2reg-
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-2independent tran-
scripts (pgp-5,ugt-29,thn-1,pqm-1, and skr-3) were similarly in-
duced by PA14 and BpR15 (Fig. 2B). To conrm 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 signicant re-
duction in worm survival on BpR15 (Fig. S4A) but did not
signicantly 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 prole
(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).
| Lee et al.
were not signicantly 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 uorescence (Fig. 3
BD)andenumeratedthenumberofuorescent nuclei in the
worm intestine (Fig. 3E). ELT-2::GFP uorescence 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
uorescence 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
asignicant 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 specictoB. pseudomallei infection and not
a consequence of intestinal damage. To further conrm 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 specic 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 signicant loss of ELT-2::GFP
over the course of infection (Fig. 4B). Infections by other Bur-
kholderia speciesB. 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 (lled columns).
Shown are fold relative to uninfected animals. Columns rep-
resent mean ±SD; n=2. (A) ELT-2 and (B) nonELT-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 specic RNA levels in BpR15-infected ani-
mals relative to uninfected animals in elt-2 RNAi animals (lled
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. (BD) Representative
uorescence 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-2specic mAb and
anti-GFP antibody. Anti-actin antibody was used as loading control.
Lee et al. PNAS
September 10, 2013
vol. 110
no. 37
The Loss of Nuclear Protein in the Intestine Is Specic 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 specic response to B. pseudomallei infection and not to
general stress. To test whether B. pseudomallei specically 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
rst 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 uorescence, DAF-16::GFP uores-
cence remained nuclear localized and at a constant intensity over
the course of a 24-h infection (Fig. S6C). Heat treatment did
not signicantly 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 specictoELT-2andspecicto
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 specic
substrates, thus transferring Ub to specic 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.
Specic E3 ligases coordinate ubiquitylation and subsequent
degradation of targeted proteins. Given that only ELT-2 is spe-
cically degraded, we hypothesized that this process may involve
specic 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 signicantly 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 sufcient 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 signicantly 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 specic. (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.
| 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
signicantly 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-
specic as well as nonimmune-related genes. A unique feature
revealed from these studies is the repression of ELT-2regulated
transcripts and the concomitant loss of ELT-2 protein in infected
worms. The loss of nuclear protein appears to be specicto
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 specic 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 specictoB. 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 specically de-
grade the intestinal transcription factor ELT-2, which has a sig-
nicant 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 ndings 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 identied 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 ndings 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-2regulated transcripts in wild-type worms are
consistent with B. pseudomallei using the T3SS to induce specic
host E3 ligases to target ELT-2 for destruction by UPS, resulting
ultimately in the down-regulation of ELT-2regulated 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
degrade ELT-2.
Our study also identied 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
ubiquitinproteosome system. (AC) 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 1720 animals per group.
Lee et al. PNAS
September 10, 2013
vol. 110
no. 37
p38 MAPKs (40), respectively, implicates the involvement of the
p38 MAPK signaling pathway during B. pseudomallei infection.
Interestingly, several known DAF-2/DAF-16regulated immune
effectors such as lys-7,spp-1, and thn-2 are signicantly 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 identied 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 magnication
using an upright uorescent microscope. For immunoblot analyses, ELT-2
specic 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 antiELT-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 InstituteStanford 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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| Lee et al.
... This Gram-negative bacterium is the causative agent of melioidosis and C. elegans animals fed with B. pseudomallei suffer from a lethal infection [130][131][132]. ELT-2 is implicated in host defence against B. pseudomallei as RNAi against elt-2 reduces the survival of infected animals [133]. Further, ELT-2 has been found to be specifically targeted by B. pseudomallei, but not by other pathogens, as a strategy to suppress C. elegans immunity [133]. ...
... ELT-2 is implicated in host defence against B. pseudomallei as RNAi against elt-2 reduces the survival of infected animals [133]. Further, ELT-2 has been found to be specifically targeted by B. pseudomallei, but not by other pathogens, as a strategy to suppress C. elegans immunity [133]. Whole-genome transcriptome analysis revealed that a set of ELT-2-dependent genes is progressively down-regulated over a time course of B. pseudomallei infection. ...
... Lee et al. identified two host genes encoding RING-finger proteins with putative E3 ligase activity whose expression was induced during infection and required for ELT-2::GFP degradation. Direct involvement of these putative E3 ligases in ELT-2 ubiquitination was not established but these results suggested a mechanism used by B. pseudomallei to degrade actively ELT-2 to counteract the immune response of C. elegans [133]. Additionally, degradation of the ELT-2::GFP protein was only observed when animals were infected with wild-type bacteria but not with type III secretion system (T3SS)-deficient strains, indicating that injected bacterial effectors might be required for ELT-2 degradation. ...
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Innate immunity is an evolutionary ancient defence strategy that serves to eliminate infectious agents while maintaining host health. It involves a complex network of sensors, signaling proteins and immune effectors that detect the danger, then relay and execute the immune programme. Post-translational modifications relying on conserved ubiquitin and ubiquitin-like proteins are an integral part of the system. Studies using invertebrate models of infection, such as the nematode Caenorhabditis elegans, have greatly contributed to our understanding of how ubiquitin-related processes act in immune sensing, regulate immune signaling pathways, and participate to host defence responses. This review highlights the interest of working with a genetically tractable model organism and illustrates how C. elegans has been used to identify ubiquitin-dependent immune mechanisms, discover novel ubiquitin-based resistance strategies that mediate pathogen clearance, and unravel the role of ubiquitin-related processes in tolerance, preserving host fitness during pathogen attack. Special emphasis is placed on processes that are conserved in mammals.
... There was also an overlap with lists corresponding to genes induced upon exposure to 2 concentrations of the bioactive polyphenol quercetin acid (Pietsch, et al., 2012), in part explicable by the >90% overlap between these 2 latter classes. Perhaps more interestingly, this representation revealed an overlap between the Drechmeria-induced genes and genes up-regulated by Burkholderia pseudomallei infection (Lee, et al., 2013) but repressed by allantoin (Calvert, et al., 2016). The commonly regulated genes include a hallmark of epidermal antifungal immunity, 8 antimicrobial peptide genes ( Figure 4B). ...
... The commonly regulated genes include a hallmark of epidermal antifungal immunity, 8 antimicrobial peptide genes ( Figure 4B). B. pseudomallei degrades ELT-2, an intestinal GATA transcription factor, and so switches off defence gene expression in the gut (Lee, et al., 2013). How this would switch on immune gene expression in the epidermis is not known. ...
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Generating meaningful interpretations of gene lists remains a challenge for all large-scale studies. Many approaches exist, often based on evaluating gene enrichment among pre-determined gene classes. Here, we conceived and implemented yet another analysis tool (YAAT), specifically for data from the widely-used model organism C. elegans. YAAT extends standard enrichment analyses, using a combination of co-expression data and profiles of phylogenetic conservation, to identify groups of functionally-related genes. It additionally allows class clustering, providing inference of functional links between groups of genes. We give examples of the utility of YAAT for uncovering unsuspected links between genes and show how the approach can be used to prioritise genes for in-depth study. Our analyses revealed several limitations to the meaningful interpretation of gene lists, specifically related to data sources and the "universe" of gene lists used. We hope that YAAT will represent a model for integrated analysis that could be useful for large-scale exploration of biological function in other species.
... Prior RNA-seq and microarray studies have indicated that W06G6.11 expression may be affected by the activity of Argonaute alg-1 (Aalto et al. 2018), a member of the RNA-induced silencing complex (RISC) involved in endogenous and exogenous short RNA processing (Grishok et al. 2001), and also by exposure to pathogens (Engelmann et al. 2011;Lee et al. 2013). These studies detected W06G6.11 expression in N2, but in samples derived from older adult hermaphrodites relative to the young adults we sampled, a study that included CB4856 also confirmed significantly higher W06G6.11 ...
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Though natural systems harbor genetic and phenotypic variation, research in model organisms is often restricted to a reference strain. Focus on a reference strain yields great depth of knowledge, but potentially at the cost of breadth of understanding. Furthermore, tools developed in the reference context may introduce bias when applied to other strains, posing challenges to defining the scope of variation within model systems. Here, we evaluate how genetic differences among five wild C. elegans strains affect gene expression and its quantification, in general and after induction of the RNA interference (RNAi) response. Across strains, 34% of genes were differentially expressed in the control condition, including 411 genes that were not expressed at all in at least one strain; 49 of these were unexpressed in reference strain N2. Reference genome mapping bias caused limited concern: despite hyper-diverse hotspots throughout the genome, 92% of variably expressed genes were robust to mapping issues. The transcriptional response to RNAi was highly strain- and target gene-specific and did not correlate with RNAi efficiency, as the two RNAi insensitive strains showed more differentially expressed genes following RNAi treatment than the RNAi-sensitive reference strain. We conclude that gene expression, generally and in response to RNAi, differs across C. elegans strains such that choice of strain may meaningfully influence scientific inferences. Finally, we introduce a resource for querying gene expression variation in this dataset at
... It is well known that host-adapted pathogens can also subvert defence pathways, rendering them partially or fully ineffective; several examples have been reported for C. elegans (e.g. (Lee et al. 2013;Vasquez-Rifo et al. 2020;Zhang et al. 2021b). Such diminished utility will lessen the selection pressure on the corresponding defence genes. ...
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The natural environment of the free-living nematode Caenorhabditis elegans is rich in pathogenic microbes. There is now ample evidence to indicate that these pathogens exert a strong selection pressure on C. elegans, and have shaped its genome, physiology, and behaviour. In this short review, we concentrate on how C. elegans stands out from other animals in terms of its immune repertoire and innate immune signalling pathways. We discuss how C. elegans often detects pathogens because of their effects on essential cellular processes, or organelle integrity, in addition to direct microbial recognition. We illustrate the extensive molecular plasticity that is characteristic of immune defences in C. elegans and highlight some remarkable instances of lineage-specific innovation in innate immune mechanisms.
... elegans infection model and performed microarray analysis of the infected worms. Expression profiling of infected worms identified an increasing number of host genes that were suppressed significantly over time and within the genes that were modulated, a large fraction of them were genes under the control of the worm GATA transcription factor [13] which was shown to be a key transcription factor for activation of the host immune response during a P. aeruginosa infection [14]. Our further analysis demonstrated that the down-regulation of GATA targets was not due to the absence of the GATA transcription fac-tor but rather, B. pseudomallei secretes an effector molecule through its Type-3 Secretion System that induces the host ubiquitin proteosomal system to degrade its own GATA transcription factor, thereby repressing the activation of the immune response. ...
... This observation might suggest that vertebrate GATA factors are remarkably well insulated from the influence of microorganisms, but this hypothesis remains to be tested directly. Indeed, multiple studies have implicated the Caenorhabditis elegans GATA homolog, ELT-2, in intestinal response to pathogenic and commensal microorganisms [118][119][120] . These observations provide early hints that even the transcription factors that seem to be most central to IEC identity might also be sensitive to microbial and other environmental factors. ...
The intestinal epithelium serves the unique and critical function of harvesting dietary nutrients, while simultaneously acting as a cellular barrier separating tissues from the luminal environment and gut microbial ecosystem. Two salient features of the intestinal epithelium enable it to perform these complex functions. First, cells within the intestinal epithelium achieve a wide range of specialized identities, including different cell types and distinct anterior-posterior patterning along the intestine. Second, intestinal epithelial cells are sensitive and responsive to the dynamic milieu of dietary nutrients, xenobiotics and microorganisms encountered in the intestinal luminal environment. These diverse identities and responsiveness of intestinal epithelial cells are achieved in part through the differential transcription of genes encoded in their shared genome. Here, we review insights from mice and other vertebrate models into the transcriptional regulatory mechanisms underlying intestinal epithelial identity and microbial responsiveness, including DNA methylation, chromatin accessibility, histone modifications and transcription factors. These studies are revealing that most transcription factors involved in intestinal epithelial identity also respond to changes in the microbiota, raising both opportunities and challenges to discern the underlying integrative transcriptional regulatory networks.
... It also identified a number of other categories previously noted to be enriched for innate immune genes. This includes genes regulated by nhr-25 and acs-3 37 , sam-10 38 or dapk-1 39 , induced by infection with Burkholderia pseudomallei 40 and in a series of mutants with an alteration of their cuticle and/or osmotic balance (osm-7, osm-8, osm-11) 41 . As expected, there was also a very significant enrichment (p < 10 -28 ) for genes characterized as being expressed in the epidermis 42 (Table S1). ...
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In addition to performing digestion and nutrient absorption, the intestine serves as one of the first barriers to the external environment, crucial for protecting the host from environmental toxins, pathogenic invaders, and other stress inducers. The gene regulatory network (GRN) governing embryonic development of the endoderm and subsequent differentiation and maintenance of the intestine has been well-documented in C. elegans. A key regulatory input that initiates activation of the embryonic GRN for endoderm and mesoderm in this animal is the maternally provided SKN-1 transcription factor, an ortholog of the vertebrate Nrf1 and 2, which, like C. elegans SKN-1, perform conserved regulatory roles in mediating a variety of stress responses across metazoan phylogeny. Other key regulatory factors in early gut development also participate in stress response as well as in innate immunity and aging and longevity. In this review, we discuss the intersection between genetic nodes that mediate endoderm/intestine differentiation and regulation of stress and homeostasis. We also consider how direct signaling from the intestine to the germline, in some cases involving SKN-1, facilitates heritable epigenetic changes, allowing transmission of adaptive stress responses across multiple generations. These connections between regulation of endoderm/intestine development and stress response mechanisms suggest that varying selective pressure exerted on the stress response pathways may influence the architecture of the endoderm GRN, thereby leading to genetic and epigenetic variation in early embryonic GRN regulatory events.
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Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. Much remains to be known about the contributions of genotypic variations within the bacteria and the host, and environmental factors that lead to the manifestation of the clinical symptoms of melioidosis. In this study, we showed that different isolates of B. pseudomallei have divergent ability to kill the soil nematode Caenorhabditis elegans. The rate of nematode killing was also dependent on growth media: B. pseudomallei grown on peptone-glucose media killed C. elegans more rapidly than bacteria grown on the nematode growth media. Filter and bacteria cell-free culture filtrate assays demonstrated that the extent of killing observed is significantly less than that observed in the direct killing assay. Additionally, we showed that B. pseudomallei does not persistently accumulate within the C. elegans gut as brief exposure to B. pseudomallei is not sufficient for C. elegans infection. A combination of genetic and environmental factors affects virulence. In addition, we have also demonstrated that a Burkholderia-specific mechanism mediating the pathogenic effect in C. elegans requires proliferating B. pseudomallei to continuously produce toxins to mediate complete killing.
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The DAF-16/FOXO transcription factor is the major downstream output of the insulin/IGF1R signaling pathway controlling C. elegans dauer larva development and aging. To identify novel downstream genes affecting dauer formation, we used RNAi to screen candidate genes previously identified to be regulated by DAF-16. We used a sensitized genetic background [eri-1(mg366); sdf-9(m708)], which enhances both RNAi efficiency and constitutive dauer formation (Daf-c). Among 513 RNAi clones screened, 21 displayed a synthetic Daf-c (SynDaf) phenotype with sdf-9. One of these genes, srh-100, was previously identified to be SynDaf, but twenty have not previously been associated with dauer formation. Two of the latter genes, lys-1 and cpr-1, are known to participate in innate immunity and six more are predicted to do so, suggesting that the immune response may contribute to the dauer decision. Indeed, we show that two of these genes, lys-1 and clc-1, are required for normal resistance to Staphylococcus aureus. clc-1 is predicted to function in epithelial cohesion. Dauer formation exhibited by daf-8(m85), sdf-9(m708), and the wild-type N2 (at 27°C) were all enhanced by exposure to pathogenic bacteria, while not enhanced in a daf-22(m130) background. We conclude that knockdown of the genes required for proper pathogen resistance increases pathogenic infection, leading to increased dauer formation in our screen. We propose that dauer larva formation is a behavioral response to pathogens mediated by increased dauer pheromone production.
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Bone morphogenetic proteins (BMPs) are members of the conserved transforming growth factor beta (TGFbeta superfamily, and play many developmental and homeostatic roles. In C. elegans, a BMP-like pathway, the DBL-1 pathway, controls body size and is involved in innate immunity. How these functions are carried out, though, and what most of the downstream targets of this pathway are, remain unknown. We performed a microarray analysis and compared expression profiles of animals lacking the SMA-6 DBL-1 receptor, which decreases pathway signaling, with animals that overexpress DBL-1 ligand, which increases pathway signaling. Consistent with a role for DBL-1 in control of body size, we find positive regulation by DBL-1 of genes involved in physical structure, protein synthesis and degradation, and metabolism. However, cell cycle genes were mostly absent from our results. We also identified genes in a hedgehog-related pathway, which may comprise a secondary signaling pathway downstream of DBL-1 that controls body size. In addition, DBL-1 signaling up-regulates pro-innate immunity genes. We identified a reporter for DBL-1 signaling, which is normally repressed but is up-regulated when DBL-1 signaling is reduced. Our results indicate that body size in C. elegans is controlled in part by regulation of metabolic processes as well as protein synthesis and degradation. This supports the growing body of evidence that suggests cell size is linked to metabolism. Furthermore, this study discovered a possible role for hedgehog-related pathways in transmitting the BMP-like signal from the hypodermis, where the core DBL-1 pathway components are required, to other tissues in the animal. We also identified the up-regulation of genes involved in innate immunity, clarifying the role of DBL-1 in innate immunity. One of the highly regulated genes is expressed at very low levels in wild-type animals, but is strongly up-regulated in Sma/Mab mutants, making it a useful reporter for DBL-1/BMP-like signaling in C. elegans.
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Burkholderia pseudomallei is a Gram-negative saprophyte that is the causative agent of melioidosis, a severe infectious disease endemic in Northern Australia and Southeast Asia. This organism has sparked much scientific interest in the West because of its classification as a potential bioterrorism agent by the U.S. Centers for Disease Control and Prevention. However, relatively little is known about its pathogenesis. We demonstrate that B. pseudomallei actively inhibits NF-kappaB and type I IFN pathway activation, thereby downregulating host inflammatory responses. We found the virulence factor TssM to be responsible for this activity. TssM interferes with the ubiquitination of critical signaling intermediates, including TNFR-associated factor-3, TNFR-associated factor-6, and IkappaBalpha. The expression but not secretion of TssM is regulated by the type III secretion system. We demonstrate that TssM is important for B. pseudomallei infection in vivo as inflammation in the tssM mutant-infected mice is more severe and corresponds to a more rapid death compared with wild-type bacteria-infected mice. Abs to TssM can be detected in the sera of melioidosis patients, indicating that TssM is functionally expressed in vivo and thus could contribute to bacterial pathogenesis in human melioidosis.
Pathogens commonly disrupt host cell processes or cause damage, but the surveillance mechanisms used by animals to monitor these attacks are poorly understood. Upon infection with pathogenic Pseudomonas aeruginosa, the nematode C. elegans upregulates infection response gene irg-1 using the zip-2 bZIP transcription factor. Here we show that P. aeruginosa infection inhibits mRNA translation in the intestine via the endocytosed translation inhibitor Exotoxin A, which leads to an increase in ZIP-2 protein levels. In the absence of infection we find that the zip-2/irg-1 pathway is upregulated following disruption of several core host processes, including inhibition of mRNA translation. ZIP-2 induction is conferred by a conserved upstream open reading frame in zip-2 that could derepress ZIP-2 translation upon infection. Thus, translational inhibition, a common pathogenic strategy, can trigger activation of an immune surveillance pathway to provide host defense.
Intestinal epithelial cells are exposed to both innocuous and pathogenic microbes, which need to be distinguished to mount an effective immune response. To understand the mechanisms underlying pathogen recognition, we investigated how Pseudomonas aeruginosa triggers intestinal innate immunity in Caenorhabditis elegans, a process independent of Toll-like pattern recognition receptors. We show that the P. aeruginosa translational inhibitor Exotoxin A (ToxA), which ribosylates elongation factor 2 (EF2), upregulates a significant subset of genes normally induced by P. aeruginosa. Moreover, immune pathways involving the ATF-7 and ZIP-2 transcription factors, which protect C. elegans from P. aeruginosa, are required for preventing ToxA-mediated lethality. ToxA-responsive genes are not induced by enzymatically inactive ToxA protein but can be upregulated independently of ToxA by disruption of host protein translation. Thus, C. elegans has a surveillance mechanism to recognize ToxA through its effect on protein translation rather than by direct recognition of either ToxA or ribosylated EF2.
The unfolded protein response (UPR), which is activated when unfolded or misfolded proteins accumulate in the endoplasmic reticulum, has been implicated in the normal physiology of immune defense and in several human diseases, including diabetes, cancer, neurodegenerative disease, and inflammatory disease. In this study, we found that the nervous system controlled the activity of a noncanonical UPR pathway required for innate immunity in Caenorhabditis elegans. OCTR-1, a putative octopamine G protein-coupled catecholamine receptor (GPCR, G protein-coupled receptor), functioned in sensory neurons designated ASH and ASI to actively suppress innate immune responses by down-regulating the expression of noncanonical UPR genes pqn/abu in nonneuronal tissues. Our findings suggest a molecular mechanism by which the nervous system may sense inflammatory responses and respond by controlling stress-response pathways at the organismal level.
A major and critical virulence determinant of many Gram-negative bacterial pathogens is the Type III Secretion Systems (T3SS). T3SS3 in Burkholderia pseudomallei is critical for bacterial virulence in mammalian infection models but its regulation is unknown. B. pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic in Southeast Asia and northern Australia. While screening for bacterial transposon mutants with a defective T3SS function, we discovered a TetR family regulator (bspR) responsible for the control of T3SS3 gene expression. The bspR mutant exhibited significant virulence attenuation in mice. BspR acts through BprP, a novel transmembrane regulator located adjacent to the currently delineated T3SS3 region. BprP in turn regulates the expression of structural and secretion components of T3SS3 and the AraC family regulator bsaN. BsaN and BicA likely form a complex to regulate the expression of T3SS3 effectors and other regulators which in turn affect the expression of Type VI Secretion Systems (T6SS). The complete delineation of the bspR initiated T3SS regulatory cascade not only contributes to the understanding of B. pseudomallei pathogenesis but also provides an important example of how bacterial pathogens could co-opt and integrate various regulatory motifs to form a new regulatory network adapted for its own purposes.
Very little is known about how animals discriminate pathogens from innocuous microbes. To address this question, we examined infection-response gene induction in the nematode Caenorhabditis elegans. We focused on genes that are induced in C. elegans by infection with the bacterial pathogen Pseudomonas aeruginosa, but are not induced by an isogenic attenuated gacA mutant. Most of these genes are induced independently of known immunity pathways. We generated a GFP reporter for one of these genes, infection response gene 1 (irg-1), which is induced strongly by wild-type P. aeruginosa strain PA14, but not by other C. elegans pathogens or by other wild-type P. aeruginosa strains that are weakly pathogenic to C. elegans. To identify components of the pathway that induces irg-1 in response to infection, we performed an RNA interference screen of C. elegans transcription factors. This screen identified zip-2, a bZIP transcription factor that is required for inducing irg-1, as well as several other genes, and is important for defense against infection by P. aeruginosa. These data indicate that zip-2 is part of a specialized pathogen response pathway that is induced by virulent strains of P. aeruginosa and provides defense against this pathogen.