Submitted to: International Journal for Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/30/2002
Publication Date: 8/15/2002
Citation: Zarlenga, D.S., Boyd, P., Lichtenfels, J.R., Hill, D.E., Gamble, H.R. 2002. Identification and partial characterization of a cdna sequence encoding a glutamic acid-rich protein specifically transcribed in trichinella spiralis newborn larvae and recognized by swine infection serum. International Journal for Parasitology. Interpretive Summary: A recent review indicates that trichinellosis is both an emerging as well as reemerging disease whereby the incidence derived from sylvatic sources is on the rise in both developing and developed countries. Infections from domestic pork continue to rise in Asia, East and Central Europe and South America, where the prevalence in swine herds can reach as high as 50%. Presently, little is known of the mechanisms by which intracellular parasites penetrate and reprogram cells to protect themselves from the host immune system without compromising the viability of the cell. Understanding one or more of these mechanisms has far reaching implications in both Agricultural and human research. To this end, a library was constructed from the T. spiralis newborn larvae and screened for sequences specific to this stage. One group of clones was identified encoding a glutamic acid rich family of proteins where similarly characterized proteins from protozoan parasites have been linked to cell membrane attachment and the infection process. This is the first report of identifying gene products that maybe associated with the infection process within parasitic nematodes. Given that state- of-the-art investigations on xenotransplantation use swine as the donor host for organs, and that degenerative muscle diseases have received a high degree of attention in recent years, delineating changes in gene expression associated with muscle atrophy will assist not only in comprehending mechanisms involved in parasite invasion of the cell, but may serve also as a model for animal and human degenerative muscle diseases.
Technical Abstract: A PCR-derived cDNA expression library was constructed using 0.5 ug of total RNA from T. spiralis newborn larvae (NBL). The library consisted of >125,000 insert-containing clones. Approximately 40-50 x 103 clones were screened immunologically using sera from pigs experimentally infected with 7000 Trichinella L1. Multiple clones reacting positively with the swine infection serum and encoding portions of a glutamic acid-rich (GAR) protein were identified. Northern and Southern blots indicated at least 2 distinct genes that encoded for the GAR proteins and that these genes were transcribed specifically in the NBL stage. cDNA sequence data predicted open reading frames of 1497 bp (NBL1500) and 1716 bp (NBL1700) generating proteins of 498 aa and 571 aa, respectively. Both sequences consisted of approximately 39% glutamic acid and 16% serine residues, and differed by the presence of a 219 bp fragment present in the 1716 bp sequence that was absent from the 1497 bp sequence. PCR data indicated that additional isoforms exist within this gene family that are different in length form those described above. In addition, it was found that more than one isoform can exist within a single worm and that this pattern can vary between individual worms within a population. Mouse antibodies to recombinant antigen localized the GAR proteins to the periphery of the developing stichocyte cells within the NBL.