Identification of a Microsporidian Polar Tube Protein Reactive Monoclonal Antibody

Abstract

The microsporidia are characterized by spores containing a single polar tube that coils around the sporoplasm. When triggered by appropriate stimuli, the polar tube rapidly discharges out of the spore forming a hollow tube. The sporoplasm passes out of the spore through this tube serving as a unique vehicle of infection. Due to the unusual functional and solubility properties of the polar tube, the proteins comprising it are likely to be members of a protein family with a highly conserved amino acid composition among the various microsporidia. Polar tube proteins were separated from the majority of other proteins in glass bead disrupted spores of Glugea americanus using sequential 1% sodium dodecyl sulfate (SDS) and 9M urea extractions. The resultant spore pellet demonstrated broken, empty spore coats and numerous polar tubes in straight and twisted formations by negative stain transmission electron microscopy. After subsequent incubation of the pellet with 2% dithiothreitol (DTT), empty spore coats were still observed but the polar tubes were no longer present in the pellet. The DTT supernatant demonstrated four major protein bands by SDS-PAGE: 23, 27, 34 and 43 kDa. Monoclonal antibodies were produced to these proteins using Hunter's Titermax adjuvant. Mab 3C8.23.1 which cross-reacted with a 43-kDa antigen by immunoblot analysis, demonstrated strong reactivity with the polar tube of G. americanus spores by immunogold electron microscopy. This antibody will be useful in further characterization of polar tube proteins and may lead to novel diagnostic and therapeutic reagents.

The microsporidian polar tube: A highly specialised invasion organelle

“…It has also been observed that the portion of the tube already everted remains unchanged while the tube elongates and even changes direction at the tip (Weidner, 1982;Frixione et al, 1992). It should be noted that spores can be broken by mechanical pressure or by glass bead disruption, thus releasing polar tubes from the sides of the spores (Kudo, 1921;Keohane et al, 1994Keohane et al, , 1996a. It has been reported that polar tubes had a similar ultrastructural appearance regardless of whether they were triggered to activate and evert from the apical pole, or if they passively burst through the lateral walls of the spores (Weidner, 1982).…”

“…Techniques have been described that permit the purification of spore coat proteins as well as polar tube proteins (Keohane et al, 1994(Keohane et al, , 1996a(Keohane et al, , 1999. With the publication of the genome of E. cuniculi this has permitted investigators to initial proteomic studies of the composition of this structure (Texier et al, 2003;Weiss L.M., unpublished data).…”

“…Polar tubes have been observed to be rapidly digested after extrusion in digestive fluid or in the midgut of insects (Ohshima, 1937;Undeen and Epsky, 1990). The polar tube resists dissociation in 1-3% sodium dodecyl sulfate (SDS), 5-8 N H 2 SO 4 , 1-2 N HCl, chloroform, 1% guanidine HCl, 0.1 M proteinase K and 8-10 M Urea, but is soluble in 50% 2-mercaptoethanol (2-ME) or 1% dithiothreitol (DTT) (Weidner, 1972(Weidner, , 1976Keohane et al, 1994Keohane et al, , 1996aKeohane et al, , 1998aKeohane et al, , 1999. The polar tube inside the spore has been found to react in the same manner to reducing agents and detergents as everted polar tubes (Weidner, 1976(Weidner, , 1982.…”

Monoclonal Antibodies to Cytoplasmic Antigens of Nosema locustae (Microsporida: Nosematidae)

“…There was no reaction of either antibody with lysates from insect tissues (Culex restuans, Helicovera zea, Lymentra dispar, or Acheta domesticus) under reducing or non-reducing conditions. These mAbs were further characterized using immunogold electron microscopy employing protocols previously described (Keohane et al, 1996). Both mAbs demonstrated a generalized localization to antigens in the spores of N. locustae and cross-reacted with antigens in E. cuniculi and a Pleistophora sp.…”

“…Fusion of mouse spleen cells with myeloma cells was performed 12 weeks postimmunization using standard techniques. Supernatants from hybridomas were screened by immunoblotting using N. locustae spore lysate as the antigen as previously described (Keohane et al, 1996). Two monoclonal antibodies [mAbs] were identified that reacted with N. locustae spore lysate by immunoblotting: 3B1.23, an IgM mAb that recognized a 40-kDa antigen, and 19 F9.24, an IgG 3 mAb that recognized a group of three antigens of 12-18 kDa ( Fig.…”