The SKPO-1 Peroxidase Functions in the Hypodermis to Protect Caenorhabditis elegans From Bacterial Infection

Abstract

In recent years, the synergistic relationship between NADPH Oxidases (NOX)/Dual Oxidases (DUOX) enzymes and peroxidases has received increased attention. Peroxidases utilize NOX/DUOX-generated H2O2 for a myriad of functions including, but not limited to thyroid hormone biosynthesis, cross-linking extracellular matrices (ECM) and immune defense. We postulated that one or more peroxidases produced by Caenorhabditis elegans would act in host defense, possibly in conjunction with BLI-3, the only NOX/DUOX enzyme encoded by the genome that is expressed. Animals exposed to RNAi of the putative peroxidase genes were screened for susceptibility to the human pathogen Enterococcus faecalis. One of three genes identified, skpo-1 (ShkT-containing peroxidase), was studied in-depth. Animals mutant for this gene were significantly more susceptible to E. faecalis, but not Pseudomonas aeruginosa. A slight decrease in longevity was also observed. The skpo-1 mutant animals had a dumpy phenotype of incomplete penetrance; half the animals displayed a dumpy phenotype ranging from slight to severe, and half were morphologically wild type. The SKPO-1 protein contains the critical catalytic residues necessary for peroxidase activity, and in a whole animal assay more H2O2 was detected from the mutant compared to the wild type, consistent with the loss of an H2O2 sink. By using tissue-specific skpo-1 RNAi and immunohistochemical localization with an anti-SKPO-1 antibody, it was determined that the peroxidase is functionally and physically present in the hypodermis. In conclusion, these results characterize a peroxidase that functions protectively in the hypodermis during exposure to E. faecalis.

Full text

GENETICS OF IMMUNITY
The SKPO-1 Peroxidase Functions in the Hypodermis
to Protect Caenorhabditis elegans From
Bacterial Infection
George R. Tiller and Danielle A. Garsin
1
Department of Microbiology and Molecular Genetics, University of Texas Health Science Center and Graduate School of Biomedical
Sciences, Houston, Texas 77030
ABSTRACT In recent years, the synergistic relationship between NADPH oxidase (NOX)/dual oxidase (DUOX) enzymes and peroxidases
has received increased attention. Peroxidases utilize NOX/DUOX-generated H
2
O
2
for a myriad of functions including, but not limited to,
thyroid hormone biosynthesis, cross-linking extracellular matrices (ECM), and immune defense. We postulated that one or more
peroxidases produced by Caenorhabditis elegans would act in host defense, possibly in conjunction with BLI-3, the only NOX/DUOX
enzyme encoded by the genome that is expressed. Animals exposed to RNA interference (RNAi) of the putative peroxidase genes were
screened for susceptibility to the human pathogen Enterococcus faecalis. One of three genes identified, skpo-1 (ShkT-containing
peroxidase), was studied in depth. Animals mutant for this gene were significantly more susceptible to E. faecalis, but not Pseudo-
monas aeruginosa. A slight decrease in longevity was also observed. The skpo-1 mutant animals had a dumpy phenotype of in-
complete penetrance; half the animals displayed a dumpy phenotype ranging from slight to severe, and half were morphologically wild
type. The SKPO-1 protein contains the critical catalytic residues necessary for peroxidase activity, and in a whole animal assay, more
H
2
O
2
was detected from the mutant compared to the wild type, consistent with the loss of an H
2
O
2
sink. By using tissue-specificskpo-
1RNAi and immunohistochemical localization with an anti-SKPO-1 antibody, it was determined that the peroxidase is functionally and
physically present in the hypodermis. In conclusion, these results characterize a peroxidase that functions protectively in the hypodermis
during exposure to E. faecalis.
HEME-containing peroxidases play critical, wide-ranging
roles in biological systems. Once called the “animal
heme peroxidases,”members of the peroxidase–cyclooxgenase
superfamily are actually found in all kingdoms of life (Zamocky
et al. 2008). The roles of most are poorly characterized, but
some are clearly involved in immune defense. For example,
the most famous and best-studied member of this group,
myeloperoxidase (MPO), is found in the granulocytes of
neutrophils where it catalyzes the formation of the potent
oxidant HOCl from H
2
O
2
and Cl
2
to kill invading microbes
(reviewed by Klebanoff 2005). Another is lactoperoxidase
(LPO), which is found on mucosal surfaces and generates
the protective oxidant hypothiocyanite (OSCN
2
)from
H
2
O
2
and thiocyanate (SCN
2
).Thisprocessisimpaired
in the lungs of patients with cystic fibrosis (CF), contributing
to the poor clearance of pathogens such as Pseudomonas
aeruginosa and Staphylococcus aureus, the most common
causes of lung infection in patients with CF (Conner et al.
2002; Geiszt et al. 2003; Forteza et al. 2005; Moskwa et al.
2007).
Many of these peroxidases are functionally associated
with members of the NADPH oxidase (NOX)/dual oxidase
(DUOX) family of proteins. These enzymes generate the
H
2
O
2
required as substrate for the peroxidases. For example,
Nox2 is the source of H
2
O
2
for MPO. DUOXs differ from
NOXs in that they encode a peroxidase domain in addition
to the oxidant-generating NADPH oxidase domain (reviewed
by Rada et al. 2008; Sumimoto 2008). Despite the fact that
they have a peroxidase domain, DUOXs appear to still asso-
ciate with separate peroxidases. For example, LPO utilizes
H
2
O
2
generated by Duox1 or Duox2 (Conner et al. 2002;
Geiszt et al. 2003; Forteza et al. 2005). In fact, the peroxidase
Copyright © 2014 by the Genetics Society of America
doi: 10.1534/genetics.113.160606
Manuscript received December 10, 2013; accepted for publication March 9, 2014;
published Early Online March 12, 2014.
Supporting information is available online at http://www.genetics.org/lookup/suppl/
doi:10.1534/genetics.113.160606/-/DC1.
1
Corresponding author: University of Texas Health Science Center at Houston,
Department of Microbiology and Molecular Genetics, 6431 Fannin St., MSB 1.168,
Houston, TX 77030. E-mail: Danielle.A.Garsin@uth.tmc.edu
Genetics, Vol. 197, 515–526 June 2014 515

domains of human Duox1/2 are reported to lack peroxi-
dase activity and may have other functions, such as serving
as an interaction domain for separate, active peroxidases
(Meitzler and Ortiz de Montellano 2009, 2011; Meitzler
et al. 2013).
Caenorhabditis elegans has been used as a model host to
study various aspects of the innate immune response, in-
cluding the purposeful generation of reactive oxygen spe-
cies (ROS) as a defense mechanism (Chavez et al. 2007,
2009). The animal encodes only one functional NADPH
oxidase, a dual oxidase called BLI-3 (Edens et al. 2001).
When exposed to human pathogens such as the Gram-
positive, opportunistic bacterium, Enterococcus faecalis,H
2
O
2
is released (Chavez et al. 2007). The response appears pro-
tective, as its loss by reducing the expression of bli-3 by
RNA interference (RNAi) renders the animals more sensi-
tive to killing by the pathogen (Chavez et al. 2009). Using
indirect immunofluorescence, BLI-3 was localized to the
hypodermis, which is essentially the “skin”of C. elegans
(Edens et al. 2001). There is also speculation that BLI-3
maybepresentintheintestinalcells(Chavezet al. 2007,
2009). In addition to playing a role in innate immunity,
BLI-3 is essential to the normal development of the worm
because it contributes to the generation of the tyrosine-
linked collagen necessary for proper biogenesis of the cu-
ticle, i.e., the exoskeleton of the animal. Its role appears
twofold. First, it generates the necessary substrate H
2
O
2
,
and then it oxidizes the tyrosines of the collagen proteins
that then combine to form di- and trityrosines, thereby
cross-linking the cuticle (Edens et al. 2001). Unlike human
Duox1/2, the peroxidase domain of BLI-3 has low levels of
peroxidase activity that is essential to this process, and
mutations in the peroxidase domain that disrupt this activ-
ity result in a “blistered”(bli) phenotype (Brenner 1974;
Simmer et al. 2003; Meitzler and Ortiz de Montellano
2009, 2010, 2011). However, more recent work has dem-
onstrated that the peroxidase domain of BLI-3 is not the
only peroxidase involved in this process. A separate perox-
idase, MLT-7,alsocontributestocuticlecross-linkingand
loss of this activity results in the same bli phenotype as loss
of the BLI-3 peroxidase domain. Additionally, loss of both
peroxidase activities greatly increases the severity of cuti-
cle blistering (Thein et al. 2009).
Because of the prevalent involvement of peroxidases in
immune responses, we hypothesized that C. elegans might
also utilize a peroxidase(s) in host defense, perhaps in con-
junction with its NADPH oxidase, BLI-3. The results of an
earlier investigation indicated that the peroxidase domain of
BLI-3 is not involved, as point mutants in this domain had
wild-type resistance to E. faecalis, despite their blistered phe-
notype (Chavez et al. 2009). In this study, we characterized
the effects of reducing the expression of putative peroxi-
dases encoded by the C. elegans genome on susceptibility
to E. faecalis. We identified three genes whose loss resulted
in susceptibility. One gene, named skpo-1,forShkT-containing
peroxidase, was investigated in depth.
Materials and Methods
Creation of complementary DNA pL4440 constructs for
double-stranded RNA production in Escherichia coli
A standard RNA isolation procedure was used to procure
total RNA from healthy N2 adults grown on Escherichia coli
OP50. We followed the first-strand complementary DNA
(cDNA) synthesis protocol from the Life Technologies Super-
Script III Synthesis system to produce first-strand total
cDNA. We then followed a standard PCR protocol using
primers specifictoF32A52.A,F32A52.B, and pxn-1, respec-
tively (Supporting Information,Table S5). PCR products
were digested and ligated into pL4440 and transformed into
chemically competent E. coli HT115 to produce double-
stranded RNA (dsRNA) to the gene of interest, respectively.
C. elegans strains and growth conditions
C. elegans strains were grown and maintained as previously
described (Hope 1999). The strains used for the hypodermal
and intestinal RNAi studies are as follows: rde-1(ne219);
Is[wrt-2prom::RDE-1::unc54 39utr;myo2p::RFP3] and sid-
1(qt9); Is[vha-6::sid-1], respectively. The RB1437 [skpo-1
(ok1640) II] strain is a partial deletion mutant for skpo-1
and was verified via sequencing (www.wormbase.org). The
RB1437 [skpo1 (ok1640) II] strain was created by the
C. elegans Reverse Genetics Core Facility at the University of
British Columbia, part of the International C. elegans Gene
Knockout Consortium and obtained through the Caenorhab-
ditis Genetics Center. The independent lines of skpo-1
strains, GF89–GF91, were obtained through backcrossing
the RB1437 [skpo-1 (ok1640) II] strain with the wild-type
N2 Bristol strain five times (backcrossing, BC 35).
RNAi
RNAi was performed by exposing L1–L4 stage larvae to
E. coli HT115-expressing dsRNA to target genes. RNAi clones
were obtained from the C. elegans library (Fraser et al. 2000;
Kamath et al. 2003). skpo-1 worms exposed to pathogens
were prone to a maternal bagging phenotype; therefore, cdc-
25.1 RNAi was used to induce sterility in some experiments
(van der Hoeven et al. 2011).
Survival and longevity assays
Unless otherwise indicated, the following bacterial strains
were used: OP50 (E. coli) (Brenner 1974), OG1RF (E. fae-
calis) (Dunny et al. 1978), and PA14 (P. aeruginosa) (Rahme
et al. 1995). Exposure to RNAi, survival assays, and longev-
ity assays were performed as previously described (Garsin
et al. 2001, 2003; Kim et al. 2002; van der Hoeven et al.
2011). Briefly, for E. faecalis survival assays, E. faecalis
grown in brain heart infusion (BHI) medium for 5 hr was
seeded (10 ml) onto BHI plates (gentamycin 50 mg/ml) and
incubated at 37°for 24 hr, whereas for P. aeruginosa survival
assays, P. aeruginosa was cultured in Luria broth (LB) over-
night at 37°, seeded (10 ml) onto slow-killing plates, and
incubated first for 24 hr at 37°and then for the duration of
516 G. R. Tiller and D. A. Garsin

the experiment at 25°. For the E. coli longevity assays, 203
E. coli was seeded (100 ml) onto nematode growth (NG)
medium plates supplemented with 5-Fluoro-29-deoxyuridine
(25 mg/ml) and streptomycin (25 mg/ml). Seeded NG plates
were incubated at 25°for 24 hr. In all assays, a total of 90 L4
larvae were transferred to three replica plates and the assay
was performed at 25°. Worms were scored as live and dead
at various time points.
Amplex Red assay for H
2
O
2
measurements
The Amplex Red hydrogen peroxide/peroxidase kit (Invi-
trogen Molecular Probes, Eugene, OR) was previously
adapted to C. elegans to measure pathogen-stimulated hy-
drogen peroxide release (Chavez et al. 2007, 2009). The
same protocol was followed with the following modifica-
tions: L4 worms were exposed to a bacterial strain for 12
hr, and the fluorescence of 30 worms per well was measured
after 30 min of incubation with Amplex Red (540/590 nm
excitation and emission, respectively). A total of 80 mM
diphenyleneiodinium chloride (DPI) (TCI, Tokyo) was
added to some wells and allowed to incubate for 15 min
prior to addition of Amplex Red, HRP, and Na
2
PO
4
(pH
7.4), and fluorescence was measured as above.
Indirect immunofluorescence
Immunofluorescence for C. elegans was performed according
to Seydoux and Dunn (1997) on young adult animals. Cus-
tom peptide synthesis, rabbit immunization, and affinity pu-
rification of the SKPO-1 polyclonal antibody was performed
by LifeTein (South Plainfield, NJ). The CRVGRRAFDIENGSR
peptide corresponds to the C terminus of the C. elegans pro-
tein SKPO-1. Adult N2 and GF89 worms were imaged using
an Olympus IX81 automated inverted microscope and Slide-
book (version 5.0) software along with the SKPO-1 poly-
clonal primary antibody and Alexa Fluor 488 goat
antirabbit IgG secondary antibody to localize SKPO-1 in
C. elegans.
Immunogold labeling and transmission
electron microscopy
Approximately 1000 N2 and GF89 animals were raised un-
der standard conditions to the young adult stage. They were
rinsed off of the propagation plates with M9 and spun down
at 2000 rpm for 1 min. The worms were sequentially resus-
pended and pelleted three times in 1 ml M9 (Hope 1999).
After the final wash, they were resuspended in 1 ml 3%
formalin, 0.15% glutaraldehyde in Millonig’s buffer, pH
7.45, and fixed for 3 days at 40°. Then the buffer was drawn
off and the samples were incubated in 0.1% fresh made
sodium borohydride in Millonig’s buffer (room temperature,
RT, for 10 min). This was followed by incubations in Mill-
onig’s buffer (RT, 10 min, repeated twice). The samples
were then dehydrated at room temperature by incubating
in 50% ethanol (10 min, twice) and 70% ethanol, (15 min,
twice). The samples were then permeated with 50% LR
White resin and 50% ethanol for 60 min, followed by an
overnight incubation at room temperature in 50% LR White
resin sealed on a rotator. The samples were then incubated
at room temperature for 4 hr in 70% LR White resin and
then for 5 hr in 100% LR White resin, while on a rotator. The
pellets were then sealed into BEEM capsules and allowed to
polymerize in a 53°oven overnight. Thin sections (120 nm)
of the LR White blocks were cut using a DiATOME diamond
knife and a Leica Ultracut R microtome. Sections were
floated onto 200 mesh nickel grids. The grids were then
floated in 50 mM glycine (RT, 15 min) and then washed
twice by floating in drops of PBS. The samples were blocked
by floating them for 30 min in Aurion Blocking Solution
followed by washes in incubation buffer (PBS with 0.1%
Aurion BSA-c) (5 min, three times). The SKPO-1 antibody
was diluted into the incubation buffer at 1:1000 and the
grids were incubated for 1.5 hr at RT. This was followed
by washes in incubation buffer (10 min, four times) and
then incubation with the secondary antibody (EMS goat
antirabbit IgG with 10 nm gold) diluted 1:20 in incubation
buffer. The samples were washed again in incubation buffer
(10 min, five times) followed by PBS (10 min, three times).
A postfix treatment of 3% glutaraldehyde was applied for
10 min followed by a final wash with distilled water (5 min,
two times). The grids were dried in a 70°oven and then
imaged using a JEOL 1200 transmission electron microscope
at 60 kV and captured with a 2k 32k Gatan CCD camera.
Statistical analysis
Amplex Red, survival, and longevity assays were analyzed
using GraphPad Prism version 5.0 (GraphPad Software, San
Diego). Student’s paired t-test was used to determine the
statistical significance of the Amplex Red data. P-values of
,0.05 were considered to be statistically significant. Statis-
tically significant differences are denoted in the figures with
asterisks. Kaplan–Meier log rank analysis was used to com-
pare survival and longevity curves pairwise and to calculate
the median survival. P-values of ,0.05 were considered to
be statistically significant.
Results
Host resistance to E. faecalis is affected by skpo-1
To investigate if C. elegans possesses any peroxidases in-
volved in host defense, we utilized an RNAi-killing assay
to screen candidate peroxidase genes. The candidate genes
were found using the BLI-3 peroxidase domain as the BLAST
query on www.wormbase.org. Twelve putative peroxidase-
encoding genes were identified, 9 of which were available in
the RNAi library (Fraser et al. 2000; Kamath et al. 2003),
including the previously studied mlt-7 (Thein et al. 2009).
Standard molecular biology techniques were used to gener-
ate the three missing RNAi constructs (Materials and Meth-
ods). Using a background in which RNAi efficiency is
increased (eri-1) (Kennedy et al. 2004), the expression of
these genes was reduced in C. elegans. The animals were
SKPO-1 Protects Against Infection 517

then exposed to E. faecalis and survival was scored over
time. By these means, the genes were screened for possible
roles in innate immune function. The reduced expression of
three genes—F09F3.5,R08F11.7, and F49E12.1—resulted
in a statistically significant susceptibility phenotype (Table
1). We decided to focus on F49E12.1, as it possessed high
homology to human myeloperoxidase (hMPO, 34% se-
quence identity) and a deletion mutant was available
through the Caenorhabditis Genetics Center (CGC). As de-
scribed in more detail below, the predicted protein associ-
ated with this gene contains an N-terminal metridin Shk
toxin (ShkT)-like domain and a C-terminal peroxidase do-
main and was therefore named SKPO-1 for ShkT-containing
peroxidase.
An example of the survival of skpo-1 RNAi eri-1 mutant
animals on E. faecalis compared to vector control (VC) RNAi
eri-1 mutant animals is shown in Figure 1A. skpo-1 RNAi
animals displayed an enhanced susceptibility phenotype rel-
ative to VC RNAi (P= 0.0011). The experiment was re-
peated five times and the data were tabulated in Table S1.
We also examined the lifespan of these animals to see if
skpo-1 RNAi caused a general fitness defect. However, on
E. coli OP50, lifespan of the skpo-1 RNAi animals was not
significantly different when compared to VC RNAi animals
(P= 0.3772) (Figure 1B and Table S2).
To further examine the phenotype resulting from the loss
of skpo-1, we obtained the partial deletion strain, RB1437
from the CGC. The deletion is between bases 827 and 3202
of the 5008 full-length transcript, which ablates a significant
portion of the protein, including the critical amino acid res-
idues necessary for peroxidase activity in classical animal
heme peroxidases (reviewed by Ortiz de Montellano
2008). Strain RB1437 was backcrossed five times into our
wild-type N2 strain and three lines were generated: GF89,
GF90, and GF91. As shown in Figure S1, these animals were
very susceptible to E. faecalis compared to wild-type N2
animals. Because significant differences in susceptibility
were not observed between the strains, we continued our
studies using GF89, which is referred to as skpo-1.
We observed a significant “bagging”phenotype—hatching
of embryos inside the hermaphrodite that had failed to be
expelled—when the skpo-1 mutant animals were exposed to
E. faecalis, unlike the skpo-1 RNAi animals. There was concern
that this was artificially enhancing their sensitivity to E. faecalis,
thus cdc-25.1 RNAi was employed to induce sterility, as done in
previous C. elegans pathogenesis studies (Shapira et al. 2006;
Irazoqui et al. 2008; van der Hoeven et al. 2011) (Figure 1, C–E).
skpo-1 mutant animals grown on cdc-25.1 dsRNA-expressing
E. coli HT115 did not display this maternal bagging phenotype
when they were subsequently exposed to E. faecalis.cdc-25.1
RNAi-exposed skpo-1 mutant worms retained a more pro-
nounced susceptibility phenotype to E. faecalis relative to simi-
larly exposed wild-type animals (P,0.0001) (Figure 1C and
Table S1), suggesting that the pathogen sensitivity is not com-
pletely explained by an increase in internal hatching of progeny.
Regardless of whether skpo-1 mutant worms were sterile,
i.e.,cdc-25.1-treated (Figure 1D and Table S2), or fecund
(Figure S2 and Table S2), they displayed a slight reduction
in lifespan, relative to wild type, when exposed to live E. coli
OP50 (P,0.0001, for both). Because live OP50 has been
Table 1 Susceptibility to E. faecalis following RNAi of putative peroxidase genes
Gene Susceptibility phenotype P-value Median survival (days)
mlt-7 control Wild type 0.3070 6
6
pxn-1 control Wild type 0.9218 4
4
pxn-2 control Wild type 0.8233 6
6
C16C8.2 control Wild type 0.1067 9
9
C46A5.4 control Wild type 0.7206 10
10
F09F3.5 control Susceptible 0.0058 6
7
F32A5.2a control Wild type 0.2499 5
5
F32A5.2b control Wild type 0.4115 6
6
F49E12.1 (skpo-1) control Susceptible 0.0053 5
9
R08F11.7 control Susceptible 0.0004 6
7
K10B4.1 control Wild type 0.7904 8
8
T06D8.10 control Wild type 0.6636 8
8
518 G. R. Tiller and D. A. Garsin

shown to have slightly pathogenic effects (Garigan et al.
2002), we also examined lifespan on heat-killed OP50 and
found that cdc-25.1 RNAi-exposed skpo-1 mutant worms still
displayed a reduction in lifespan (Figure S3 and Table S2).
To determine if the defect in lifespan completely accounted
for the pathogen sensitivity or not, the relative mortality of
skpo-1 mutant worms, compared to wild type, was calcu-
lated (Figure 1F). Relative mortality is the ratio of the lethal
time to kill 50% of the organisms (LT
50
) of pathogen-
infected animals to uninfected animals with the ratio of
wild-type animals normalized to one and has been used in
previous work to analyze susceptibility phenotypes (Tenor
et al. 2004; Chavez et al. 2007). The survival defect for the
skpo-1 mutant animals on E. faecalis was more severe than
on nonpathogenic E. coli compared to wild-type animals
(P= 0.0091) (Figure 1E and Table S3), arguing against
a general fitness defect.
To further examine the extent of the susceptibility-to-
pathogen phenotype and determine if it was pathogen
specific, we used P. aeruginosa instead of E. faecalis in the
killing assay (P= 0.3783) (Figure 1F and Table S4). Inter-
estingly, we observed no significant difference in survival
between cdc-25.1 RNAi skpo-1 mutants and cdc-25.1 RNAi
wild-type animals when exposed to P. aeruginosa. These
results further argue against a general fitness defect and
suggest that the function of SKPO-1 may be beneficial dur-
ing infection with some, but not all pathogens.
To determine if the susceptibility phenotype on E. faecalis
was a result of changes in pathogen burden, we assessed
how many colony-forming units (CFUs) were in the intes-
tines of the infected worms. Wild-type and skpo-1 mutant
animals were raised on E. coli OP50 until L4 and then ex-
posed to E. faecalis for 12 or 36 hr. The infected worms were
washed to remove surface bacteria, homogenized, and then
Figure 1 SKPO-1 contributes to
C. elegans resistance to E. faeca-
lis. (A) Survival of eri-1 mutant
worms on E. faecalis OG1RF fol-
lowing exposure to vector con-
trol (VC) RNAi and skpo-1 RNAi
(P= 0.0011). (B) Longevity of eri-1
mutant worms on E. coli OP50
following exposure to VC RNAi
and skpo-1 RNAi (P= 0.3772). (C)
Survival of wild-type and skpo-1
mutant worms on E. faecalis fol-
lowing exposure to cdc-25.1
RNAi (P,0.0001). (D) Longevity
of wild-type and skpo-1 mutant
worms on E. coli OP50 following
exposure to cdc-25.1 RNAi (P,
0.0001). (E) The relative mortal-
ity of skpo-1 mutant worms ex-
posed to E. faecalis (EF) is
expressedasaratioof(LT
50
of
wild-type cdc-25.1 RNAi on EF/
LT
50
of skpo-1 mutant cdc-25.1
RNAi on EF) over (LT
50
of wild-
type cdc-25.1 RNAi on E. coli/
LT
50
of skpo-1 mutant cdc-25.1
RNAi on E. coli). The average of
four independent experiments,
with 90 animals each, was used
to calculate the relative mortality. (F)
Survival of wild-type and skpo-1
mutant worms on P. aeruginosa
following exposure to cdc-25.1
RNAi (P= 0.3783). Error bars rep-
resent standard error of the mean
(SEM) and the asterisk indicates
asignificant difference between
wild-type and skpo-1 mutant
worms (P=0.0091). The P-values
were calculated using Student’s
paired t-test. The median survival
for survival and longevity assays
are listed in Table S1 and Table
S2 along with replicates of the
experiments.
SKPO-1 Protects Against Infection 519

serial dilutions of the homogenates were plated. Interest-
ingly, we observed no significant difference in CFUs per
worm between the wild-type and skpo-1 mutant animals
at either time point (Figure S4). The result suggests that
the susceptibility phenotype of the skpo-1 mutant cannot
be explained by an increased load of bacteria in the
intestine.
To test for alterations in the immune response between
the skpo-1 and wild-type animals a small panel of genes
known to be upregulated in response to E. faecalis were
examined, clec-35,-42,-60, and -71. These genes encode
C-type lectins (clec), many of which are upregulated in re-
sponse to pathogens (Irazoqui et al. 2010; Engelmann et al.
2011). Sterile (by exposure to cdc-25 RNAi) wild-type and
skpo-1 mutant animals at the L4 stage were exposed to E.
faecalis or E. coli for 18 hr, at which point the animals were
lysed and RNA was extracted. By qRT-PCR, no significant
difference in the expression of clec-35 and -71 was observed
between the wild type and skpo-1 mutants exposed to E.
faecalis. However, we observed clec-60’s expression was sig-
nificantly higher in the skpo-1 mutant and there was a trend
toward increased expression of clec-42, which was not sta-
tistically significant (Figure S5). These data suggest that the
immune response is altered in the skpo-1 mutant.
Morphological characterization of skpo-1
mutant animals
Animals deficient in the peroxidase mlt-7 displayed altered
morphology, including a bli (blistered) phenotype, indicative
of incomplete cross-linking of the cuticle (Thein et al. 2009).
The phenotype strongly suggests that MLT-7’s peroxidase
activity contributes to cuticle formation (Thein et al.
2009). Interestingly, despite this rather dramatic cuticle de-
fect, an increase in susceptibility to E. faecalis was not ob-
served (Table 1). To determine if SKPO-1 is also involved in
cuticle biogenesis, we observed the morphology of skpo-1
RNAi and mutant animals. RNAi of skpo-1 did not result in
any visible morphological change in the eri-1 mutant ani-
mals (Figure 2B). However, the skpo-1 mutant did display
some morphological phenotypes. We observed young adult
animals under the dissecting microscope (N= 300) and
found that they ranged from very dumpy (16.5%), to
slightly dumpy (32.9%), to wild type (50.6%) in appearance
(Figure 2, C–E). No blistering of the cuticle was ever ob-
served. Because a dumpy phenotype can be associated with
cuticle defects (Page and Johnstone 2007), these results
suggest that SKPO-1 may have some role in cuticle biogene-
sis, but one that is different than MLT-7. Additionally, we
observed no significant difference in susceptibility between
skpo-1 mutant morphotypes, i.e., all were equally hypersuscep-
tible to E. faecalis relative to wild-type animals (Figure S1).
SKPO-1 features and activity
As mentioned, SKPO-1 contains a ShkT-like domain at its
N terminus from residues 21–56 (www.wormbase.org)in
addition to the predicted peroxidase domain (Figure 3A).
It shares this feature with MLT-7 (Thein et al. 2009), and
putative peroxidases C16C8.2 and F32A5.2 (Table 1). For this
reason we propose naming F49E12.1,SKPO-1, and C16C8.2
and F32A5.2,SKPO-2,andSKPO-3 for ShkT-containing
peroxidase. In addition to the ShkT-like domain, the very 59
end contains a predicted signal sequence for secretion, and
cleavage is predicted to occur at Ser19 (www.predisi.de).
To examine SKPO-1’s peroxidase domain, we aligned it
with other well-characterized peroxidase domains of inter-
est, those contained in hMPO, BLI-3, and MLT-7. Upon
alignment, we observed SKPO-1 possesses the catalytic triad
(distal histidine, H
222
; arginine, R
332
; and proximal histi-
dine, H
428
) necessary for peroxidase activity (reviewed by
Ortiz de Montellano 2008). However, SKPO-1 lacks the
Figure 2 skpo-1 mutant displays multiple morphotypes.
(A) Wild-type young adult representing the approximate
average size and morphology of a typical C. elegans. (B)
skpo-1 RNAi young adult raised on skpo-1 dsRNA-expressing
E. coli HT115 from L1–L4 stages. (C–E) Young adult skpo-1
mutants ranged from very dumpy to wild type in size. The
310 microscopy images are representative of .100 wild-
type, skpo-1 RNAi, and skpo-1 worms observed, respectively.
520 G. R. Tiller and D. A. Garsin

residues necessary for covalent heme binding, characteristic
of the animal heme peroxidase family (reviewed by Ortiz de
Montellano 2008). Interestingly, human Duox1/2 (hDuox1/
2) also lack the conserved aspartic and glutamic residues
required for covalent linkage of the heme prosthetic group;
however, both of these recombinant peroxidase domains still
bind heme, albeit weakly (Meitzler and Ortiz de Montellano
2009, 2011). This noncovalent binding may explain MLT-7’s
peroxidase activity even though it lacks these covalent
heme-binding residues (Thein et al. 2009).
By comparison, SKPO-1 possesses all of the highlighted
residues in MLT-7 (Figure 3A) and shares significant identity
with hMPO (34% identity). Thus, we decided to indirectly
assay SKPO-1’s potential peroxidase activity using an
Amplex Red assay, modified for whole animals, that detects
H
2
O
2
(Chavez et al. 2007, 2009). Previously, we observed
that exposure to E. faecalis causes a significant release in
H
2
O
2
dependent on the NADPH oxidase, BLI-3 (Chavez
et al. 2007, 2009). We reasoned that loss of an important
peroxidase during this release might increase the amount of
H
2
O
2
detected. After infecting VC and skpo-1 RNAi eri-1
mutant animals with E. faecalis for 12 hr, released H
2
O
2
was measured (P= 0.0091) (Figure 3B). skpo-1 RNAi eri-1
mutant animals released significantly greater amounts of
H
2
O
2
, relative to VC RNAi eri-1 mutants, consistent with
the loss of a predicted H
2
O
2
sink. The same result was ob-
served with the skpo-1 mutants compared to wild-type ani-
mals (P,0.0001) (Figure 3C). The difference required E.
faecalis, and the concomitant release of H
2
O
2
, as no signifi-
cant differences were observed between wild-type and skpo-
1-deficient animals on E. coli (Figure 3, B and C).
To investigate if the increased amount of H
2
O
2
produced
by the skpo-1 mutant was dependent on BLI-3 activity, we
added the NADPH oxidase inhibitor, DPI, to the assay. We
previously demonstrated that BLI-3 is the only expressed
NADPH oxidase encoded by the genome (Chavez et al.
2009), and DPI can be utilized to reduce its activity in
wild-type animals (Chavez et al. 2007). In Figure 3C, DPI
also abrogated the enhanced H
2
O
2
generation observed in
skpo-1 mutant animals. Taken together, Figure 3 supports
the hypothesis that SKPO-1 is a peroxidase that utilizes
H
2
O
2
produced by BLI-3 during infection. Unfortunately,
attempts to purify the protein to demonstrate definitive per-
oxidase activity in vitro were not successful.
SKPO-1 localizes to the hypodermis
C. elegans interfaces with E. faecalis at the cuticle, synthe-
sized by the underlying hypodermis, as it crawls through the
pathogen lawn, and in its intestine, due to ingestion of the
bacteria (Garsin et al. 2001). It has previously been demon-
strated that C. elegans mounts immune responses at these
host–pathogen boundaries, depending on the pathogen and
the nature of the infection (Wong et al. 2007; Pujol et al.
2008; Irazoqui et al. 2010). To address what tissue SKPO-1’s
activity is required for normal levels of resistance to E. fae-
calis, a tissue-specific RNAi approach was employed. RNAi-
defective strains of C. elegans were used in which RNAi
activity was genetically restored to specific tissues through
intestinal or hypodermal-specific promoters (vha-6::SID-1
and wrt-2::RDE-1, respectively) (Melo and Ruvkun 2012).
In Figure 4, A and C (and Table S1), we tested the suscep-
tibility of the hypodermal and intestinal-specificRNAi
Figure 3 Evidence that skpo-1 is a potential peroxidase.
(A) Peroxidase domain sequences were aligned against
the putative peroxidase domain of SKPO-1. SKPO-1 pos-
sesses the distal histidine (H
222
), catalytic arginine (R
332
),
and proximal histidine (H
428
), which are necessary for
peroxidase activity. However, SKPO-1 lacks covalent
heme-binding residues (S
221
and L
335
) that are character-
istic of mammalian peroxidases (Ortiz de Montellano
2008). (B) eri-1 mutant worms were grown on VC RNAi
or skpo-1 RNAi prior to exposure with either E. coli or E.
faecalis for 12 hr at 25°. (C) Wild-type and skpo-1 mutant
worms were grown on cdc-25.1 RNAi prior to exposure
with either E. coli or E. faecalis for 12 hr at 25°. (B and C)
Following exposure to E. coli or E. faecalis, the amount of
H
2
O
2
produced per minute was determined using the
Amplex Red assay. Error bars represent the SEM and the
asterisks indicate significant differences between eri-1
worms exposed to VC RNAi or skpo-1 RNAi that were
infected with E. faecalis as well as between wild-type and
skpo-1 mutant worms exposed to cdc-25.1 RNAi prior to
infection with E. faecalis [P=0.0091 (B) and P,0.0001
(C)]. Additionally, wild-type and skpo-1 mutant worms were
exposedto80mM diphenyleneiodinium chloride (DPI) and
H
2
O
2
levels were calculated for both E. coli-andE. faecalis-
exposed animals (EC, P= 0.0752; EF, P= 0.4161, respec-
tively) P-values were calculated via Student’spairedt-test.
Data in B and C are representative of at least two indepen-
dent replicates.
SKPO-1 Protects Against Infection 521

strains to E. faecalis.AsinFigure1,thesestrainswere
exposed to VC and skpo-1 RNAi prior to infection. In the
hypodermal RNAi strain, we observed an enhanced suscep-
tibility phenotype to E. faecalis in the skpo-1 RNAi animals
relative to VC RNAi (P= 0.0002); however, in the intesti-
nalRNAistrain,nosignificant difference between skpo-1
andVCRNAianimalswasobservedwhenonE. faecalis
(P= 0.9435). On E. coli,nosignificant difference was
observed between VC and skpo-1 RNAi for either the hypo-
dermal or intestinal RNAi line (P= 0.9997 and P=0.6379;
Figure 4, B and D, respectively, and Table S2). From these
experiments we conclude that SKPO-1’s functional activity
during pathogen exposure is required in the C. elegans
hypodermis.
To determine in which tissue SKPO-1 is produced, we
used indirect immunofluorescence to visualize SKPO-1 local-
ization in young adult animals raised under standard con-
ditions. Using rabbits, a polyclonal primary antibody was
raised against a chemically synthesized SKPO-1 peptide
(see Materials and Methods). A freeze-cracking methodology
was used to disrupt the cuticle of the animal and allow for
internal fixation and staining (Seydoux and Dunn 1997).
Following fixation, the samples were double stained with
the polyclonal primary antibody to SKPO-1 and an Alexa
Fluor 488 secondary antibody. Localization was clearly ob-
served in the hypodermis of wild-type animals, and an ex-
ample is shown in Figure 5, A–C and D–F (40 times and 10
times, respectively). Note the strong fluorescent staining just
under the outermost layer of the animal. No internal organs
showed evidence of staining, including the gonad and the
intestine, which in this animal had become partially sepa-
rated from the rest of the body. In contrast to wild type,
skpo-1 mutant animals showed no evidence of staining in
any organ (Figure 5, G–I). We also examined wild-type and
skpo-1 mutant animals that had been exposed to E. faecalis
for 24 hr prior to staining and did not observe any differ-
ences in the localization of SKPO-1 (data not shown).
By using a secondary antibody conjugated to 10 nm gold
particles for immunogold labeling, we examined localization
at higher resolution by transmission electron microscopy
(TEM). As seen in Figure 5J, the black dots, indicating the
gold particles, were located just under the cuticle layer in
association with the hypodermal cells of wild-type animals.
Hardly any particles were observed in the skpo-1 mutant
animals (Figure 5K). The few observed were randomly scat-
tered. In total, these results demonstrate SKPO-1 is both
physically present and functionally active in the C. elegans
hypodermis.
Discussion
In this work, we demonstrated that a previously unstudied
C. elegans protein with a peroxidase domain, F49E12.1,
plays a protective role during infection with E. faecalis.We
named this protein SKPO-1 for ShkT-containing peroxidase,
because it contains an N-terminal ShkT-like domain. The
ShkT domain family was originally defined as a potassium
channel blocker in the sea anemone (Metridium senile).
Figure 4 SKPO-1 is necessary in
the hypodermis for resistance to
E. faecalis infection. (A) Survival
of the hypodermal RNAi strain
on E. faecalis following exposure
to VC RNAi or skpo-1 RNAi (P=
0.0002). (B) Longevity assay on
E. coli OP50 of the hypodermal
RNAi strain following exposure
to VC RNAi or skpo-1 RNAi (P=
0.9997). (C) Survival of the intes-
tinal RNAi strain on E. faecalis fol-
lowing exposure to VC RNAi or
skpo-1 RNAi (P= 0.9435). (D)
Longevity assay of the intestinal
RNAi strain on E. coli OP50 fol-
lowing exposure to VC RNAi or
skpo-1 RNAi (P= 0.6379).
522 G. R. Tiller and D. A. Garsin

Binding to the potassium channel requires two conserved
residues that are not found in this particular ShkT-like do-
main of SKPO-1 or others from C. elegans (data not shown).
It is postulated that the more general function of ShkT-like
domains are as contact surfaces for protein interactions
(Tsang et al. 2007). For this reason, SKPO-1 and other ShkT-
containing peroxidases may be most closely related to the
peroxidasins, subfamily 2 of the peroxidase-cyclooxygenase
superfamily, and in fact, SKPO-1 was placed in this family
by phylogenetic analysis (Soudi et al. 2012). These peroxi-
dase domain-containing proteins also have protein interaction
domains, but they are typically type C-like immunoglobulin
domains, leucine-rich repeats, orvonWillebrandfactorCmod-
ules. This is in contrast to the mammalian peroxidases, MPO,
LPO, and eosinophil peroxidase that lack these extra domains
and belong to subfamily 1 (Zamocky et al. 2008). Additionally,
it is thought that subfamily 1 evolved from subfamily 2
(Zamocky et al. 2008). Other C. elegans peroxidase do-
main-containing proteins that have a ShkT-like domain in-
clude MLT-7 (Thein et al. 2009), C16C8.2,andF32A5.2,
but no study has yet addressed this domain’s function in
the context of a peroxidase.
An unexpected finding was the discovery that SKPO-1 is
located in the hypodermis and is functionally protective in
this tissue against E. faecalis infection. Infection of C. elegans
with E. faecalis results in colonization of the gut, leading to
distension of the intestinal lumen and clear signs of physical
damage, such as effacement of the microvilli (Garsin et al.
2001; Cruz et al. 2013). Though the worm is exposed to E.
faecalis on its outer surface as it moves through the patho-
gen lawn, there is no notable colonization or characterized
physical damage to the cuticle or hypodermis. In contrast,
Figure 5 SKPO-1 localizes to the
C. elegans hypodermis. (A–F and
J) Wild-type and (G–I and K)
skpo-1 mutant worms were immu-
nostained with anti-SKPO-1 poly-
clonal antibodies and imaged
using fluorescence (A–I) or trans-
mission electron microscopy (J and
K), respectively. (A–C) The 340
magnification of a wild-type worm
shows hypodermal SKPO-1 lo-
calization. (D–F) The 310 mag-
nification of a wild-type worm.
(G–I) The 310 magnification of
askpo-1 worm shows loss of
SKPO-1 staining. (J) In wild-type
worms, the black dots, indica-
tive of immunogold labeling,
are localized beneath the cuticle
layer, but external to the outer
hypodermal cell surface. (K) In
skpo-1 mutant worms, very few
black dots are observed. White
arrowhead, apical hypodermal
surface; black arrowhead, 10 nm
gold-labeled secondary to SKPO-1).
Microscopy images are representa-
tive of .100 (fluorescent) or .10
(TEM) wild-type and skpo-1 mu-
tant worms observed.
SKPO-1 Protects Against Infection 523

bacterial pathogens such as Microbacterium nematophilum,
Xenorhabdus nematophila, and Yersinia pestis adhere to and
colonize the cuticle surface (Hodgkin et al. 2000; Couillault
and Ewbank 2002; Darby et al. 2002). The natural fungal
pathogen Drechmeria coniospora initially adheres to the cu-
ticle and then penetrates the hypodermis, whereas the hu-
man fungal pathogen Candida albicans first colonizes the
intestine and eventually penetrates the cuticle from the in-
side out (Jansson et al. 1985; Breger et al. 2007). We pre-
viously showed that tissue-specific loss of bli-3 in the
hypodermis also increased susceptibility of C. elegans to E.
faecalis (Chavez et al. 2009). Based on the protective effects
of SKPO-1 and BLI-3 in this tissue, we postulate that a hypo-
dermal immune response does play some role in protecting
C. elegans during infection with E. faecalis. The question is by
what mechanism?
Several models for how these proteins might exert their
protective effects can be imagined. It could be that loss of
skpo-1 results in a weaker cuticle barrier that increases sus-
ceptibility. Alternatively, SKPO-1 could use H
2
O
2
produced
by BLI-3 to form more potent antioxidants, analogous to the
human Duox/LPO system on mucosal surfaces (Conner et al.
2002; Geiszt et al. 2003; Forteza et al. 2005). Or the H
2
O
2
produced by BLI-3 could act as a signaling molecule in either
an autonomous or noncell-autonomous fashion. Unlike
many signaling molecules, H
2
O
2
is highly diffusible, and it
could directly move into other tissues, such as the intestine,
to signal responses. Interestingly, in zebrafish, H
2
O
2
pro-
duced by a Duox in response to wounding acts as an attrac-
tant in a diffusion gradient to draw leukocytes to the area of
damage (Niethammer et al. 2009). Clearly, the amount of
H
2
O
2
is important. We previously showed that loss of BLI-3
and the resulting decrease in H
2
O
2
production caused an
increase in susceptibility to infection (Chavez et al. 2009),
whereas in this work we show that loss of a peroxidase and
a concurrent increase in H
2
O
2
also increases susceptibility. A
similar situation is apparent from studies of infection using
the model host Drosophila melanogaster (Ha et al. 2005a,b).
Loss of an intestinal DUOX enzyme or an intestinal catalase
both increase susceptibility to infection, but with opposite
effects on ROS levels in this tissue. It is possible that SKPO-1
is catalyzing the degradation of excess H
2
O
2
to prevent host
damage. We formerly demonstrated, by a variety of means,
that infection causes oxidative stress in C. elegans (Chavez
et al. 2007; Mohri-Shiomi and Garsin 2008; van der Hoeven
et al. 2011), much of it dependent on BLI-3 activity (van der
Hoeven et al. 2011).
Interestingly, there is evidence for a hypodermal response
to several pathogens that are thought to mainly cause
infection in the intestine. Microarray studies that examined
the transcriptional responses of C. elegans to E. faecalis,Ser-
ratia marcescens,Erwinia carotovora,Photorhabdus lumines-
cens,S. aureus, and P. aeruginosa all noted a dramatic
downregulation in the expression of genes related to cuticle
biosynthesis, such as those encoding collagens (Wong et al.
2007; Irazoqui et al. 2010). The response is not thought to
be due to a general reduction of gene transcription in this
tissue and may be indicative of several possibilities (Wong
et al. 2007). The changes in transcription of the genes could
be part of a protective response that is occurring in the
hypodermis to protect against pathogens. Or the changes
could be purposely caused by the pathogens as part of their
virulence programs to damage the host. Alternatively, the
changes in expression of the cuticle biosynthetic genes could
be a neutral side effect resulting from alterations in signal-
ing due to pathogen exposure. Another microarray study
observed an increase in the expression of a large number
of genes related to cuticle biosynthesis in an arr-1 mutant
that was found to have altered susceptibility to pathogens
(Singh and Aballay 2012). arr-1 encodes for arrestin-1,
a G-protein coupled receptor (GPCR) adaptor protein that
is necessary for GPCR signaling in several neurons of
C. elegans. Whether these changes in resistance had anything
to do with alterations in the amount of cuticle biosynthetic
proteins being produced was not investigated (Singh and
Aballay 2012). Overall, these studies suggest that pathogen
exposure, even to those pathogens not thought to directly
affect the cuticle, cause major changes in the expression of
the cuticle biosynthetic genes that might be indicative of a re-
sponse to the infection in the hypodermis.
In conclusion, we uncovered putative peroxidases that
affected susceptibility to E. faecalis in C. elegans. We focused
our characterization on F49E12.1, named SKPO-1 because it
contains a ShkT-like domain and a peroxidase domain. In
addition to the pathogen susceptibility phenotype, loss of
skpo-1 resulted in a dumpy phenotype of uneven pene-
trance, suggestive of some role in cuticle biosynthesis (Page
and Johnstone 2007). In support of a functional peroxidase
domain, we noted that SKPO-1 shares the same critical res-
idues as MLT-7 in its active site (Thein et al. 2009), and
animals mutant for skpo-1 produce significantly more
H
2
O
2
during infection. The functional and physical location
for the protein was shown to be the hypodermis, which was
surprising since E. faecalis has been characterized as infect-
ing the intestine of C. elegans (Garsin et al. 2001; Cruz et al.
2013). This result, along with other evidence from the liter-
ature (Wong et al. 2007; Irazoqui et al. 2010), suggests that
the hypodermis plays an important role during exposure of
C. elegans to many human pathogens that do not obviously
colonize or damage the cuticle, warranting further
investigation.
Acknowledgments
For the TEM imaging, we thank S. Kolodziej and P. Navarro
(Department of Pathology and Laboratory Medicine EM
Laboratory, University of Texas Health Science Center at
Houston). We thank G. Ruvkun for the tissue-specific RNAi
C. elegans strains and K. McCallum, S. Arur, K. Park, T. Furata,
T. Leto, A. Page, M. Cruz, and W. Nauseef for helpful input on
this project. We acknowledge the Caenorhabditis Genetics
Center for providing us with many of the C. elegans strains
524 G. R. Tiller and D. A. Garsin

used in the study, J. Ahringer and Geneservice for the bacte-
rial strains for RNAi, and WormBase for their help and advice
in naming genes. This work was supported by Public Health
Service grants R01AI076406 to D.A.G. and T32AI55449 to
G.R.T. from the National Institute of Allergy and Infectious
Diseases.
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Communicating editor: B. Lazzaro
526 G. R. Tiller and D. A. Garsin

GENETICS
Supporting Information
http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.160606/-/DC1
The SKPO-1 Peroxidase Functions in the Hypodermis
to Protect Caenorhabditis elegans From
Bacterial Infection
George R. Tiller and Danielle A. Garsin
Copyright © 2014 by the Genetics Society of America
DOI: 10.1534/genetics.113.160606

G.R.TillerandD.A.Garsin
 
2SI
FileS1
SUPPLEMENTARYMATERIALSANDMETHODS
RNAisolationandqRT‐PCR
PriortoRNAisolation,wildtypeandskpo‐1mutantanimalsweregrownoncdc‐25.1dsRNAexpressingE.coliHT115
fromtheeggtoL4larvalstage.L4animalswerethenexposedtoeithercdc‐25.1RNAiorE.faecalisOG1RFfor18hours.The
RNAwasisolatedusingTrizol(Invitrogen)accordingtothemanufacturer.RNAsamplesweretreatedwithDNaseItoeliminate
contaminatingDNAbytheTurboDNAfreekit(AppliedBiosystem)accordingtothemanufacturer.qRT‐PCRwasperformedas
described(VANDERHOEVENetal.2011).PrimersusedarelistedinTableS6(act‐1servedasthereferencegene).
BacterialColonization
TheCFUanalysiswasconductedinamannersimilartopastwork(GARSINetal.2001).Briefly,L4wildtypeandskpo‐1
mutantanimalsgrownonE.coliOP50wereexposedon100mmplatescontainingBHI‐agarwithgentamycin(10g/mL)seeded
with100lofE.faecalisOG1RFforeither12or36hoursat25˚C.Wormswerewashed3timeswithM9bufferat1.4rpm.
Wormswerethenwashedtwicewith25mMtetramisolehydrochloridetopreventingestionoftheantibiotictreatment.
Wormswereincubatedatroomtemperaturefor60minutesin25mMtetramisolehydrochloridesupplementedwithampicillin
andkanamycin,bothat1mg/mL,tokillsurface‐attachedE.faecalis.Wormswerecollectedat1.4rpmandwashedtwicewith
25mMtetramisolehydrochloridepriortogrinding.Tenwormsin10lweretransferredto200lofM9andgroundfor1
minuteusingamotorizedpestle(Kontescordlesscat#K749540‐0000andpestlescat#K749521‐1590).Serialdilutionswere
performedand100lofeachdilutionplatedonto100mmBHIgentamycin10g/mLplatesfor24hoursat37˚C.




G.R.TillerandD.A.Garsin
 
3SI

FigureS1Survivalofcdc‐25.1RNAi‐treatedskpo‐1animallinesonE.faecalis.Wildtypeandskpo‐1wormsweregrownoncdc‐
25.1dsRNAexpressingE.coliHT115fromL1throughL4larvalstagepriortoexposuretoE.faecalisOG1RF.Theskpo‐1worms
(GF89,GF90andGF91)weremoresusceptibletoE.faecalisrelativetowildtype(P‐values<0.0001,0.0110,and<0.0001,
respectively).



G.R.TillerandD.A.Garsin
 
4SI
FigureS2Lifespandefectofskpo‐1mutantsonE.coliOP50withoutpriorcdc‐25.1exposure.Wildtypeandskpo‐1worms
weregrownonE.coliOP50fromL1throughL4larvalstagepriortothelongevityassayinwhichtheyweretransferredtoNGM
plateswithFUDR(100µg/ml)seededwithconcentrated(20X)E.coliOP50.Theaveragelifespanoftheskpo‐1wormswas
significantlyshorterthantheirwildtype,counterparts(P‐value<0.0001).


G.R.TillerandD.A.Garsin
 
5SI
FigureS3skpo‐1mutantsdisplayalifespandefectonheat‐killedE.coliOP50.Wildtypeandskpo‐1wormsweregrownoncdc‐
25.1dsRNAexpressingE.coliHT115fromL1throughL4larvalstagepriortothelongevityassay.Thewormsweretransferredto
NGMplateslikeinFigureS2;however,theE.coliOP50hadbeenheat‐killed.Weobservedthattheskpo‐1wormsdisplayeda
longevitydefectonheat‐killedOP50,relativetowildtypeworms(P‐value<0.0001).

G.R.TillerandD.A.Garsin
 
6SI
FigureS4Theskpo‐1mutantdoesnothaveincreasedintestinalbacterialloadduringE.faecalisinfection.L4wildtypeand
skpo‐1mutantanimalswereexposedtoE.faecalisat25˚Cfor12or36hours,respectively.CFUvaluesforbothwildtypeand
skpo‐1animalsindicatedthatthereisnosignificantdifferenceinintestinalcolonizationbyE.faecalis.Threebiologicalreplicates
foreachstrainwereusedpertimepointandtheexperimentrepeatedtwice—i.eatotaln=60perstrain.P‐valueswere0.3913
and0.3592for12and36hours,respectively.

G.R.TillerandD.A.Garsin
 
7SI
FigureS5clec‐60expressioniselevatedinskpo‐1mutantanimals,relativetowildtype,inresponsetoE.faecalisinfection.L4
wildtypeandskpo‐1mutantanimalswereexposedtoE.faecalisfor18hoursat25˚C.Threetechnicalreplicatesperstrainper
genewereassayedforrelativeexpressionofinnateimmuneresponsegenesandtheexperimentwasrepeated,independently,
twice.ThesegeneswerepreviouslyfoundtodisplayincreasedexpressioninwildtypeanimalsinresponsetoE.faecalis
(ENGELMANNetal.2011).Ofthegenessurveyed,clec‐60wastheonlygenetodisplayasignificantincreaseinexpressionduring
infection,relativetoact‐1.P‐valueswere0.1912,0.1768,0.04432,and0.4684forclec‐35,42,60and71,respectively.


G.R.TillerandD.A.Garsin
 
8SI
TableS1MediansurvivalandP‐valuesofE.faecalisOG1RFkillingassays
FigurenumberExperimentnumberStrain,exposureconditionsduring
development
Median
survival(days)
P
‐value
1A1wildtype,VCRNAi
wildtype,skpo‐1RNAi
8
7
C
=0.0404
2wildtype,VCRNAi
wildtype,skpo‐1RNAi
7
6
C
=0.0331
3er
i
‐1,VCRNAi
eri‐1,skpo‐1RNAi
8
7
C
=0.001
4er
i
‐1,VCRNAi
eri‐1,skpo‐1RNAi
7
5
C
=0.0053
5er
i
‐1,VCRNAi
eri‐1,skpo‐1RNAi
9
8
C
=0.0037
1C1wildtype,cdc‐25.1 RNAi
skpo‐1,cdc‐25.1RNAi
10
6
C
<0.0001
2wildtype,cdc‐25.1 RNAi
skpo‐1,cdc‐25.1RNAi
10
8
C
<0.0001
3wild type,cdc‐25.1 RNAi
skpo‐1,cdc‐25.1RNAi
13
8
C
=0.0016
4wildtype,cdc‐25.1 RNAi
skpo‐1,cdc‐25.1RNAi
13
8
C
=0.0005
5wildtype,cdc‐25.1 RNAi
skpo‐1,cdc‐25.1RNAi
13
7
C
<0.0001
4A1wrt‐2,VCRNAi
wrt‐2,skpo‐1RNAi
5
4
C
=0.0002
2wrt‐2,VCRNAi
wrt‐2,skpo‐1RNAi
4
3
C
=0.0176
3wrt‐2,VCRNAi
wrt‐2,skpo‐1RNAi
5
4
C
=0.0376
4wrt‐2,VCRNAi
wrt‐2,skpo‐1RNAi
4
4
C
=0.0058
4C1vha‐6,VCRNAi
vha‐6,skpo‐1RNAi
11
11
C
=0.6379
2vha‐6,VCRNAi
vha‐6,skpo‐1RNAi
11
11
C
=0.735
S11wildtype,OP50
skpo‐1*,OP50
8
3
C
<0.0001
2wildtype,OP50
skpo‐1*,OP50
8
3
C
<0.0001
C=control,*DataonlyshownforGF89skpo‐1strain.

G.R.TillerandD.A.Garsin
 
9SI


TableS2MediansurvivalandP‐valuesofE.coliOP50longevityassays
FigurenumberExperiment
number
Strain,exposureconditionsduring
development
Mediansurvival
(Days)
P
‐value
1B1er
i
‐1,VCRNAi
eri‐1,skpo‐1RNAi
10
10
C
=0.3772
2er
i
‐1,VCRNAi
eri‐1,skpo‐1RNAi
10
10
C
=0.8948
1D1wildtype,cdc‐25.1 RNAi
skpo‐1,cdc‐25.1RNAi
10
8
C
<0.0001
2wildtype,cdc‐25.1 RNAi
skpo‐1,cdc‐25.1RNAi
14
11
C
<0.0001
3wildtype,cdc‐25.1 RNAi
skpo‐1,cdc‐25.1RNAi
14
11
C
<0.0001
4B1vha‐6,VCRNAi
vha‐6,skpo‐1RNAi
10
10
C
=0.9997
2vha‐6,VCRNAi
vha‐6,skpo‐1RNAi
11
10
C
=0.4484
4D1wrt‐2,VCRNAi
wrt‐2,skpo‐1RNAi
11
11
C
=0.6379
2wrt‐2,VCRNAi
wrt‐2,skpo‐1RNAi
11
11
C
=0.7235
S21wildtype,OP50
skpo‐1,OP50
14
11
C
<0.0001
2wildtype,OP50
skpo‐1,OP50
14
11
C
<0.0001
S3*1N2,cdc‐25.1 RNAi*
skpo‐1,cdc‐25.1RNAi*
16
13
C
<0.0001
2N2,cdc‐25.1 RNAi*
skpo‐1,cdc‐25.1RNAi*
14
12
C
<0.0001
C=control,*Assayedonheat‐killedOP50










G.R.TillerandD.A.Garsin
 
10SI

TableS3DataforRelativeMortalityCalculation
FigurenumberExperiment
number
Strain Relativemortality
P
‐value
1FAverageof
experiments1‐4
N2




skpo‐1
1
1
1
1
Avg=1
1.312
1.452
1.654
1.253
Avg=1.418



C




=0.0091
C=control








G.R.TillerandD.A.Garsin
 
11SI
TableS4MediansurvivalandP‐valuesofP.aeruginosaPA14killingassays
FigureNumberExperiment
number
Strain,exposureconditions
duringdevelopment
Mediansurvival
(Hours)
P
‐value
1E1N2,cdc‐25.1 RNAi
skpo‐1,cdc‐25.1RNAi
91
88
C
=0.3783
2N2,cdc‐25.1 RNAi
skpo‐1,cdc‐25.1RNAi
89
69
C
=0.0639
3N2,cdc‐25.1 RNAi
skpo‐1,cdc‐25.1RNAi
72
72
C
=0.2613
































G.R.TillerandD.A.Garsin
 
12SI
TableS5PrimersforcDNAinsertsligatedintopL4440constructfordsRNAproductioninE.coli
OligonameSequence
F32A52.A5F5’TTGCGGCCGCAATTCACCAGGGAATTTACACC3’
F32A52.A3R5’CCTCGAGGGTGGTCTTTTCACATTCTGG3’
F32A52.B5F5’TTGCGGCCGCAACAACCACTCATTTCACCAGG3’
F32A52.B3R5’CCTCGAGGCTGTAGTTGTTACTCTTTGTGG3’
p
xn‐15F5’TTGCGGCCGCAAGGACTCTGGAAGGTACAC3’
p
xn‐13R5’CCTCGAGGATACTTGGCACTTCCACTCT3’
































G.R.TillerandD.A.Garsin
 
13SI
TableS6qRT‐PCRprimersforassessingexpressionofinnateimmuneresponsegenesinresponsetoE.faecalis
OligonameSequence
clec‐355F5’AGATGCTGGACAGTGGAAAAG3’
clec‐353R5’GTGCGGAGTATTGTAGCGTAG3’
clec‐425F5’GTAACTCCGTATTGGCTGGG3’
clec‐423R5’GTAAACGCAGCTTCCAATCTC3’
clec‐605F5’TGTAAGAGAACAGTTGGAACCC3’
clec‐603R5’TATGTGCATGGGTACTGATCG3’
clec‐715F5’ACGACAGGAAGTGATGTATTGG3’
clec‐713R5’TTGACGGACTTTAGCCACTG3’
act‐15F5’ACCATGTACCCAGGAATTGC3’
act‐13R5’TGGAAGGTGGAGAGGGAAG3’




G.R.TillerandD.A.Garsin
 
14SI
SUPPLEMENTARYLITERATURECITED

Engelmann,I.,A.Griffon,L.Tichit,F.Montanana‐Sanchis,G.Wangetal.,2011Acomprehensiveanalysisofgeneexpression
changesprovokedbybacterialandfungalinfectioninC.elegans.PLoSOne6:e19055.
Garsin,D.A.,C.D.Sifri,E.Mylonakis,X.Qin,K.V.Singhetal.,2001AsimplemodelhostforidentifyingGram‐positivevirulence
factors.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica98:10892‐10897.
vanderHoeven,R.,K.C.McCallum,M.R.CruzandD.A.Garsin,2011Ce‐Duox1/BLI‐3GeneratedReactiveOxygenSpecies
TriggerProtectiveSKN‐1Activityviap38MAPKSignalingduringInfectioninC.elegans.PLoSPathog7:e1002453.
