Lettuce-derived secretory IgA specifically neutralizes the Shiga toxin 1 activity.

MAIN CONCLUSION: An edible plant was tested as a host for the production of secretory monoclonal IgA against Shiga toxin 1 (Stx1). The lettuce-derived IgA completely protected Vero cells from Stx1. Secretory immunoglobulin A (SIgA) is thought to control mucosal infections and thus it may be applicable to oral passive immunotherapy. Edible plants are candidate hosts for producing oral formulations with SIgA against pathogenic agents. We previously established a recombinant IgA specific for the B subunit of Shiga toxin 1 (Stx1B) consisting of the Fab fragment of Stx1B-specific monoclonal IgG and the Fc region of IgA (hyIgA). Here, we developed transgenic lettuce (Lactuca sativa) that produces hyIgA in a secretory form (S-hyIgA). An Arabidopsis-derived light-harvesting complex II (LHCB) promoter was used for the expression of all four transgenes (hyIgA heavy, light and j chains, and secretory component). Agrobacterium-mediated transformation was carried out to introduce genes into lettuce leaf discs by means of a single vector harboring all four transgenes. Consistent with the tissue specificity of the LHCB promoter, the expression of hyIgA transgenes was observed in leaf and stem tissues, which contain chloroplasts, at the mRNA and protein levels. The leaves produced hyIgA in a more than tenfold higher yield as compared with stems. The lettuce-derived S-hyIgA was found to bind to Stx1B in a dose-dependent manner by means of ELISA. A leaf extract of the transgenic lettuce completely neutralized the cytotoxicity of Stx1 against Vero cells, which are highly susceptible to Stx1. In conclusion, we established a transgenic lettuce producing a secretory form of hyIgA that can bind bacterial toxin. The results indicate that edible practical plants containing S-hyIgA will provide a possible means for immunotherapy for food poisoning.

Plant-derived secretory component forms secretory IgA with shiga toxin 1-specific dimeric IgA produced by mouse cells and whole plants.

KEY MESSAGE: A key module, secretory component (SC), was efficiently expressed in Arabidopsis thaliana. The plant-based SC and immunoglobulin A of animal or plant origin formed secretory IgA that maintains antigen-binding activity. Plant expression systems are suitable for scalable and cost-effective production of biologics. Secretory immunoglobulin A (SIgA) will be useful as a therapeutic antibody against mucosal pathogens. SIgA is equipped with a secretory component (SC), which assists the performance of SIgA on the mucosal surface. Here we produced SC using a plant expression system and formed SIgA with dimeric IgAs produced by mouse cells as well as by whole plants. To increase the expression level, an endoplasmic reticulum retention signal peptide, KDEL (Lys-Asp-Glu-Leu), was added to mouse SC (SC-KDEL). The SC-KDEL cDNA was inserted into a binary vector with a translational enhancer and an efficient terminator. The SC-KDEL transgenic Arabidopsis thaliana produced SC-KDEL at the level of 2.7% of total leaf proteins. In vitro reaction of the plant-derived SC-KDEL with mouse dimeric monoclonal IgAs resulted in the formation of SIgA. When reacted with Shiga toxin 1 (Stx1)-specific ones, the antigen-binding activity was maintained. When an A. thaliana plant expressing SC-KDEL was crossed with one expressing dimeric IgA specific for Stx1, the plant-based SIgA exhibited antigen-binding activity. Leaf extracts of the crossbred transgenic plants neutralized Stx1 cytotoxicity against Stx1-sensitive cells. These results suggest that transgenic plants expressing SC-KDEL will provide a versatile means of SIgA production.

Draft Genome Sequence of Pandoravirus japonicus Isolated from the Sabaishi River, Niigata, Japan.

"Pandoraviridae" is a proposed family of the phylum Nucleocytoviricota Its features include an amphora-shaped capsid and the largest genome among all viruses. We report the isolation and genome sequencing of a new member of this family, named Pandoravirus japonicus, the third strain discovered in Japan.

Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size.

Autophagy is an intracellular bulk degradation system. We established mouse fibroblast lines coupling the Tet-off system with an Atg5-/- mouse embryonic fibroblast line to artificially regulate autophagic ability. In the presence of doxycycline (Dox), Atg5 expression was completely suppressed and these cells were autophagy-defective. After removal of Dox, autophagic ability was restored within 6 h. Very low levels of Atg5 could induce an autophagy competent state. We applied this novel system to examine the contribution of autophagy to controlling cell size. Cell size reduction in response to starvation was significantly inhibited in cells unable to undergo autophagy. The generated cell lines will be useful reagents for future mechanistic studies into the regulation and physiologic significance of autophagy.

Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size.

Autophagy is an intracellular bulk degradation system. We established mouse fibroblast lines coupling the Tet-off system with an Atg5(-/-) mouse embryonic fibroblast line to artificially regulate autophagic ability. In the presence of doxycycline (Dox), Atg5 expression was completely suppressed and these cells were autophagy-defective. After removal of Dox, autophagic ability was restored within 6h. Very low levels of Atg5 could induce an autophagy competent state. We applied this novel system to examine the contribution of autophagy to controlling cell size. Cell size reduction in response to starvation was significantly inhibited in cells unable to undergo autophagy. The generated cell lines will be useful reagents for future mechanistic studies into the regulation and physiologic significance of autophagy.

Atg101, a novel mammalian autophagy protein interacting with Atg13.

Autophagy is a major route by which cytoplasmic contents are delivered to the lysosome for degradation. Many autophagy-related (ATG) genes have been identified in yeast. Although most of them are conserved in human, the molecular composition of the Atg1 complex appears to differ between yeast and mammals. In yeast, Atg1 forms a complex with Atg11, Atg13, Atg17, Atg29 and Atg31, whereas mammalian Atg1 (ULK1/2) interacts with Atg13 and FIP200. Here, we identify a novel mammalian Atg13 binding protein, named Atg101. Atg101 shows no homology with other Atg proteins, and is conserved in various eukaryotes, but not in Saccharomyces cerevisiae. Atg101 associates with the ULK-Atg13-FIP200 complex, most likely through direct interaction with Atg13. In Atg13 siRNA-treated cells, Atg101 is present solely as a monomer. Interaction between Atg101 and the ULK-Atg13-FIP200 complex is stable, and is not regulated by nutrient conditions. GFP-Atg101 localizes to the isolation membrane/phagophore. GFP-LC3 dot formation is suppressed and endogenous LC3-I accumulates in Atg101 siRNA-treated cells, suggesting that Atg101 is a critical factor for autophagy. Furthermore, Atg101 is important for the stability and basal phosphorylation of Atg13 and ULK1. These data suggest that Atg101 is a novel Atg protein that functions together with ULK, Atg13 and FIP200.

Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy.

Autophagy is an intracellular degradation system, by which cytoplasmic contents are degraded in lysosomes. Autophagy is dynamically induced by nutrient depletion to provide necessary amino acids within cells, thus helping them adapt to starvation. Although it has been suggested that mTOR is a major negative regulator of autophagy, how it controls autophagy has not yet been determined. Here, we report a novel mammalian autophagy factor, Atg13, which forms a stable approximately 3-MDa protein complex with ULK1 and FIP200. Atg13 localizes on the autophagic isolation membrane and is essential for autophagosome formation. In contrast to yeast counterparts, formation of the ULK1-Atg13-FIP200 complex is not altered by nutrient conditions. Importantly, mTORC1 is incorporated into the ULK1-Atg13-FIP200 complex through ULK1 in a nutrient-dependent manner and mTOR phosphorylates ULK1 and Atg13. ULK1 is dephosphorylated by rapamycin treatment or starvation. These data suggest that mTORC1 suppresses autophagy through direct regulation of the approximately 3-MDa ULK1-Atg13-FIP200 complex.