List of primers used for PCR-amplification of full-size genes encoding the studied proteins.

a<p>restriction enzyme sites are underlined in the primer sequences.</p>b<p>to express hexokinase in <i>Pichia pastoris</i>, the forward primer with the <i>Bgl</i>II site instead of <i>Xho</i>I was used for PCR amplification of gene copies.</p

Immunolocalization of <i>A. locustae</i> hexokinase in infected cells.

<p>A. The presence of microsporidial protein in infected cytoplasm and its accumulation in host nuclei was demonstrated using wide field immunofluoresence microscopy. The top row demonstrates accumulation of hexokinase in three nuclei of infected host cells. A nucleus of an uninfected cell is located in the bottom left corner. The bottom row shows accumulation of hexokinase in the single nucleus of an infected cell surrounded by a number of uninfected ones. The thick and thin arrows on the DAPI images indicate nuclei of infected and uninfected host cells, respectively. The nucleus of infected cells on the periphery of the invasion region (top row) is marked by the middle arrow. Scale bars, 10 μm. B. Colocalization of <i>A. locustae</i> hexokinase and Hsp70 were analyzed by confocal microscopy. Scale bars, 5 μm.</p

Western blot assay of <i>A. locustae</i> proteins.

<p>Samples of <i>A. locustae</i> intracellular stages isolated in Percoll density gradient (lane St), infected host cytoplasm obtained by gentle homogenization of locust fat bodies following sedimentation of parasites (lane M), and cytoplasm of uninfected (control) fat bodies prepared in the same manner (lane C) were equalized in protein concentration. The proteins transferred on nitrocellulose membranes were stained either by Ponceau S or by Abs against <i>A. locustae</i> Hsp70, hexokinase, α/β-hydrolase, two LRR-proteins, and trehalase. This experiment identified parasite proteins in the infected host cytoplasm.</p

Heterologous expression of <i>A. locustae</i> proteins in yeast <i>P. pastoris</i> cells.

<p>A. Expression of microsporidian proteins without any exogenous signal peptides in yeast <i>P. pastoris</i> led to the secretion of two LRR-proteins, hexokinase and trehalase. Lane 1, yeast cells before methanol induction; lane 2, cells expressing the studied protein after adding methanol; lane 3, culture medium concentrated approximately 50 times; lane 4, yeast cells expressing another parasite protein after methanol induction (control cells); lane 5, concentrated medium after cultivation of cells expressing another parasite protein (control medium). Samples were analyzed by immunoblotting with Abs specific to the expressed protein. B. Though α/β-hydrolase was not found in the culture medium, a significant amount of enzyme accumulated in the insoluble fraction of <i>P. pastoris</i> cell homogenate. Yeast cells were broken in the presence of 0.3 M sucrose and homogenate was cleared at 270 <i>g</i> for 4 minutes and then centrifuged at 18,000 <i>g</i> for 20 minutes. Western blot analysis of the supernatant (lane “sup”) and pellet (lane “pel”) showed precipitation of the enzyme, which suggests its association with cell membranes.</p

Analysis of ORFs encoding <i>Antonospora locustae</i> proteins.

<p>A. ClustalW alignment demonstrated that most genes (ORFs) encoding LRR-proteins in <i>A. locustae</i> genome form two large families. The location of the new LRR-protein of family A found in the microsporidian genome and expressed in this study is marked with two asterisks. The position of the protein encoded by ORF 515 of family B is indicated by one asterisk. Proteins with secretion signal peptides predicted by SignalP server are indicated by a plus sign. B. The amino acid sequence of the novel <i>A. locustae</i> LRR-protein of family A. C. Compared to NCBI data, two extra C-terminal repeats VPENPLVSTLSVP(E/D)DLP(A/T)CTQH were found in cloned α/β-hydrolase.</p

Construction of scFv Antibodies against the Outer Loops of the Microsporidium <i>Nosema bombycis</i> ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth

Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis. This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite β-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or “camelization” of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs