Spodoptera frugiperda X-tox protein, an immune related defensin rosary, has lost the function of ancestral defensins.
BACKGROUND
X-tox proteins are a family of immune-related proteins only found in Lepidoptera and characterized by imperfectly conserved tandem repeats of several defensin-like motifs. Previous phylogenetic analysis of X-tox genes supported the hypothesis that X-tox have evolved from defensins in a lineage-specific gene evolution restricted to Lepidoptera. In this paper, we performed a protein study in which we asked whether X-tox proteins have conserved the antimicrobial functions of their ancestral defensins and have evolved as defensin reservoirs.


RESULTS
We followed the outcome of Spod-11-tox, an X-tox protein characterized in Spodoptera frugiperda, in bacteria-challenged larvae using both immunochemistry and antimicrobial assays. Three hours post infection, the Spod-11-tox protein was expressed in 80% of the two main classes of circulating hemocytes (granulocytes and plasmatocytes). Located in secretory granules of hemocytes, Spod-11-tox was never observed in contact with microorganisms entrapped within phagolyzosomes showing that Spod-11-tox is not involved in intracellular pathogen killing. In fact, the Spod-11-tox protein was found to be secreted into the hemolymph of experimentally challenged larvae. In order to determine antimicrobial properties of the Spod-11-tox protein, it was consequently fractionated according to a protocol frequently used for antimicrobial peptide purification. Over the course of purification, the anti-Spod-11-tox immunoreactivity was found to be dissociated from the antimicrobial activity. This indicates that Spod-11-tox is not processed into bioactive defensins in response to a microbial challenge.


CONCLUSIONS
Altogether, our results show that X-tox proteins have not evolved as defensin reservoirs and have lost the antimicrobial properties of the ancestral insect defensins. The lepidopteran X-tox protein family will provide a valuable and tractable model to improve our knowledge on the molecular evolution of defensins, a class of innate immune effectors largely distributed over the three eukaryotic kingdoms.
Destoumieux-Garzón
Delphine
D
CNRS, UMR5119 Laboratoire Ecosystèmes Lagunaires, CC80, Montpellier, France.
Brehelin
Michel
M
Bulet
Philippe
P
Boublik
Yvan
Y
Girard
Pierre-Alain
PA
Baghdiguian
Stephen
S
Zumbihl
Robert
R
Escoubas
Jean-Michel
JM
D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018613:Microscopy, Confocal; D010587:Phagocytosis; D018411:Spodoptera / Q000276:immunology / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D019476:Insect Proteins