Construction of scFv Antibodies against the Outer Loops of the Microsporidium Nosema bombycis 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 ß-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.

Heterologous expression of scFv fragment against Vairimorpha (Nosema) ceranae hexokinase in Sf9 cell culture inhibits microsporidia intracellular growth.

Secretion of hexokinase (HK) by microsporidia into infected cells suggests an important role for this enzyme for the intracellular development of parasites. To verify whether the expression of HK-specific antibodies in the host cell cytoplasm can suppress the growth of microsporidia, we constructed an immune library of recombinant scFv fragments against the enzyme of the honey bee pathogen Vairimorpha (Nosema) ceranae (VcHK) with a representativeness of about 5 million bacterial transformants. Two variants of VcHK-specific recombinant antibodies were selected by library panning and expressed in lepidopteran Sf9 cell line. Infecting of cells expressing two selected and control scFv fragments with V. ceranae spores was followed by their cultivation for 4 days. Analysis of parasite ß-tubulin as well as spore wall protein SWP32 transcripts in infected cultures by reverse transcription PCR and real-time qPCR showed (1) V. ceranae growth in cells heterologous to bee pathogens, (2) its inhibition by one of the selected VcHK-specific recombinant antibodies. The latter result once again emphasizes an important role of microsporidia hexokinases in their relationships with infected host cells and suggests further focusing on the mechanisms of such suppression, as well as on the search for new V. ceranae - inhibiting scFv fragments.

Construction and heterologous overexpression of two chimeric proteins carrying outer hydrophilic loops of Vairimorpha ceranae and Nosema bombycis ATP/ADP carriers.

Microsporidia Nosema bombycis and Vairimorpha ceranae cause destructive epizootics of honey bees and silkworms. Insufficient efficiency of the antibiotic fumagillin against V. ceranae, its toxicity and the absence of effective methods of N. bombycis treatment demand the discovery of novel strategies to suppress infections of domesticated insects. RNA interference is one such novel treatment strategy. Another one implies that the intracellular development of microsporidia may be suppressed by single-chain antibodies (scFv fragments) against functionally important parasite proteins. Important components of microsporidian metabolism are non-mitochondrial, plastidic-bacterial ATP/ADP carriers. These membrane transporters import host-derived ATP and provide the capacity to pathogens for energy parasitism. Here, we analyzed membrane topology of four V. ceranae and three N. bombycis ATP/ADP transporters to construct two fusion proteins carrying their outer hydrophilic loops contacting with infected host cell cytoplasm. Interestingly, full-size genes of N. bombycis transporters may be derived from the Asian swallowtail Papilio xuthus genome sequencing project. Synthesis of the artificial genes was followed by overexpression of recombinant proteins in E. coli as insoluble inclusion bodies. The gene fragments encoding the loops of individual transporters were also effectively expressed in bacteria. The chimeric antigens may be used to construct immune libraries or select microsporidia-suppressing scFv fragments from synthetic, semisynthetic, naïve and immune antibody libraries. A further expression of such antibodies in insect cells may increase their resistance to microsporidial infections.

Antibodies raised against a Sunn bug (Eurygaster integriceps Put.) recombinant protease, rGHP3p2, can inhibit gluten-hydrolyzing activity.

Sunn pest or Sunn bug, Eurygaster integriceps Put., salivary gland proteases are responsible for the deterioration of wheat flour quality during dough mixing, resulting from gluten hydrolysis. These proteases are highly heterogeneous and show low sensitivity to most types of proteinaceous inhibitors, meaning that such inhibitors cannot be used to prevent gluten damage. The present study describes the generation of a specific peptide antibody, raised against the active center of the recombinant gluten-hydrolyzing protease (GHP3). The recombinant protein, encoding two repeats of the GHP3 sequence element involved in forming the S4 pocket and binding of substrate at position P4, was designed and expressed in Escherichia coli. The antibodies raised to this recombinant protein showed inhibitory activity against the GHP3 protease. The results indicate that it is possible to design specific antibodies to inhibit wheat-bug gluten-hydrolyzing proteases.

Heterologous overexpression of active hexokinases from microsporidia Nosema bombycis and Nosema ceranae confirms their ability to phosphorylate host glucose.

The secretion of hexokinases (HKs) by microsporidia followed by their accumulation in insect host nuclei suggests that these enzymes play regulatory and catalytic roles in infected cells. To confirm whether HKs exert catalytic functions in insect cells, we expressed in E. coli the functionally active HKs of two entomopathogenic microsporidia, Nosema bombycis and Nosema ceranae, that cause silkworm and honey bee nosematoses. N. bombycis HK with C-terminal polyHis tag and N. ceranae enzyme with N-terminal polyHis tag were cloned into pOPE101 and pRSET vectors, respectively, and overexpressed. Specific activities of N. bombycis and N. ceranae enzymes isolated by metal chelate affinity chromatography were 29.2 ± 0.5 and 60.2 ± 1.2 U/mg protein at an optimal pH range of 8.5-9.5. The kinetic characteristics of the recombinant enzymes were similar to those of HKs from other parasitic and free-living organisms. N. bombycis HK demonstrated Km 0.07 ± 0.01 mM and kcat 1726 min-1 for glucose, and Km 0.39 ± 0.05 mM and kcat 1976 min-1 for ATP, at pH 8.8. N. ceranae HK showed Km 0.3 ± 0.04 mM and kcat 3293 min-1 for glucose, and Km 1.15 ± 0.11 mM and kcat 3732 min-1 for ATP, at the same pH value. These data demonstrate the capability of microsporidia-secreted HKs to phosphorylate glucose in infected cells, suggesting that they actively mediate the effects of the parasite on host metabolism. The present findings justify further study of the enzymes as targets to suppress the intracellular development of silkworm and honey bee pathogens.