Serine protease-mediated host invasion by the parasitic nematode Steinernema carpocapsae.
Steinernema carpocapsae is an insect parasitic nematode used in biological control, which infects insects penetrating by mouth and anus and invading the hemocoelium through the midgut wall. Invasion has been described as a key factor in nematode virulence and suggested to be mediated by proteases. A serine protease cDNA from the parasitic stage was sequenced (sc-sp-1); the recombinant protein was produced in an Escherichia coli system, and a native protein was purified from the secreted products. Both proteins were confirmed by mass spectrometry to be encoded by the sc-sp-1 gene. Sc-SP-1 has a pI of 8.7, a molecular mass of 27.3 kDa, a catalytic efficiency of 22.2 × 10(4) s(-1) m(-1) against N-succinyl-Ala-Ala-Pro-Phe-pNA, and is inhibited by chymostatin (IC 0.07) and PMSF (IC 0.73). Sc-SP-1 belongs to the chymotrypsin family, based on sequence and biochemical analysis. Only the nematode parasitic stage expressed sc-sp-1. These nematodes in the midgut lumen, prepared to invade the insect hemocoelium, expressed higher levels than those already in the hemocoelium. Moreover, parasitic nematode sense insect peritrophic membrane and hemolymph more quickly than they do other tissues, which initiates sc-sp-1 expression. Ex vivo, Sc-SP-1 was able to bind to insect midgut epithelium and to cause cell detachment from basal lamina. In vitro, Sc-SP-1 formed holes in an artificial membrane model (Matrigel), whereas Sc-SP-1 treated with PMSF did not, very likely because it hydrolyzes matrix glycoproteins. These findings highlight the S. carpocapsae-invasive process that is a key step in the parasitism thus opening new perspectives for improving nematode virulence to use in biological control.
Toubarro
Duarte
D
Departamento de Biologia, Universidade dos Açores, Centro de Investigação de Recursos Naturais, Apartado 1422, Ponta Delgada 9501-801, Portugal.
Lucena-Robles
Miguel
M
Nascimento
Gisela
G
Santos
Romana
R
Montiel
Rafael
R
Veríssimo
Paula
P
Pires
Euclides
E
Faro
Carlos
C
Coelho
Ana V
AV
Simões
Nelson
N
D000595:Amino Acid Sequence; D000818:Animals; D015801:Helminth Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006458:Hemolymph / Q000469:parasitology; D007313:Insecta / Q000469:parasitology*; D008969:Molecular Sequence Data; D009348:Nematoda / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D009842:Oligopeptides / Q000737:chemistry; D010572:Pest Control, Biological / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057057:Serine Proteases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D015801:Helminth Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; C002101:chymostatin; D057057:Serine Proteases