Serine Protease Promotes S. carpocapsae Host Invasion
imply that Sc-SP-1 has a somewhat different substrate prefer- has been shown to cause cell death (20), and to Sc-Trypsin and
ence. Similar to most invertebrate serine proteases, the pres- Sc-Chymotrypsin, which have been shown to correlate with
ence of six cysteine residues indicates three disulfide linkages insect immune depression (55, 56). The identification of these
(
39). Nevertheless, two of them are located close to the carbox- serine proteases in excreted/secreted products from the S. car-
ylic end of the protein and therefore we presume close to the pocapsae parasitic stage strongly contributes to our under-
42
58
168
182
well conserved Cys –Cys and Cys –Cys
disulfide standing of the pathogenic process in this insect parasitic nem-
bonds. Nevertheless, the Sc-SP-1 activity was not affected by atode. Furthermore, the involvement of Sc-SP-1 in host
reducing agents.
invasion makes this gene a useful candidate to improve nema-
Our work demonstrates that Sc-SP-1 is part of the excreted/ todes to use in the biological control of insects.
secreted products of the parasitic stage of S. carpocapsae. The
analysis of the mode of action for Sc-SP-1 supports our hypoth- Acknowledgment—We thank J. Medeiros for the expertise in S.E.
esis that this protein is involved in invasion of the insect gut
wall. The gut wall is a physical barrier that opposes pathogen
REFERENCES
invasion very efficiently in insects (47). The first component of
1
2
3
4
. Akhurst, R. J., and Dunphy, G. B. (1993) in Parasites and Pathogen of
Insects (Beckage, N. E., Thompson, S. N., and Federici, B. A., eds) Vol. 2,
pp. 1–23, Academic Press, New York
this wall is the peritrophic membrane that is a mesh of proteo-
glycans that avoid pathogen contact with cells. The basal lam-
ina, which is composed of collagen, elastin, glucosaminogly-
cans, and glycoproteins, serves as a filter to protect adjacent
tissues (48). Our assays show that Sc-SP-1 is highly efficient at
destroying both peritrophic and basal lamina. Additionally, the
columnar cells detach after treatment with Sc-SP-1 that binds
to the host basal lamina. Using a matrix membrane model to
mimic basal lamina, which has been used before to study patho-
gen host tissue invasion (49) and invasive tumor cells (50), we
proved that Sc-SP-1 is able to open holes in the matrix gel mem-
brane, very likely creating passages by which nematodes can
reach the insect hemocoelium. We suggest that Sc-SP-1 hydro-
lyzes fibronectin and laminin, and to a lesser extent collagen IV,
which are the most abundant proteins of the basal lamina. The
interaction of Sc-SP-1 with basal lamina glycoproteins, such as
fibronectin and laminin, can be explained by the predicted
B_lectin domain that was identified in sc-sp-1. This domain is
known to enable binding with glycoproteins, thus mediating a
wide variety of biological processes, particularly host-pathogen
interactions (51).
. Klein, M. G. (1990) in Entomopathogenic Nematodes in Biological Control
(Gaugler, R., and Kaya, H. K., eds) pp. 195–214, CRC Press, Inc., Boca
Raton, FL
. Begley, J. W. (1990) in Entomopathogenic Nematodes in Biological Control
(
Gaugler, R., and Kaya, H. K., eds) pp. 233–246, CRC Press, Inc., Boca
Raton, FL
. Burnell, A. (2002) in Entomopathogenic Nematology (Gaugler, R., ed) pp.
241–264, CABI Publishing, Oxon, UK
5. Vellai, T., Moln a´ r, A., Lakatos, L., Banfalvi, T., Fodor, A., and S a´ ringer, G.
(
1999) in COST 819 Entomopathogenic Nematodes: Survival of Ento-
mopathogenic Nematodes (Glazer, I., Richardson, P., Boemare, N., and
Coudert, F., eds) pp. 105–119, EUR 18855 EN, Office for Official Publica-
tions of the EC, Luxembourg
6
. Grewal, P. S., Lewis, E. E., Gaugler, R., and Campbell, J. F. (1994) Parasi-
tology 108, 207–215
7. Hao, Y. J., Montiel, R., Abubucker, S., Mitreva, M., and Sim o˜ es, N. (2010)
Mol. Biochem. Parasitol. 169, 79–86
8
9
. Dowds, B. C., and Peters, A. (2002) in Entomopathogenic Nematology
(
Gaugler, R., ed) pp. 79–98, CABI Publishing, Oxon, UK
. Ishibashi, N., and Kondo, E. (1990) in Entomopathogenic Nematodes in
Biological Control (Gaugler, R., and Kaya, H. K., eds) pp. 139–150 CRC
Press, Inc., Boca Raton, FL
The involvement of Sc-SP-1 in the parasitic process was also 10. Sim o˜ es, N., and Rosa, J. S. (1996) Biocontrol Sci. Technol. 6, 403–411
1
1. Sim o˜ es, N., Caldas, C., Rosa, J. S., Bonifassi, E., and Laumond, C. (2000)
J. Invertebr. Pathol. 75, 47–54
in accordance with the expression analysis of the encoding gene
during the nematode life cycle. Nematodes in the arrested stage
do not express sc-sp-1; however, this gene was overexpressed in
recovered nematodes, particularly those in the gut lumen pre-
paring to invade the insect hemocoelium. In other parasitic
nematodes, the role for serine proteases in tissue invasion was
inferred based on expression analysis (52–54). In vitro assays
showed that, although the resistant stage does not express sc-
sp-1, the nematodes initiate expression shortly after stimula-
tion with insect tissues. Peritrophic membrane and hemo-
lymph induce sc-sp-1 expression more rapidly than other
tissues such as fat bodies and gut epithelium. This immediate
response of the nematode is probably related to the route it
normally uses to reach the insect hemocoelium, first contacting
the peritrophic membrane that lines the gut lumen and then
with various hemolymph components, which represent the
first lines of defense. The Sc-SP-1 expression time frame and its
ability to destroy proteins in the midgut, particularly those of
the basal lamina, are in agreement with the development of
parasitism.
1
1
1
2. Dzik, J. M. (2006) Acta Biochim. Pol. 53, 33–64
3. Trap, C., and Boireau, P. (2000) Vet. Res. 31, 461–471
4. McKerrow, J. H., Pino-Heiss, S., Lindquist, R., and Werb, Z. (1985) J. Biol.
Chem. 260, 3703–3707
1
1
5. Sakanari, J. A., and McKerrow, J. H. (1990) J. Parasitol. 76, 625–630
6. Haffner, A., Guilavogui, A. Z., Tischendorf, F. W., and Brattig, N. W.
(
1998) Exp. Parasitol. 90, 26–33
1
1
7. Todorova, V. K. (2000) Folia Parasitol. 47, 141–145
8. Romaris, F., North, S. J., Gagliardo, L. F., Butcher, B. A., Ghosh, K., Beiting,
D. P., Panico, M., Arasu, P., Dell, A., Morris, H. R., and Appleton, J. A.
(2002) Mol. Biochem. Parasitol. 122, 149–160
1
2
2
9. Bedding, R. A., Molyneux, A. S., and Akhurst, R. J. (1983) Exp. Parasitol.
55, 249–257
0. Toubarro, D., Lucena-Robles, M., Nascimento, G., Costa, G., Montiel, R.,
Coelho, A. V., and Sim o˜ es, N. (2009) Int. J. Parasitol. 39, 1319–1330
1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A
Laboratory Manual, 2nd Ed., pp. 9.31–9.58, Cold Spring Harbor Labora-
tory Press, Cold Spring Harbor, NY
2. Emanuelsson, O., Brunak, S., von Heijne, G., and Nielsen H. (2007) Nat.
Protoc. 2, 953–971
2
2
3. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan,
P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R.,
Thompson, J. D., Gibson, T. J., and Higgins, D. G. (2007) Bioinformatics
23, 2947–2948
The identification of Sc-SP-1 adds to our knowledge of pro-
tease family members in S. carpocapsae such as Sc-SP-3, which
3
0674 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 40•OCTOBER 1, 2010