WWP-1 is a novel modulator of the DAF-2 insulin-like signaling network involved in pore-forming toxin cellular defenses in Caenorhabditis elegans.

Abstract

Pore-forming toxins (PFTs) are the single largest class of bacterial virulence factors. The DAF-2 insulin/insulin-like growth factor-1 signaling pathway, which regulates lifespan and stress resistance in Caenorhabditis elegans, is known to mutate to resistance to pathogenic bacteria. However, its role in responses against bacterial toxins and PFTs is as yet unexplored. Here we reveal that reduction of the DAF-2 insulin-like pathway confers the resistance of Caenorhabditis elegans to cytolitic crystal (Cry) PFTs produced by Bacillus thuringiensis. In contrast to the canonical DAF-2 insulin-like signaling pathway previously defined for aging and pathogenesis, the PFT response pathway diverges at 3-phosphoinositide-dependent kinase 1 (PDK-1) and appears to feed into a novel insulin-like pathway signal arm defined by the WW domain Protein 1 (WWP-1). In addition, we also find that WWP-1 not only plays an important role in the intrinsic cellular defense (INCED) against PFTs but also is involved in innate immunity against pathogenic bacteria Pseudomonas aeruginosa and in lifespan regulation. Taken together, our data suggest that WWP-1 and DAF-16 function in parallel within the fundamental DAF-2 insulin/IGF-1 signaling network to regulate fundamental cellular responses in C. elegans.

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WWP-1 Is a Novel Modulator of the DAF-2 Insulin-Like
Signaling Network Involved in Pore-Forming Toxin
Cellular Defenses in Caenorhabditis elegans
Chang-Shi Chen1,2, Audrey Bellier3, Cheng-Yuan Kao3, Ya-Luen Yang2, Huan-Da Chen1, Ferdinand C. O.
Los3, Raffi V. Aroian3*
1Department of Biochemistry and Molecular Biology, National Cheng Kung University, Tainan, Taiwan, 2Institute of Basic Medical Sciences, National Cheng Kung
University, Tainan, Taiwan, 3Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America
Abstract
Pore-forming toxins (PFTs) are the single largest class of bacterial virulence factors. The DAF-2 insulin/insulin-like growth
factor-1 signaling pathway, which regulates lifespan and stress resistance in Caenorhabditis elegans, is known to mutate to
resistance to pathogenic bacteria. However, its role in responses against bacterial toxins and PFTs is as yet unexplored. Here
we reveal that reduction of the DAF-2 insulin-like pathway confers the resistance of Caenorhabditis elegans to cytolitic
crystal (Cry) PFTs produced by Bacillus thuringiensis. In contrast to the canonical DAF-2 insulin-like signaling pathway
previously defined for aging and pathogenesis, the PFT response pathway diverges at 3-phosphoinositide-dependent
kinase 1 (PDK-1) and appears to feed into a novel insulin-like pathway signal arm defined by the WW domain Protein 1
(WWP-1). In addition, we also find that WWP-1 not only plays an important role in the intrinsic cellular defense (INCED)
against PFTs but also is involved in innate immunity against pathogenic bacteria Pseudomonas aeruginosa and in lifespan
regulation. Taken together, our data suggest that WWP-1 and DAF-16 function in parallel within the fundamental DAF-2
insulin/IGF-1 signaling network to regulate fundamental cellular responses in C. elegans.
Citation: Chen C-S, Bellier A, Kao C-Y, Yang Y-L, Chen H-D, et al. (2010) WWP-1 Is a Novel Modulator of the DAF-2 Insulin-Like Signaling Network Involved in Pore-
Forming Toxin Cellular Defenses in Caenorhabditis elegans. PLoS ONE 5(3): e9494. doi:10.1371/journal.pone.0009494
Editor: Alejandro Aballay, Duke University Medical Center, United States of America
Received December 21, 2009; Accepted February 8, 2010; Published March 2, 2010
Copyright: ? 2010 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by NIH grant AI056189 (CSC, AB), NSF grant MCB0517718 (CSC), and NIH grant GM071603 (CYK, FCOL) to RVA. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: raroian@ucsd.edu
Introduction
Pore-forming toxins (PFTs) are bacterial toxins that damage the
plasma membrane of host cells and play important roles in the
pathogenesis of many important human pathogens including
Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, and
Enterococcus faecalis [1,2,3]. Crystal (Cry) toxins produced by the
Gram-positive spore-forming soil bacterium Bacillus thuringiensis (Bt)
are a large family of PFTs [4]. Over 100 phylogenetically related
three-domain Cry toxins are known [4]. Although known
primarily for their ability to kill insects [5], we have reported
that some Cry toxins, including Cry5B and Cry21A, can
intoxicate a wide range of plant-parasitic, animal-parasitic, and
free-living nematodes including the standard laboratory nematode
species, Caenorhabditis elegans [6,7,8].
This C. elegans–Cry toxin interaction system opened up the first,
and to date the only, whole-animal genetic model for studying
PFTs in vivo and led to the discovery of signal transduction
pathways that protect cells against PFTs, including the p38
mitogen-activated protein kinase (MAPK) pathway and the
unfolded protein response (UPR) pathway, which are also
protective against and/or activated by PFTs in mammalian cells
[9,10,11]. More recently, we have shown that the hypoxia
response pathway also protects C. elegans against PFTs [12].
Although significant and extensive responses to PFTs at the
molecular level (e.g., modulation of signal transduction cascades)
have been recorded, the roles of these responses in coping with
PFTs as yet poorly understood [13]. Thus, functional analyses of
innate immune responses to the single largest class of bacterial
protein virulence factors remains a largely understudied area.
The DAF-2 insulin/insulin-like growth factor 1 (IGF-1) receptor
pathway is part of a neuroendocrine system that regulates
longevity, metabolism, and development in C. elegans and is
homologous to the mammalian insulin and IGF-1 signaling
pathway [14,15]. The C. elegans daf-2 gene encodes the worm
homolog of the insulin/IGF-1 receptor. C. elegans strains that carry
reduction-of-function or loss-of-function mutations in daf-2 or the
downstream phosphoinositol 3-kinase (PI3K) age-1 are long-lived
and are also resistant to a variety of stresses and bacterial
pathogens [16,17,18].
Here we demonstrate that reduction of the DAF-2 insulin/IGF-
1 signaling pathway confers resistance to Bt Cry PFTs in C. elegans.
Unexpectedly, this resistance does not solely rely on the canonical
DAF-2 insulin/IGF-1 signaling pathway through AKT/PKB and
DAF-16 but that rather, at least in part, deviates form the main
pathway at PDK-1 and that includes WWP-1. We demonstrate
that WWP-1 may be a novel signaling arm that diverges from
PDK-1 and functions in parallel to DAF-16 in the DAF-2 insulin/
IGF-1 signaling network. Furthermore WWP-1 is functionally
important for the intrinsic cellular defenses (INCED) against
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pathogenic attacks since loss of this pathway leads to animals
hypersensitive to PFTs and pathogenic bacteria Pseudomonas
aeruginosa in C. elegans.
Results
Reduction of the DAF-2 Insulin-Like Receptor Signal
Confers Resistance to Bt Cry PFTs in C. elegans
To address whether the DAF-2 insulin/IGF-1 signaling
pathway plays a role in C. elegans against the PFTs, C. elegans daf-
2 reduction-of-function mutant daf-2(e1370) were qualitatively
compared to wild-type N2 animals in their susceptibilities to the
nematicidal PFT, Cry5B (Figure 1A). Fourth larval (L4) stage
worms were fed for 48 hours either on control plates with E. coli
strain JM103 that did not express Cry5B or plates prepared with
E. coli JM103 expressing Cry5B. Specifically, the relative health of
each worm was evaluated qualitatively by comparing body size,
darkness of the intestine as an indicator of feeding, and activity,
including pharyngeal pumping and whether the worm demon-
strated spontaneous movement. In the absence of Cry5B, the wild-
type and mutant worms are healthy adults with similar
appearance. In the presence of the Cry5B, wild-type N2 worms
were severely intoxicated compared to those found on control no-
toxin plates, as evidenced by their smaller sizes and paler
appearances (Figure 1A). However, under the same conditions,
the daf-2(e1370) mutant animals were qualitatively healthier and
appeared resistant to Cry5B.
The sensitivity to Cry5B PFT of animals mutant for the DAF-2
insulin/IGF-1 receptor was also quantitatively assessed using a
dose-dependent mortality assay (Figure 1B) [19]. Form these data,
LC50(lethal concentration at which 50% of the animals die) values
were obtained (Table 1). These quantitative results confirm that
daf-2(e1370) mutant animals are at least an order of magnitude
more resistant to Cry5B than wild-type N2 animals (P,0.01). In
order to test whether daf-2(e1370) mutant animals were also
resistant to another Cry PFT, we also quantitative analyzed the
LC50values of daf-2(e1370) animals to the nematicidal Cry PFT,
Cry21A [7,12]. These data show that daf-2(e1370) animals are 7X
more resistant to Cry21A than wild type N2 animals (Figure 1C
and Table 1; P,0.01).
To independently confirm the importance of DAF-2 in PFT
responses, DAF-2 function was reduced using RNA interference
(RNAi) in the RNAi-sensitive strain rrf-3(pk1426) (Figure 1D). The
resistant to Cry5B and Cry21A resulting from knockdown of daf-2
was also seen, confirming the Cry PFT resistant phenotype is
caused by reduction of DAF-2 function. Interestingly, knock down
of bre-3 gene induced resistance to Cry5B but not to Cry21A at the
level detectable by this qualitative assay, although more quanti-
tative data indicate that mutants lacking bre-5 that functions in the
same pathway as bre-3 show partial resistance to Cry21A (data not
shown). This result implies that Cry21A at least partly might
require a different receptor than Cry5B for intoxication of C.
elegans.
Resistance of daf-2 Mutant to Cry Toxins Is, in Part,
through a daf-16-Independent Manner
In fertile animals, the extended lifespan and enhanced stress and
pathogen resistance phenotypes associated with reduction-of-
function mutations in daf-2 are totally suppressed by loss-of-function
mutations in daf-16, which encodes a Forkhead transcription factor
[17,20,21,22,23]. To test whether DAF-16 is also functionally
downstream of DAF-2 with regards to Cry PFT resistance, animals
containing the partial lost-of-function daf-2(e1370) mutant, the daf-
16nullmutant,mu86[24],and thedaf-2(e1370);daf-16(mu86)double
mutant were exposed to Cry5B and Cry21A and scored for viability
at the single dose of 40 mg/ml purified Cry5B [8]. Under these
conditions, daf-2(e1370) animals are significantly more resistant
(8.5X more alive animals) to Cry5B than wild-type N2 animals
(P,0.01) whereas loss-of-function daf-16(mu86) mutant animals are
as sensitive as N2 (Figure 2A, Table 2; P=0.683). daf-2(e1370);daf-
16(mu86) double mutant animals had an intermediate phenotype
that was statistically more sensitive than daf-2(e1370) animals
(P,0.001) but statistically more resistant (respectively 4.3X and
3.7X more alive animals) than daf-16(mu86) or wild-type animals in
response to Cry5B (P,0.001 and P,0.001). Similar results were
also seen in the animals treated with 8 mg/ml of the Cry21A. daf-
2(e1370);daf-16(mu86) double mutant animals also had an interme-
diate phenotype that was statistically more sensitive than daf-
2(e1370) animals (P,0.05) but statistically more resistant (respec-
tively 2.3X and 2.4X more alive animals) than daf-16(mu86) or wild-
type animals in response to Cry21A (P,0.05 and P,0.05)
(Figure 2B, Table 2). These results indicate that DAF-16 is required
forsome,butnotall,ofthePFTresistance conferredbyreductionof
DAF-2 function.
The Resistance of Cry Toxins Signal in DAF-2 Signaling
Pathway in Part Deviates from PDK-1
In the canonical DAF-2 insulin/IGF-1 signaling pathway for
the regulation of lifespan, energy metabolism, and dauer
development in C. elegans, DAF-2 regulates DAF-16 through the
activation of PI3K, encoded by age-1 and aap-1 for the catalytic
subunit and the regulatory subunit respectively. PI3K potentiates
the activity of PDK-1, which in turn activates the three down-
stream serine/threonine kinases: the Akt/PKB homologs AKT-1
and AKT-2, and the serum- and glucocorticoid-inducible kinase
homolog, SGK-1. These three serine/threonine kinases than
inhibit the nuclear translocation and transcriptional activity of
DAF-16 by phosphorylating DAF-16 at different serine/threonine
residues [25]. In order to identify where in the canonical DAF-2
insulin-like signaling pathway a DAF-16-independent arm might
branch off for responding to Cry PFTs, we obtained available
mutants in this pathway from the Caenorhabditis Genetic Center
(CGC) and exposed them to E. coli expressing Cry5B in liquid
medium. In this Cry5B toxicity assay, the daf-2(e1370), age-
1(hx546), aap-1(m889),pdk-1(sa680),
2(e1370);daf-16(mu86) animals are all statistically resistant to
Cry5B compared to the wild-type N2 animals (Figure 2C,
Table 2). Consistent with these results, daf-18(e1375) mutant
animals (DAF-18 encodes the mammalian PTEN lipid phospha-
tase homolog that antagonizes PI3K), are hypersensitive to Cry5B
compare to wild-type animals. Thus, animals from the insulin-
receptor down to PDK-1 behave as expected from what is known
about the pathway. However, animals with mutations in the three
serine/threonine kinases immediately downstream of PDK-1,
including akt-1(sa573), akt-1(ok525), akt-1(mg144), akt-2(ok393), and
sgk-1(ok538), were not resistant to Cry5B (Figure 2C, Table 2), and
in fact in two cases (akt-1(ok525) and akt-2(ok393)) were
hypersensitive to the PFT.
To independently test these results, the sensitivity to Cry5B of
animals with mutations in the pdk-1, akt-1, akt-2 and sgk-1 genes
were quantitatively assessed using the Cry5B dose-dependent
mortality assay (Figure 2D and 2E). Our quantitative results
comparing LC50values confirmed that animals with reduction-of-
function mutations in pdk-1, including pdk-1(sa680) and pdk-
1(sa709), were statistically more resistant to Cry5B than wild type
N2 animals. A gain-of-function mutation in pdk-1(mg142) was, as
predicted, sensitive to Cry5B. As above, we found animals with
the loss-of-function mutations in akt-1, akt-2 and sgk-1, including
pdk-1(sa709),
and
daf-
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Figure 1. Reduction of the DAF-2 insulin-like receptor signal confers resistance to Cry toxins. (A) Comparison of the reduction-of-
function daf-2(e1370) mutant animals to wild-type N2 animals and the known Cry5B resistant bre-3(ye28) animals on Cry5B-expressing E. coli plates
indicates daf-2(e1370) animals are resistant to Cry5B. The experiment was performed three times. Two representative worms are shown for each strain
48 hours after feeding either on E. coli with (Cry5B) or without Cry5B (No Toxin). (B) A dose-dependent mortality assay was performed using purified
Cry5B toxin to quantitatively compare sensitivities of wild-type N2 to the bre-3(ye28) and daf-2(e1370) mutants. Lethality was determined after 6 days.
This semi-log graph represents three independent experiments, and each data point is the mean and standard deviations of the experiments. Note,
that although daf-2 mutant animals are highly resistant based on their ability to stay alive, they clearly are not as resistant as bre-3 animals since the
latter are alive and robustly active and healthy whereas the former are alive but sickly at the end of this 6 day assay. (C) The dose-dependent mortality
assay for Cry21A spore-crystal lysates. The fact that spores are present in these assays is due to the fact we currently do not have purified Cry21A. The
presence of spores increases the toxicity of the Cry protein. Hence, the curves in (B) and (C) cannot be directly compared. (D) RNA interference E. coli-
expressed Cry5B toxin plate assay. RNAi sensitive rrf-3(pk1426) worms after feeding on pL4440 (empty vector control), bre-3, or daf-2 genes dsRNA
expressing E. coli were transferred to the E. coli-expressing either Cry5B (Cry5B), Cry21A (Cry21A) or without toxin (No Toxin) bacterial lawns for 48
hours. Knock down of the daf-2 gene leads to resistant to both Cry5B and Cry21A toxins. The experiment was performed three times and two
representative worms are shown for each RNAi knock down strain.
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akt-1(sa573), akt-1(ok525), akt-2(ok393), and sgk-1(ok538) were
either as sensitive or hypersensitive to Cry5B compared to N2
animals (Figure 2D and Table 1). Since there is some redundancy
between akt-1, akt-2, and sgk-1 mutant animals, it is possible that
the triple mutant (which is lethal and therefore not possible to test;
[26]) might be partly resistant. However, it should be noted that
sgk-1 mutant animals on their own have a long-lived phenotype
[27]. The fact that sgk-1 animals were not resistant to Cry5B and
that no level of resistance was seen with akt-1 or akt-2 suggested
that there is a divergence of the insulin pathway with regards to
Cry5B response upstream of these genes and branching off at
PDK-1.
WWP-1 Is a Novel PDK-1 Interacting Protein Involved in
Cry5B Defense
In order to identify the novel signaling arm that branches off from
PDK-1, we searched the C. elegans Interactome database (http://
vidal.dfci.harvard.edu/interactomedb/i-View/interactomeCurrent.
pl) for novel PDK-1 interacting proteins [28]. Two novel PDK-1
interacting proteins, WWP-1(WW domain Protein) and PCM-
1(Protein CarboxyMethyltransferase), were identified from high-
throughput yeast two-hybrid screens using H42K12.1(PDK-1) as the
bait (Figure 3A). The wwp-1 gene encodes a putative E3 ubiquitin
ligase orthologous to budding yeast Rsp5, Drosophila Su(dx), and
human WWP1 and WWP2 [29] (Figure 3B), and the pcm-1 gene
encodes an L-isoaspartate O-methyltransferase orthologous to human
PCMT1 [30]. We requested all available mutants of these two genes
from CGC and exposed them to Cry5B. The sensitivity to Cry5B of
animals with mutations in the wwp-1 and pcm-1 genes were
quantitatively assessed using the Cry5B dose-dependent lethality assay
and LC50values were obtained (Figure 3C and Table 1). The results
showed that wwp-1(ok1102) loss-of-function mutant animals are
significantly hypersensitive to Cry5B PFT compared to wild type N2
animals (.6 fold; P,0.01), but the sensitivity to Cry5B PFT of pcm-
1(qa201) mutant animals are statistically indistinguishable from N2
animals. Two additional wwp-1 mutant alleles, wwp-1(gk372) and wwp-
1(gk397) (Figure 3B), were also tested. All three wwp-1 mutant alleles
are significantly hypersensitive to Cry5B PFT (Figure 3D, Table 1).
Table 1. Data analysis of the quantitative Crystal toxins lethal concentration assays.
Toxin and Strain
LC50
(m mg/ml){
Standard
Deviation
p value relative
to N2
Relative sensitivity
(LC50mutant/LC50N2)
Cry5B LC50assay (Figure 1B)
N2 wild-type 11.241.86
bre-3(ye28)
.12011
,0.01
.10
daf-2(e1370)
.120
1
,0.01
.10
Cry21A LC50assay (Figure 1C)
N2 wild-type 3.731.03
daf-2(e1370) 26.108.03p,0.01 7.00
Cry5B LC50assay (Figure 2D)
N2 wild-type10.42 1.09
daf-2(e1370)
.120
1
p,0.01
.10
pdk-1(sa680)
.120
1
p,0.01
.10
pdk-1(sa709)
.120
1
p,0.01
.10
pdk-1(mg142)7.19 0.76p,0.050.69
Cry5B LC50assay (Figure 2E)
N2 wild-type20.556.64
akt-1(ok525)10.21 1.40p,0.05 0.50
akt-1(sa573)5.77 0.82p,0.010.28
akt-2(ok393)18.30 1.960.5890.89
sgk-1(ok538)13.95 1.49 0.1330.68
Cry5B LC50assay (Figure 3C)
N2 wild-type7.441.45
wwp-1(ok1102) 1.330.15p,0.010.18
pcm-1(qa201)6.461.920.428 0.87
Cry5B LC50assay (Figure 3D)
N2 wild-type 8.331.09
wwp-1(gk372) 2.521.03p,0.01 0.30
wwp-1(gk397)1.90 0.77p,0.01 0.23
wwp-1(ok1102)1.30 0.26p,0.010.16
{The range of the LC50values for Cry5B in N2 animals varies from 8.33 to 20.55 mg/ml, because the absolute quality and measured quantity of toxins varies from
different fermentation batches. We performed each experiment using the same batch of the purified toxins.
1The absolute LC50values of these mutant worms can not be calculated by Probit, because the limitation of the solubility of Cry5B and therefore we cannot achieve a
100% killing concentration for these mutants. We used 120 mg/ml, the maximum dose of Cry5B, for the statistical calculations of these resistant mutants.
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Figure2.The resistancetoCrytoxinis onlypartly dependentuponDAF-16FOXOand forksat PDK-1intheDAF-2 insulin-likenetwork.
(A, B)Comparisonsofthe daf-2(e1370), daf-16(mu86), anddaf-2(e1370);daf-16(mu86) mutantanimals towild-type N2animals in40 mg/ml purifiedCry5Bor
8 mg/ml Cry21A indicate daf-2(e1370) mutant and daf-2(e1370);daf-16(mu86) double mutant are all statistically resistant to Cry5B and Cry21A compared
with N2. * indicates P,0.001 (P,0.05 in B) relative to wild-type N2.#indicates P,0.001 (P,0.05 in B) relative to daf-2(e1370);daf-16(mu86) mutant. (C)
Comparisons of the mutants in the canonical DAF-2 insulin-like signaling pathway, including daf-2(e1370), age-1(hx546), aap-1(m889), daf-18(e1375), pdk-
1(sa680), pdk-1(sa709), akt-1(sa573), akt-1(ok525), akt-1(mg144), akt-2(ok393), sgk-1(ok538), daf-16(mu86), daf-2(e1370);daf-16(mu86), to wild-type N2
animals in E. coli-expressed Cry5B liquid toxicity assay. * indicates P,0.01 relative to wild-type N2. (D, E) Dose-dependent mortality were performed using
purified Cry5Btoxin toquantitatively compare sensitivitiesofwild-type N2tothe daf-2(e1370), pdk-1(sa680), pdk-1(sa709), pdk-1(mg142),akt-1(ok525), akt-
1(sa573), akt-2(ok393), and sgk-1(ok538) mutants. This semi-log graph represents three independent experiments, and each data point shows the mean
and standard deviations from three experiments.
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To independently test that our results stem from loss of function
phenotypes, RNAi sensitive rrf-3(pk1426);glp-4(bn2) mutant worms
were used to knock down the wwp-1 genes by two independent E.
coli RNAi feeding clones (8A5 and 8A15) from the Ahringer RNAi
library [31]. rrf-3(pk1426);glp-4(bn2) animals were used because the
rrf-3(pk1426) mutant is hypersensitive to RNAi and the glp-4(bn2)
mutant does not produce progeny that would otherwise compli-
cate the assay (via internal hatching of larvae that sometimes
occurs when adult C. elegans are intoxicated with Cry proteins). We
have demonstrated that both mutants have roughly normal
response to Cry5B [10]. DNA sequencing results demonstrated
that these two wwp-1 RNAi clones target different sequence
regions of the wwp-1 gene (Figure 3B). The resistant and
hypersensitive phenotypes to Cry5B resulting from knockdown
of daf-2 and wwp-1 respectively were seen, confirming the
phenotypes are caused by lost or reduction of function of these
genes (Figure 3E, Table 3). Overall, these data suggest that WWP-
1, a novel PDK-1 putative interacting protein, is functionally
important for defense against PFT attack since loss of this pathway
leads to animals hypersensitive to Cry5B PFT.
WWP-1 Is also Involved in Innate Immunity and Aging
Regulation
The DAF-2 signaling network in C. elegans is well documented
for essential in innate immunity against various pathogens,
including the human Gram-negative bacterial pathogens Pseudo-
monas aeruginosa [17,18]. If WWP-1 is in the DAF-2 pathway and is
antagonistic to DAF-2 function as it is relative to the Cry protein
response (i.e., DAF-2 animals are resistant whereas WWP-1 mutant
animals are hypersensitive), we predict WWP-1 would also play a
role in response to P. aeruginosa and be antagonistic to DAF-2. We
exposed wwp-1 mutants to the pathogenic bacteria P. aeruginosa
strain PA14 (Figure 4 and Table 4). As we predicted, animals
lacking WWP-1 are significantly hypersensitive to the killing by P.
aeruginosa PA14 (P,0.01; the opposite phenotype of DAF-2 mutant
animals). These data also demonstrate that WWP-1 is involved in
promoting innate immunity against pathogenic bacteria.
The DAF-2 insulin/IGF-1 signaling network as aforementioned
also plays important roles in regulation of lifespan in C. elegans. It also
has been suggested that signal transductions in the DAF-2 insulin/
IGF-1 signaling pathway for longevity and innate immunity can be
interrelated [16,18]. If WWP-1 is in the DAF-2 pathway, it might
also have a role in C. elegans lifespan, again antagonistic to DAF-2.
As predicted, we found that wwp-1(gk372) and wwp-1(ok1102)
mutant animals had statistically significant shorter lifespan compared
with wild-type N2 animals (Figure 5A, Table 4). These data indicate
that wwp-1 is also a positive regulator of lifespan in C. elegans.
To confirm that the hypersensitivity of WWP-1 mutant animals
to aging, P. aeruginosa, and Cry proteins is not due to general ill
health of these animals, we tested whether wwp-1 mutant animals
are hypersensitive to two other toxic chemical compounds, the
heavy metal CuSO4(a toxic insult that kills with kinetics similar to
Table 2. Data analysis of the toxins toxicity assays.
Cry5B 40 m mg/ml (Figure 2A)Survival (%)Standard Deviation p value relative to N2 p values relative to daf-2;daf-16
N2 wild-type11.529.47p,0.001
daf-2(e1370)97.91 3.45p,0.001p,0.001
daf-16(mu86)9.81 7.72 p=0.663p,0.001
daf-2(e1370);daf-16(mu86) 42.1610.69p,0.001
Cry21A 8 m mg/ml (Figure 2B) Survival (%)Standard Deviation p value relative to N2 p values relative to daf-2;daf-16
N2 wild-type20.21 2.67P,0.05
daf-2(e1370) 79.07 11.65p,0.01P,0.05
daf-16(mu86)20.23 5.59p=0.996P,0.05
daf-2(e1370);daf-16(mu86) 47.51 15.93p,0.05
E. coli-expressed Cry5B liquid
toxicity assay (Figure 2C)Survival (%) Standard Deviationp value relative to N2
Relative sensitivity (mutant/wild
type)
N2 wild-type19.82 3.44
daf-2(e1370)90.911.90p,0.014.59
age-1(hx546)83.75 9.32p,0.014.23
aap-1(m889)81.088.39p,0.014.09
daf-18(e1375)3.34 2.04p,0.010.17
pdk-1(sa680)90.151.80p,0.014.55
pdk-1(sa709) 96.110.13p,0.014.85
akt-1(sa573)20.533.94 p=0.8221.04
akt-1(ok525) 3.765.32p,0.010.19
akt-1(mg144)12.505.73 p=0.080.63
akt-2(ok393)7.411.48p,0.010.59
sgk-1(ok538)23.44 4.00p=0.2791.18
daf-16(mu86) 25.612.77 p=0.0921.29
daf-2(e1370);daf-16(mu86)79.9113.59p,0.014.03
doi:10.1371/journal.pone.0009494.t002
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Figure 3. WWP-1 is a novel PDK-1 interacting protein involved in Cry5B defense. (A) Two novel PDK-1 interacting protein identified by the
Worm Interactome Database. Two bait-prey interactions, including H42K12.1(pdk-1)-Y65B4BR.4(wwp-1) and H42K12.1(pdk-1)-C10F3.5(pcm-1), were
identified by using H42K12.1(pdk-1) as the bait in the high-throughput yeast two hybrid screens [28]. (B) Predicted genomic structure of wwp1. Boxes
and lines denote exons and introns respectively. The predicted functional domains of WWP1, including a C2 domain, four WW domains and a HECT
domain, are indicated. The regions corresponding to the interfering dsRNA clones, 8A5 and 8A15 are underlined. The mutation regions of the three
wwp-1 mutant alleles used in this study are indicated as gray boxes. (C) A Cry5B dose-dependent mortality assay was performed to quantitatively
compare sensitivities of wild-type N2 to the wwp-1(ok1102) and pcm-1(qa201) mutants. Only wwp-1(ok1102) mutant animals showed statistically
(p,0.01) hypersensitivity to Cry5B compared to N2. (D) A Cry5B dose-dependent mortality assay was performed to quantitatively compare
sensitivities of wild-type N2 to three wwp-1 mutants, including wwp-1(gk372), wwp-1(gk397), wwp-1(ok102). All wwp-1 mutants showed statistically
hypersensitive to Cry5B. (E) RNAi sensitive rrf-3(pk1426);glp-4(bn2) mutant worms after feeding on pL4440 (empty vector control), daf-2, or two wwp-
1(8A5 and 8A15) dsRNA expressing E. coli were exposed to purified Cry5B at 25uC. Lethality was determined after 6 days. Animals knocked down for
the daf-2 gene were statistically resistant to Cry5B whereas animals knocked down for the wwp-1 gene by two independent wwp-1 RNAi clones were
statistically hypersensitive to Cry5B.
doi:10.1371/journal.pone.0009494.g003
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Cry5B) [11] and the oxidative stress agent H2O2(a toxic insult that
kills much more rapidly). All wwp-1 mutants, including wwp-
1(gk372), wwp-1(gk397), and wwp-1(ok1102), have the similar
sensitivity as wild type to killing by either CuSO4 or H2O2
(Figure 5B and 5C; Table 4). These data argue against the
supposition that these mutants are hypersensitive to the PFT and
P. aeruginosa tested above merely because they are generally
unhealthy. Taken together, the above results suggest that WWP-1
is not only a positive regulator in the longevity regulation but also
specifies in the intrinsic cellular defense (INCED) against PFTs
and the innate immune response against pathogenic bacteria in C.
elegans.
Table 3. Data analysis of the RNA interference assays.
RNAi in rrf-3(pk1426);
glp-4(bn2) (Figure 3E)
Survival
(%)
Standard
Deviation
p value relative to RNAi empty
vector control (pL4440)
pL4440 (vector control)22.553.00
daf-2 RNAi (pAD48) 47.914.11p,0.01
wwp-1 RNAi (8A5) 7.970.98p,0.01
wwp-1RNAi (8A15) 5.432.15p,0.01
wwp-1 RNAi
(Figure 6)
Survival
(%)
Standard
Deviation
p value relative to RNAi empty
vector control (pL4440)
Relative sensitivity
(wwp-1 RNAi/vector control)
p values relative to
daf-2;daf-16/wwp-1 RNAi
N2/pL4440 41.87 12.25 0.12
N2/wwp-1 4.845.32p,0.01 0.120.06
daf-2/pL444099.221.35
daf-2/wwp-175.87 5.52p,0.01 0.76
daf-2;daf-16/pL4440 69.211.62
daf-2;daf-16/wwp-1 23.1511.23p,0.01 0.33
wwp-1/pL44408.062.17
wwp-1/wwp-16.253.27 p=0.471 0.78
doi:10.1371/journal.pone.0009494.t003
Figure 4. WWP-1 is involved in the innate immunity against P.
aeruginosa PA14. A lifespan assay was used to compare the wwp-1
mutants to slow killing by P. aeruginosa PA14. This graph represents
combined data from three experiments. The lifespan of wwp-1 mutants,
including wwp-1(gk372) and wwp-1(ok1102), feeding on P. aeruginosa
PA14 bacteria are statistically shorter than wild-type N2 animals.
doi:10.1371/journal.pone.0009494.g004
Table 4. Data analysis of the lifespan and general stresses
assays.
Life span assay
(Figure 5A) and
strain
Median
survival
(days)
p value
relative
to N2
N2 wild-type17.00
wwp-1(gk372) 12.00p,0.01
wwp-1(ok1102) 10.50p,0.01
CuSO4
(Figure 5B)
and strain
LC50
(mM)
Standard
Deviation
p value
relative
to N2
N2 wild-type 2.59 0.57
wwp-1(gk372) 3.55 0.930.094
wwp-1(gk397)3.450.930.146
wwp-1(ok1102)1.93 0.390.306
H2O2
(Figure 5C)
and strain
LC50
(mM)
Standard
Deviation
p value
relative
to N2
N2 wild-type1.260.25
wwp-1(gk372)1.570.420.144
wwp-1(gk397)1.620.390.076
wwp-1(ok1102)1.380.280.788
PA14 killing
assay (Figure 4)
and strain
Median
survival
(hours)
p value
relative
to N2
N2 wild-type 75
wwp-1(gk372) 50p,0.01
wwp-1(gk397) 50p,0.01
wwp-1(ok1102) 50p,0.01
doi:10.1371/journal.pone.0009494.t004
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WWP-1 Works Downstream of DAF-2 and in Parallel to
DAF-16 in the DAF-2 Insulin/IGF-1 Signaling Network
The two-hybrid interactome data and the Cry5B/aging/P.
aeruginosa results are consistent with WWP-1 acting in the DAF-2
pathway antagonistic to and downstream of DAF-2. If true, then
we predict that knock-down of WWP-1 should suppress a DAF-2
mutant phenotype. We therefore knocked down wwp-1 in daf-
2(e1370) and daf-2(e1370);daf-16(mu86) mutant animals and
exposed these wwp-1 knockdown animals (and control ‘‘no knock
down’’ animals in which the animals were fed empty RNAi vector)
to purified Cry5B (Figure 6, Table 3). Our results showed that
wwp-1 RNAi can partly suppress the daf-2(e1370) resistant
phenotype and that the combination of daf-16 knock out (via
mu86 mutation) and wwp-1 knock down (via RNAi) can completely
suppress daf-2(e1370) resistance back to a response similar to wild-
type (p=0.12 daf-2;daf-16/wwp-1 RNAi vs. N2/L4440 vector
control; note, although the response of daf-2;daf-16/wwp-1 RNAi
animals looks less hypersensitive than that of N2/wwp-1 RNAi
animals, the two are actually statistically similar; P=0.06). These
data are consistent with both daf-16 and wwp-1 acting in parallel
daf-2-dependent pathways to mediate resistance to Cry5B PFT.
Furthermore, that wwp-1 RNAi can not further sensitize the wwp-
1(ok1102) worms in this experiment also confirmed that this mutant
allele is a total loss-of-function mutant.
Discussion
Here we demonstrate for the first time that the DAF-2 insulin/
IGF-1 pathway is involved in intrinsic cellular defenses (INCED)
against PFTs. Furthermore, our data suggest that the daf-2 PFT
defense pathway bifurcates at PDK-1 into DAF-16-dependent and
DAFT-16-independent branches. This is the first report of a
bifurcation of the daf-2 insulin/IGF-1 pathway at this junction.
We furthermore find a protein, WWP-1, that appears to be
involved in the DAF-16 –independent branch of the INCED/
Figure 5. WWP-1 is a positive regulator of lifespan in C. elegans.
(A) A lifespan assay was used to compare the normal lifespan of wwp-1
mutant worms and the wild-type N2 worms. This graph represents
combined data from three independent experiments. The lifespan of
wwp-1 mutants, including wwp-1(gk372) and wwp-1(ok1102), are
statistically shorter than wild-type N2 animals. (B) A dose-dependent
mortality assay comparing sensitivity to CuSO4 revealed the wwp-1
mutants, wwp-1(gk372), wwp-1(gk397), and wwp-1(ok1102) are not
hypersensitive compared to wild-type N2. Lethality was determined
after 6 days of CuSO4exposure, the same time frame as the Cry5B
lethality assay. Data, plotted semi-log, are the mean and standard
deviation of three independent experiments. (C) A dose- dependent
mortality assay comparing sensitivity to H2O2revealed wwp-1 mutants
are not hypersensitive to this toxic insult compared to wild-type N2.
Lethality was determined after 4 hours of H2O2exposure. Data, plotted
semi-log, are the mean and standard deviation of three independent
experiments.
doi:10.1371/journal.pone.0009494.g005
Figure 6. WWP-1 is a downstream signal of DAF-2 and in
parallel to DAF-16 in response to Cry5B. daf-2(e1370), daf-
16(mu86), daf-2(e1370);daf-16(mu86) and wwp-1(ok1102) mutant worms
were fed dsRNA to wwp-1 (clone 8A15) to knock down wwp-1 gene in
all these strains (plaid boxes). The worms fed on E. coli HT115
transformed with the RNAi empty vector (pL4440 vector) were used as
controls (solid boxes). After developing to L4 stage, all RNAi knock
down worms are exposed to purified Cry5B at 25uC. Lethality was
determined after 6 days. Knockdown of wwp-1 gene in animals,
including N2, daf-2(e1370), daf-16(mu86), and daf-2(e1370);daf-16(mu86),
increased their sensitivity to Cry5B statistically significant. However, the
sensitivity of wwp-1(ok1102) animals to Cry5B cannot be further
enhanced by wwp-1 RNAi.
doi:10.1371/journal.pone.0009494.g006
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innate immune response. Loss of WWP-1, a protein in the
ubiquitin E3 ligase family, leads to hypersensitivity to the PFT
Cry5B and the pathogen P. aeruginosa and that, based on double
and triple knock out/knock down analyses with daf-2 and daf-16
mutants, appears to work in parallel with DAF-16. A model
summarizing our findings is shown in Figure 7.
The assignment of WWP-1 as downstream of the DAF-2
insulin/IGF-1 pathway is based on two-hybrid interactome data
with PDK-1, on phenotypic analyses of responses to PFT, P.
aeruginosa, and aging, and on our genetic pathway data. A similar
conclusion is evidenced from a previous genome-wide RNAi
screen in which it was shown that knockdown of wwp-1 gene
decreased median lifespan by 24% in daf-2(e1370) mutants, 5% in
daf-2(e1370);daf-16(mgDf47) mutants, and 9% in wild-type N2
animals [32]. The knockdown of wwp-1 gene more significantly
shortened the lifespan of daf-2 animals but still decreased the
lifespan of daf-2;daf-16 mutant and wild-type N2 animals. This
suggested that wwp-1 functions in parallel to daf-16 and converging
within the DAF-2 insulin/IGF-1 signaling network to regulate
longevity, which is reminiscent of our data. In addition, recently
ubiquitin ligase activity has been shown for WWP-1, as well as
demonstration that WWP-1 is involved in a daf-16-independent
life span extension in response to diet restriction [33]. Taken
together, our and others’ results imply that WWP-1 functions in
parallel to DAF-16 and converging within the fundamental DAF-2
insulin/IGF-1 signaling network to regulate the INCED against
PFTs attack, innate immune responses against P. aeruginosa, as well
as longevity in C. elegans.
Our results also show that regulation of PFT defense and
lifespan can clearly be decoupled. First, we note that lifespan
extension of daf-2 mutants is completely dependent upon daf-16
[34] but response to PFT is not. Second, we note that where sgk-1
mutants are long-lived [27], mutation in this gene alone is not
resistant to PFTs. The hypersensitivity of akt-1 and possibly akt-2
mutant animals is intriguing and suggests that they might also have
other roles outside of the DAF-2 insulin/IGF-1 pathway in PFT
defenses. Interestingly along these lines, we note that in
mammalian cells a role of AKT in response to PFTs has already
been noted [35].
That DAF-2 signal can be decoupled from the conical pathway
is reminiscent of several recent reports. Firstly, it has been
demonstrated that EAK-3 (Enhancer of AKT-1 null) functions in
parallel to AKT-1 to inhibit the expression of Forkhead
transcription factor DAF-16 target genes involved in C. elegans
dauer development. That Eak-3 mutants have normal lifespan
indicates EAK-3 decouples insulin-like regulation of development
and longevity [36,37]. Secondly, it has been reported that long-
lived mutants of genes downstream of daf-2, such as pdk-1 and sgk-
1, show wild-type resistance to the human opportunistic pathogen,
Pseudomonas aeruginosa strain PA14. However, mutants of akt-1 and
akt-2 show enhanced resistance to P. aeruginosa PA14 [26]. Thirdly,
it has also bee reported that some akt-1 and pdk-1 alleles can
uncouple the dauer arrest, adult longevity and stress resistance
phenotypes of age-1(mg109) mutants [38]. They also demonstrated
reproductive development in age-1(mg109); mg227 animals re-
quired only akt-1, and pdk-1 activity was dispensable in this
background. These findings suggested larval and adult phenotypes
of DAF-2 signaling are fully separable in these mutants. Finally,
the other possible PDK-1 interacting protein identified in the
interactome database, PCM-1, has also been demonstrated to
participate in the repair of age-damaged proteins and overexpres-
sion of PCM-1 increases adult life span [39,40]. However in our
results, PCM-1 is not involved in Cry5B INCED. All of these data
suggest that dauer formation, lifespan regulation, stress response,
and pathogen resistance signals can be intertwined and in some
cases decoupled within the DAF-2 insulin/iGF-1 signaling
network in C. elegans.
In summary, we have identified specifically WWP-1 and the
DAF-2 insulin/IGF-1 signaling network as components of INCED
against PFTs. The biological functions of the DAF-2/DAF-16
signaling pathway in longevity and pathogens resistance have been
extensively studied for years [15,18,41]. Our current hypothesis is
that the DAF-2 signaling network has split off a separate arm at
PDK-1/WWP-1 not only involved in longevity regulation and the
innate immunity against P. aeruginosa PA14 but also the INCED
against PFTs as well as perhaps some yet unknown responses.
Materials and Methods
C. elegans and Bacterial Strains
Some Caenorhabditis elegans strains used in this work were
provided by the Caenorhabditis Genetics Center (CGC), which is
funded by the NIH National Center for Research Resources
(NCRR). C. elegans strains were maintained on NG plates using
Escherichia coli strain OP50 as the food source [42]. Strains used in
this study were wild-type Bristol strain N2, daf-2(e1370), age-
1(hx546), aap-1(m889), daf-18(e1375), pdk-1(sa680), pdk-1(sa709),
pdk-1(mg142), akt-1(ok525), akt-1(sa573), akt-1(mg144), akt-2(ok393),
Figure 7. Schematic illustrating relationship between WWP-1
and DAF-2 insulin-like signal network. In the canonical DAF-2
insulin/IGF-1 signaling pathway in C. elegans, DAF-2 regulates DAF-16
through the activation of PI3K, composed of AGE-1 and AAP-1 for the
catalytic subunit and the regulatory subunit respectively. PI3K activates
the activity of PDK-1, which eventually inhibits the nuclear translocation
and transcriptional activity of DAF-16. Here we demonstrate that the
attenuated DAF-2 signal can also regulate an additional DAF-16-
independent signal arm that diverges form PDK-1 to WWP-1 to defense
against Cry5B PFT.
doi:10.1371/journal.pone.0009494.g007
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sgk-1(ok538), daf-16(mu86), daf-2(e1370);daf-16(mu86), bre-3(ye28),
rrf-3(pk1426), rrf-3(pk1426);glp-4(bn2),
1(ok1102), wwp-1(gk372), and wwp-1(gk397) were each backcrossed
at least 4 times. Bacteria expressing dsRNA directed against bre-3
and wwp-1 were part of a C. elegans RNAi library in E. coli strain
HT115 (Geneservice, Cambridge U.K.). All RNAi clones have
been confirmed by plasmid DNA sequencing. Escherichia coli
HT115 transformed with the pAD48 construct, which expresses
dsRNA targeting the daf-2 gene, was kindly provided by A. Dillin
(Salk Institute, San Diego) [43]. All bacterial strains were cultured
under standard conditions.
pcm-1(qa201), wwp-
Cry Toxins Toxicity Assays and Microscopy
All assays were performed at 25uC unless indicated elsewhere.
Qualitative toxicity assays based on visual comparison of worm
intoxication were performed on plates with E. coli-expressed
Cry5B as described [19]. L4 stage worms were fed for 48 hours
either on control plates with E. coli that did not express Cry5B
(pQE9 vector control) or on plates prepared with E. coli expressing
Cry5B (pQE9-Cry5B) [7]. The relative health of each worm was
determined qualitatively by comparing body size, darkness, and
activity. Images were acquired using an Olympus SZ60 compound
microscope using the 3X magnification linked to a Canon
PowerShot A620 digital camera. Quantitative mortality assays
were performed as described [19]. Concentrations of each toxin
were set-up in triplicate for each assay with approximately 20,30
worms per well, and each assay was performed independently at
least three times. The purified Cry5B and the crystal-spore toxin
lysates of Cry21A were prepared as described [8,44]. Approxi-
mately 1500 worms were scored for each strain in the calculation
of the LC50values for each toxin. E. coli-expressed Cry5B liquid
toxicity assay: N2 wild-type and various daf-2 pathway mutant
worms were exposed to E. coli-expressed Cry5B in S media in 48-
wll plates to quantitative scored the survival. Similar conditions
were used as the quantitative mortality assays described above,
except that E. coli JM103 Cry5B expressing bacteria at 0.6 OD600
in stead of purified Cry5B and E. coli OP50 were used in this assay.
The survival rate of each well was scored after incubating at 25uC
for 6 days.
RNA Interference (RNAi)
For figures 1D, RNAi assays were carried out on E. coli-
expressed Cry5B Plates. E. coli strain HT115 transformed with
RNAi plasmids were spread on NG-IC plates [NG plates with
25 mg/ml carbenicillin and 0.1 mM isopropyl-b-D-1-thiogalacto-
pyranoside (IPTG)] and incubated at 25uC overnight to induce the
dsRNA expression. E. coli HT115 with pL4440, an empty vector,
was used as negative control of RNAi. Synchronized rrf-3(pk1426)
L1 larvae were obtained using standard protocols [19] then
cultured on pL4440, bre-3, or daf-2 genes dsRNA expressing RNAi-
plates at 20uC until L4 stage. These L4 stage worms were
transferred to either control plates with E. coli HT115 that did not
express Cry toxins (empty vector) or plates prepared with E. coli
HT115 expressing either Cry5B or Cry21A (using our standard
expression vector; [7]) together with E.coli HT115 either carrying
RNAi plasmids or the pL4440 plasmid and then incubated at 25u
for 48 h. The relative health of each worm was determined
qualitatively by its appearance as described above.
For figures 3E, synchronized L1 rrf-3(pk1426);glp-4(bn2) animals
(mutant is hypersensitive to RNAi and does not produce progeny
that would otherwise complicate the assay; both mutants have
roughly normal response to Cry5B; [10]) were cultured on the
NG-IC daf-2 or wwp-1 RNAi-plates at 20uC until L4 stage. L4
stage rrf-3(pk1426);glp-4(bn2) RNAi knock down animals were
washed out by S medium and transferred to the wells of 48-well
plate with S medium containing E. coli HT115 RNAi bacteria at
0.6 OD600 and 20 mg/ml of purified Cry5B. After incubating at
25uC for 6 days, the survival rate of each well was scored.
For figure 6, the daf-2(e1370), daf-16(mu86), daf-2(e1370);daf-
16(mu86) and wwp-1(ok1102) mutant worms were used. Synchro-
nized L1 animals were cultured on the NG-IC wwp-1 RNAi or
pL4440 E. coli HT115 plates at 20uC until L4 stage. L4 stage
RNAi knock down animals were washed out by S medium and
transferred to the wells of 48-well plate with S medium containing
with either wwp-1 RNAi or pL4440 E. coli HT115 bacteria at 0.6
OD600 and 20 mg/ml of purified Cry5B. After incubating at 25uC
for 6 days, the survival rate of each well was scored.
Lifespan Assay
Lifespan analysis was conducted according to standard
protocols [34,45]. All life span experiments were performed in
the absence of 5-fluoro-29-deoxyuridine. Briefly, to obtain a
synchronously growing population, eggs were prepared by treating
a population of C. elegans with hypochlorite/NaOH solution and
transferring the resulting eggs to NG agar plates covered with E.
coli strain OP50. When these had reached the young adults, ,150
nematodes were transferred to fresh plates, which also represents
the first day of life span analysis. Nematodes were transferred to
fresh plates daily during the progeny production period and after
that were transferred every second to third day but monitored
daily for dead animals. Nematodes that did not respond to gentle
prodding and displayed no pharyngeal pumping were scored as
dead. Animals that crawled off the plate or died due to internal
hatching or protrusion of the gonads through the vulva were
censored. Censoring describes an event where partial information
on the life span of an individual animal is lost as a consequence of
premature death. Thus, censored animals were included in
statistical analysis only until the day of the censoring event.
Survival analysis was performed using GraphPad Prism 5.0
(GraphPad Software, Inc. La Jolla, CA). The Mantel-Cox logrank
test was used to assess statistical significance of difference in
survival. Only p-values,0.01 were considered significant to
minimize type I errors.
Pseudomonas aeruginosa PA14 Killing Assay
The P. aeruginosa killing assay was performed on slow-killing
plates as described [46], with the following modifications: PA14
was cultured overnight in tryptic soy broth instead of King’s broth
and then spread on slow-killing plates complemented with 75 uM
5-fluoro-29-deoxyuridine. The experiment was performed three
times with approximately 100–150 worms total per strain at 25uC.
The Mantel-Cox logrank test was used to assess statistical
significance of difference in survival. Only p-values,0.01 were
considered significant to minimize type I errors.
General Stressors Analysis
CuSO4assay.
brief, a serial doses of CuSO4, E. coli OP50 at an optical density of
0.2–0.25 OD600, and ,30 L4 larvae were used per well in 48-well
plates. Lethality was determined after 6 days of CuSO4exposure
at 25uC.
H2O2 assay.
Assays were carried out as described [11];
lethality was determined after 4 hours of H2O2exposure at 25uC.
Assays were carried out as described [11]. In
Data Analysis
All experiments were performed a minimum of three times.
LC50 values were determined by PROBIT analysis [47]. The
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lethal concentration assays are represented graphically using
nonlinear regression performed with the software GraphPad
Prism 5.0. Statistical analysis between two values was compared
with a paired t-test. Statistical analysis among three or more values
was compared with one-way ANOVA with Dunnett adjustment.
All data analysis was performed using SPSS, ver 13.0 (SPSS,
Chicago, IL). Statistical significance was set at p,0.05.
Acknowledgments
We are grateful to the members of the Chen and Aroian labs, for critical
comments, discussions and technical advices. We thank A. Dillin (Salk
Institute, San Diego) for the daf-2 RNAi plasmid, the Caenorhabditis Genetics
Center and the C. elegans Gene Knockout Consortium for worm strains. We
are grateful to Dr. I-Hua Chu for her discussions in statistical analysis.
Author Contributions
Conceived and designed the experiments: CSC RVA. Performed the
experiments: CSC AB CYK YLY HDC FCOL. Analyzed the data: CSC
RVA. Wrote the paper: CSC RVA.
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Insulin Pathway and PF Toxins
PLoS ONE | www.plosone.org 12March 2010 | Volume 5 | Issue 3 | e9494