(Received for publication, April 19, 1995; and in revised form, January 4, 1996)
From the
The developmentally arrested third stage infective larva of hookworms resumes development upon entry into the definitive host. This transition to parasitism can be modeled in vitro by stimulating infective larvae with a low molecular weight ultrafiltrate of host serum together with methylated glutathione analogues. When stimulated to resume development in vitro, activated larvae of the hookworm Ancylostoma caninum released a 42-kDa protein, termed Ancylostoma-secreted protein (ASP). ASP was the major protein released by activated hookworm larvae. Degenerate oligonucleotide primers, based on a partial internal amino acid sequence of the protein, were used together with flanking vector sequence primers to amplify a fragment from a third stage larval cDNA library by polymerase chain reaction. The fragment was used as a probe to isolate a longer clone from the larval cDNA library. The full-length ASP cDNA was found to encode a 424-amino acid protein with homology to the antigen 5/antigen 3 family of proteins from hymenopteran venoms and a family of cysteine-rich secretory proteins. ASP was expressed in bacterial cells, and a polyclonal antiserum against purified recombinant ASP was produced. The antiserum, which was demonstrated to be specific for ASP, was used as a probe to measure the kinetics of ASP release by hookworm larvae. ASP is released within 30 min of stimulation, with the majority released by 4 h. Low levels of ASP were released continuously following activation, but only if the stimuli were present in the incubation medium. The compound 4,7-phenanthroline, previously shown to inhibit larval activation, also inhibited release of ASP. The specific, rapid release of ASP by activated infective larvae suggests that this molecule occupies a critical and central role in the transition from the external environment to parasitism.
The early events of the hookworm infectious process are poorly
understood, especially at the molecular and biochemical level. A better
understanding of the molecules released by invading larvae, and the
host's responses to them, would allow for the rational design of
immuno- and chemotherapeutic intervention strategies. However, the
inability to culture hookworms beyond the third-stage larvae
(L) (
)in vitro, together with the
difficulties associated with the isolation of sufficient parasitic
stages for molecular and biochemical studies, has, until recently,
hampered investigations of the critical first steps in the
establishment of the parasitic relationship.
The initial events of
the infectious process can now be modeled in vitro, using the
resumption of feeding as a marker for the transition from the
free-living L to the developing parasitic
L
(1, 2, 3) . When free-living
L
of the canine hookworm Ancylostoma caninum are
stimulated to resume feeding in vitro, they release several
molecules into the culture medium. Recently, a zinc metalloprotease
activity has been reported from activated A. caninum L
ES products(4) . Here we report the isolation, cloning,
and expression of a 40-kDa protein from activated ES products, referred
to as Ancylostoma-secreted protein (ASP), that is homologous
to the major venom allergen of hymenopteran insects. ASP is the major
protein released by activated hookworm L
. Its release
during invasion of the host, together with its partial homology to
previously described allergens from insect venoms, suggest that it may
orchestrate key events in the patho- and immunobiology of hookworm
infection.
Figure 1:
The complete DNA and deduced amino acid
sequence of the ASP cDNA. The sequenced peptide (amino acids
255-267) from which PCR primers were derived is in bold
type, and the sequence of the primer used for PCR is double
overlined. The location of primers used for 5` RACE are overlined, and the restriction enzyme sites EcoRI and NcoI used for subcloning are underlined. The 18-amino
acid secretory signal sequence (amino acids 1-18) is in bold
italic type. The complete cDNA sequence was submitted to
GenBank with the accession no.
U26187.
For purification of rASP, a 2-liter
culture of BL21 cells containing the recombinant plasmid pET28:ASP was
induced as described above. The cells were centrifuged, the pellet
resuspended in 60 ml of 50 mM Tris (pH 8.0), 2 mM EDTA, 0.1% Triton X-100, 6 mg of lysozyme, and incubated at 30
°C for 30 min. The solution was sonicated using a Branson 450
Sonifier (30% duty cycle, output setting 3) until it was no longer
viscous and centrifuged at 12,000 g for 15 min. The
pellet was resuspended in 60 ml of 1.0% SDS, 0.5% 2-mercaptoethanol,
boiled for 5 min, and cooled to 22 °C. The extract was dialyzed (M
= 12,000-14,000) against 2 liters
of 0.1% SDS in phosphate-buffered saline for 2 days at 22 °C, with
two changes of buffer. The cell extract was applied to a 10-ml HisBind
resin column (Novagen), and chromatography was conducted according to
the manufacturer's instruction, except that 0.1% SDS was added to
all buffers. The expressed protein, containing a 6-residue histidine
tag (His-Tag, Novagen) that bound it to the Ni
resin,
was eluted from the column with 0.1-1.0 M stepwise
gradient of imidazole, dialyzed as above, and isolated by preparative
SDS-PAGE on 11% acrylamide gels.
Specificity of the antisera was confirmed by its adsorption with purified rASP. rASP was coupled to 3 M Emphaze beads (Pierce, Rockford, IL) by incubating 0.2 g of beads with 0.27 mg of rASP in phosphate-buffered saline, 0.1% SDS, 0.8 M sodium citrate, pH 8.0, overnight at 22 °C. The beads were sedimented by gentle centrifugation, the supernatant was discarded, and the coupling reaction was stopped by adding 10 ml of 1.0 M Tris, pH 8.0, followed by incubation at 22 °C for 2.5 h. The beads were washed twice with phosphate-buffered saline and stored at 4 °C. rASP antiserum (1:500) and Vesq Ag5 antiserum (1:2000) were diluted in 5% NFDM-P, and 10 ml were incubated with 0.2 ml of ASP-conjugated beads overnight at 4 °C. The beads were removed by centrifugation, and the supernatant was used in a Western blot (see below).
To determine
if ASP was released continuously during the activation process, the ES
products of a single population of L were sampled over
time. A well containing approximately 6500 L
was incubated
under standard activation conditions. At 1, 4, and 24 h of incubation,
the L
were removed and pelleted by centrifugation, and the
supernatant was transferred to a new tube and concentrated by
ultrafiltration. The L
were washed three times with 15 ml
of RPMI and returned to a new well. Fresh medium containing the stimuli
was added, and the incubation was resumed, except at 24 h, which was
the terminal time point. A negative control (no stimuli) and a positive
control (entire 24-h activated L
ES product output) were
harvested at 24 h. The percentage of feeding L
was
determined at 24 h.
To determine whether the presence of the
stimulus was required for continuous ASP release, two wells containing
6000 L were incubated with the stimulus for 1 or 4 h. At
these times, the ES products were harvested, and the L
were
washed three times with RPMI and reincubated in RPMI alone (i.e. without the activation stimuli). At 24 h, the ES products were
harvested again, and the percentage of feeding L
was
determined. Activated and nonactivated larvae incubated for 24 h were
used as controls, with their ES products collected at 24 h only.
4,7-Phenanthroline, a known feeding inhibitor(4) , was
tested for its effect on ASP release. Approximately 6000 L were incubated with 0.5 mM 4,7-phenanthroline and
activation stimuli for 24 h. Following incubation, the ES products were
harvested and concentrated by ultrafiltration. The ES products from all
experiments were examined by Western blotting using rASP antiserum as
described above.
When A. caninum L are stimulated to
resume feeding in vitro, they release a major product of
approximately M
= 40,000, referred to as
ASP. In contrast, no proteins are detected in the ES products of
nonactivated L
(not shown). Microsequencing of a trypsin
digestion product of this protein revealed a peptide with the sequence
GLEPDALGGNAPK. A degenerate primer derived from the EPDALG portion of
this sequence, together with a primer complementary to flanking vector
sequence, amplified a fragment of approximately 550 bp from phage
DNA containing an A. caninum L
cDNA library. This
product was used as a probe to isolate a 1.2-kb cDNA clone encoding ASP
from the A. caninum L
cDNA library. The cDNA
contained a 3` poly(A) tail, but lacked a 5` initiation codon. The 5`
end containing the ATG (Met) start codon was isolated from L
cDNA by 5`-RACE(16, 17, 18) . Four 5`
end clones were sequenced, and found to encode identical sequences that
were in frame with and overlapped the cDNA clone. Unlike another
hookworm cDNA recently cloned in our laboratory(10) , none of
the 5` sequences contained the conserved nematode SL 1 or SL
2(22, 23, 24) , nor could the 5` end be
amplified from cDNA using SL 1 primer and an internal gene-specific
primer.
The full-length cDNA encodes an ORF of 424 amino acids, with a predicted molecular weight of 45,735, a pI of 7.13, and a 34-bp 3`-untranslated region (Fig. 1). The entire sequence of the original peptide purified from ES products was present in the ORF (amino acids 255-267), confirming that this cDNA encodes the ASP molecule. The amino-terminal 18 amino acids are highly hydrophobic (11/18), and a potential eukaryotic signal sequence cleavage site is located between amino acids 18 (A) and 19 (S)(25) , consistent with the secretory nature of the protein. There are no N-linked glycosylation sites present in the ORF.
Data base
comparison (SwissProt) revealed significant homology between the
COOH-terminal 215 amino acids of the ASP-deduced amino acid sequence
and the antigen 5/antigen 3 family of molecules from Hymenoptera venoms(26, 27, 28) , a family of
cysteine-rich secretory proteins, called
CRISPs(29, 30, 31, 32, 33, 34) ,
and pathogenesis-related proteins in plants (35) (Fig. 2). The amino-terminal 191 amino acids failed
to show significant homology to any proteins in the data base. Because
of the apparent homology to hymenopteran venom Ag 5, a polyclonal
antiserum against Vesq Ag5 was tested for its ability to recognize
secreted and recombinant ASP. As shown in Fig. 3b, Vesq
Ag5 antiserum cross-reacted with both the recombinant and native ASP
molecules, although recognition was not entirely specific. In addition
to ASP, the heterologous serum also recognized a protein of M
28,000 in ES products from activated
L
. The antiserum also cross-reacted with rASP, as well as a
higher molecular weight protein. However, adsorption of the Vesq Ag5
antiserum with rASP removed all reactivity with the ES products, and
decreased binding to rASP by 78%, in addition to abrogating the high M
reactivity in the rASP lane. The Vesq Ag5
antiserum failed to recognize any proteins in the ES products from
nonactivated L
.
Figure 2: Comparison of the ASP deduced amino acid sequence with the amino acid sequences of selected homologous molecules. Shaded residues are identical to those in ASP, and positions marked with an asterisk (*) are conserved in all molecules. Numbers in parentheses represent the percent identity with ASP, calculated using pairwise comparisons between ASP and the homolog using the GAP program of the Wisconsin Genetics program. Solin, antigen 3 from the red imported fire ant, Solenopsis invicta (accession no. P35778); Vessq, Vesq Ag5 from the yellow jacket V. squamosa (P35786); Htpx, human testes-specific protein (B33329); Rscg, rat sperm-coating glycoprotein (acidic epididymal glycoprotein, A24609); Helo, helothermine from Mexican beaded lizard (Heloderma horridum horridum) salivary gland (U13619); Pr1a, tobacco pathogenesis-related protein 1a precursor (S00513).
Figure 3:
Adsorption of antisera with rASP.
Concentrated ES products from 6000 nonactivated (NA) or
activated (ACT) A. caninum L, together
with 8 µg of purified rASP, were separated in an 11% polyacrylamide
gel. Following transfer to poly(vinylidene fluoride) membranes, the
blots were probed with neat antiserum or antiserum adsorbed against
rASP. a, anti-rASP antiserum, 1:500; b, anti-Vesq Ag5
antiserum, 1:2000.
In contrast, the rabbit antiserum
prepared against purified rASP exclusively recognized both the
recombinant and the native molecules. As seen in the Western blot in Fig. 3a, the antiserum recognized a band of M
40,000-42,000 in ES products from
activated L
, and a slightly lower M
band in purified rASP, but nothing in the ES products from
nonactivated L
. Adsorption of the antiserum with rASP
completely abrogated the reaction (Fig. 3a), indicating
that the antiserum was specific for ASP.
The rASP antiserum was used
investigate the kinetics and biology of ASP release. The first
experiment was designed to determine when ASP is first released by
activated L. ES products were collected at several time
points, and analyzed by Western blotting with rASP antiserum as the
probe. Infective L
released ASP as early as 30 min
following exposure to activating stimuli (Fig. 4). Image
analysis indicated that by 4 h, ASP release had reached levels equal to
that released by 24 h in the positive control (pd at 4 h = 118,
24 h = 116).
Figure 4:
Time course of ASP release. Western blots
containing ES products from 6000 activated A. caninum L were probed with rASP antiserum (1:500). Lane 1,
nonactivated L
, 24 h of incubation; lane 2, 30 min
of incubation; lane 3, 1 h of incubation; lane 4, 4 h
of incubation; lane 5, 24 h of
incubation.
A second experiment was conducted to determine
whether ASP was released continuously during the incubation, or whether
it was primarily released early in the activation process. In this
experiment, ES products were harvested from a single population of
activated L at several time points. The ES products were
first harvested at 1 h, and the L
were thoroughly washed
and returned to the tissue culture plate with fresh medium containing
the activation stimuli. This was repeated on the same population of
L
at 4 h. At 24 h, the ES products were collected, and the
L
were assayed for feeding. Therefore, the ES products
contained the ASP secreted between 0 and 1 h of incubation, 1-4 h
of incubation, and 4-24 h of incubation. As shown in Fig. 5, ASP was present in the ES products from all time
intervals, although the 0-1 h fraction contained more than the
other time intervals. Comparison of the 0-1 h band (lane
2, pd = 65.4) with the total ASP released (lanes
2-4, total pd = 111) by image analysis indicated that
nearly 60% of the total ASP released over 24 h was released in the 1st
h following exposure to stimuli. Furthermore, almost 90% of the total
ASP is released by 4 h (sum of lanes 2-3, pd =
98.9, versus lanes 2-4). The number of the manipulated
L
that were feeding at 24 h was similar to the percentage
of undisturbed, activated L
that fed (88 versus 95%), indicating that the larvae suffered no untoward effects
during harvesting of the ES products.
Figure 5:
Kinetics of ASP release by a single
population of A. caninum L. ES products from 6500
L
were collected at various time points, the L
were washed, and the incubation was continued in the presence of
activation stimuli. Lane 1, nonactivated L
, 24 h
of incubation (negative control); lane 2, ES products
harvested at 1 h of incubation (0-1-h output); lane 3,
ES products harvested at 4 h of incubation (1-4-h output); lane 4, ES products harvested at 24 h of incubation
(4-24-h output); lane 5, ES products incubated for 24 h
(0-24-h output, positive control).
A third experiment was
designed to determine if ASP release was dependent on the presence of
the activation stimuli. In this experiment, ES products were harvested
from individual populations of L at either 1 or 4 h of
incubation, and the L
were washed and reincubated in the
absence of stimuli. The ES products from the reincubated L
were harvested at 24 h and examined by Western blot with rASP
antiserum. As seen in Fig. 6, rASP antiserum recognized strong
bands in the 1-, 4-, and 24-h (positive control) ES products. However,
no further ASP was released when the ES products from L
reincubated without the stimuli were examined at 24 h, despite
the fact that significant proportions of the L
had been
triggered to resume feeding when assayed at 24 h (1 h, 51% feeding; 4
h, 68%; 24-h control, 87%).
Figure 6:
Continuous ASP release requires the
stimulus. ES products were harvested at specified times, the L were washed, and incubation was continued in the absence of the
activation stimuli. ES products were examined by Western blotting. Numbers in parentheses indicate percentage feeding when
assayed at 24 h. Lane 1, ES products from nonactivated L
incubated for 24 h; lane 2, activated L
ES
products incubated 24 h; lane 3, activated L
ES
products, 0-1-h output; lane 4, activated L
ES products, 1-24 h without stimulus; lane 5,
activated L
ES products, 0-4-h output; lane
6, activated L
ES products, 4-24 h without
stimulus.
Inclusion of 2.5 mM 4,7-phenanthroline in the incubation decreased ASP release by 58% (Fig. 7, uninhibited pd = 106.8, inhibited pd =
45.3). Feeding was decreased from 96% in uninhibited L to
13% in L
exposed to the inhibitor.
Figure 7:
Effect of 0.5 mM 4,7-phenanthroline on release of ASP by A. caninum activated L. NA, ES products from 6000
nonactivated L
; ACT, ES products from 6000
activated L
; 4,7-PHE, ES products from 6000
activated L
incubated with 2.5 mM 4,7-phenanthroline. The percentage of L
feeding at 24
h was: NA, 3.5%; ACT, 96%; 4,7-PHE, 13%.
During invasion of a definitive host, hookworm infective
L encounter signals that initiate developmental pathways
that were previously suspended in the free-living L
stage (2, 36, 37) . The resumption of feeding by
parasitic L
associated with exposure to hostlike conditions
functions as a marker for activation and the transition to
parasitism(2, 3) . Coincident with in vitro activation, L
release a 40-kDa molecule, known as ASP,
as the major component of ES products. In order to investigate its role
in the transition to parasitism, the cDNA encoding ASP was cloned and
analyzed. The cloned molecule was expressed in E. coli cells,
purified, and used to produce a polyclonal antiserum.
Sequencing of
the ASP cDNA revealed an open reading frame of 424 amino acids. The 5`
end of the cDNA encoded a highly hydrophobic (11/18) amino acid
NH terminus. The presence of a probable cleavage site (25) between Ala-18 and Ser-19 suggests that the 18
NH
-terminal amino acids represent the signal peptide that
targets ASP as a secretory protein. The calculated molecular mass of
the complete peptide encoded by the ORF is 45,735 daltons, whereas the
calculated molecular mass of the protein without the signal sequence is
43,793 daltons, which agrees more closely with the apparent M
of ASP in ES products (
40,000-42,000)
determined by gel electrophoresis (Fig. 3). Comparison of the
ASP predicted amino acid sequence to protein data bases revealed
significant homology (
50-56% similarity,
30-35%
identity) between the carboxyl-terminal 230 amino acids of ASP and the
Ag 3/Ag 5 proteins of hymenopteran venoms (Fig. 2). Venom Ag5 is
the major protein of several hymenopteran venoms and as such represents
the major antigen(38) . Its function is unknown, although it
has been hypothesized to be an invertebrate neurotoxin(39) .
The homology to venom proteins was confirmed by the ability of
antiserum directed against Ag 5 of the yellow jacket V. squamosa to recognize ASP on Western blots of ES products and expressed
rASP (Fig. 3b). The Vesq Ag5 antiserum also recognized
a protein of lower M in ES products from activated
L
that is not recognized by the rASP antiserum. Amino acid
sequencing indicated that this lower M
component
is closely related to ASP (not shown) and probably represents another
member of a family of ASP-like secretory proteins. This is supported by
the elimination of cross-reactivity by adsorption of the antiserum with
rASP, indicating that the low M
molecule contains
an epitope that is recognized by the polyclonal Vesq Ag5 antiserum, but
not by the rASP antiserum.
ASP is also homologous to a family of
CRISPs isolated from testis (mouse and human TPX-1, 29.7 and 37.7%
identity; mouse CRISP-1, 31.7%) and salivary glands of mice (CRISP-3,
29.1%) and beaded lizards (helothermine,
33.5%)(29, 30, 31, 32, 33, 34) .
The latter peptide is a salivary toxin that inhibits
Ca-induced Ca
release by blocking
the ryanodine receptor on the sarcoplasmic reticulum. ASP contains 7 of
the 16 invariant cysteine residues, but less than half of the
cysteine-rich COOH-terminal domain, that are hallmarks of the CRISP
family of proteins. However, all of the molecules, including ASP and
the insect venoms, share a common sequence (HYTQ) corresponding to
amino acids 358-361 of ASP(31) .
ASP release occurs
rapidly in the activation process, with large amounts secreted within
30 min of exposure to the activation stimuli. Nearly all of the total
ASP secreted is released by 4 h of incubation. These data suggest that
ASP might be produced and stored in secretory granules that are
released in response to activation, as occurs during activation and
molting of other parasitic nematodes(37) . Indeed, isolation of
the ASP message from a cDNA library of nonactivated L indicates that mRNA-encoding ASP is present in the free-living
stage prior to activation. However, it is possible that ASP mRNA is
masked and translated only in response to the activation signal. Also,
there is a low level of stimulus-dependent ASP released between 4 and
24 h, suggesting new, activation-associated synthesis of ASP. One
possible explanation is that the activation stimulus causes the release
of stored ASP from secretory granules, but also initiates ASP gene
expression, resulting in continued low levels of ASP production and
release. Confirmation of this hypothesis awaits the results of more
detailed analysis of ASP stage-specific expression and ASP
immunolocalization experiments.
ASP release occurs much earlier than the resumption of feeding, which typically begins at 6-8 h(6) . Although feeding and ASP release both occur following exposure to the activation stimulus, the two events differ in that, once initiated, the presence of the stimulus is not required for feeding to continue(6) , whereas the stimulus is required for continuous release of ASP. Studies are underway to determine whether ASP secretion can be uncoupled from feeding, or whether both phenomena occur in response to the same stimuli.
The rapid and constitutive
release of ASP suggests an important function early in the infective
process, perhaps as a modulator of the host immune response. There are
numerous reports of ES products from nematode parasitic stages that
alter host physiology or suppress host immunity, either by inhibiting
immune effector mechanisms (40, 41) or by direct
immunosuppression(42, 43, 44) . Recently,
adult A. caninum were shown to secrete a neutrophil inhibitory
factor that binds to CD11/CD18 receptor of neutrophils and inhibits
their ability to mediate phagocytosis(45, 46) .
Sequence comparison indicated that there is a 30.7% identity between
ASP and neutrophil inhibitory factor (not shown). Thus, ASP might serve
a similar function in pre-adult parasitic stages, thereby allowing
invading L to interfere with the host inflammatory response
and evade destruction. Because ASP also exhibits homology with the
peptide toxin helothermine (33.5% identity)(31) , it is also
possible that ASP exerts a direct toxic effect on host immune effector
cells.
Alternatively, because of its close homology to Vesq Ag5, ASP might function as an allergen. Allergens elicit immediate-type hypersensitivity inflammatory host responses comprised of IgE production and a so called TH2 cytokine profile, including IL-4, Il-5, and Il-10. It is of interest that intestinal nematode infections also bias the host inflammatory response to produce Il-4 and Il-5 for the stimulation of IgE production and eosinophilia, respectively(47) . Studies are underway to determine whether the TH2-like responses found in hookworm infection might arise in response to ASP.
Determination of the actual in vivo function of ASP will require further investigation. However,
because its release coincides with infection and the transition to
parasitism, ASP is a promising candidate for a recombinant vaccine
against hookworm disease. ES products of several nematodes elicit some
degree of host protection when used as vaccine
antigens(40, 48, 49, 50, 51, 52) .
The only successful hookworm vaccine to date, against canine hookworm,
employed radiation-attenuated infective L, and although a
commercial failure, did reduce worm burden and the resultant pathology (53) . One possibility is that the immune response against the
irradiated L
was directed against an ES product, perhaps
ASP, released during invasion. Evaluation of ASP as a vaccine candidate
is currently under way.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U26187[GenBank].