Arabidopsis Phosphomannose Isomerase 1, but Not Phosphomannose Isomerase 2, Is Essential for Ascorbic Acid Biosynthesis*S⃞

We studied molecular and functional properties of Arabidopsis phosphomannose isomerase isoenzymes (PMI1 and PMI2) that catalyze reversible isomerization between d-fructose 6-phosphate and d-mannose 6-phosphate (Man-6P). The apparent Km and Vmax values for Man-6P of purified recombinant PMI1 were 41.3 ± 4.2 μm and 1.89 μmol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372 ± 13 μm and 22.5 μmol/min/mg protein, respectively. Both PMI1 and PMI2 were inhibited by incubation with EDTA, Zn2+, Cd2+, and l-ascorbic acid (AsA). Arabidopsis PMI1 protein was constitutively expressed in both vegetative and reproductive organs under normal growth conditions, whereas the PMI2 protein was not expressed in any organs under light. The induction of PMI1 expression and an increase in the AsA level were observed in leaves under continuous light, whereas the induction of PMI2 expression and a decrease in the AsA level were observed under long term darkness. PMI1 showed a diurnal expression pattern in parallel with the total PMI activity and the total AsA content in leaves. Moreover, a reduction of PMI1 expression through RNA interference resulted in a substantial decrease in the total AsA content of leaves of knockdown PMI1 plants, whereas the complete inhibition of PMI2 expression did not affect the total AsA levels in leaves of knock-out PMI2 plants. Consequently, this study improves our understanding of the molecular and functional properties of Arabidopsis PMI isoenzymes and provides genetic evidence of the involvement of PMI1, but not PMI2, in the biosynthesis of AsA in Arabidopsis plants

C-Terminal Clostridium perfringens Enterotoxin-Mediated Antigen Delivery for Nasal Pneumococcal Vaccine.

Efficient vaccine delivery to mucosal tissues including mucosa-associated lymphoid tissues is essential for the development of mucosal vaccine. We previously reported that claudin-4 was highly expressed on the epithelium of nasopharynx-associated lymphoid tissue (NALT) and thus claudin-4-targeting using C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) effectively delivered fused antigen to NALT and consequently induced antigen-specific immune responses. In this study, we applied the C-CPE-based vaccine delivery system to develop a nasal pneumococcal vaccine. We fused C-CPE with pneumococcal surface protein A (PspA), an important antigen for the induction of protective immunity against Streptococcus pneumoniae infection, (PspA-C-CPE). PspA-C-CPE binds to claudin-4 and thus efficiently attaches to NALT epithelium, including antigen-sampling M cells. Nasal immunization with PspA-C-CPE induced PspA-specific IgG in the serum and bronchoalveolar lavage fluid (BALF) as well as IgA in the nasal wash and BALF. These immune responses were sufficient to protect against pneumococcal infection. These results suggest that C-CPE is an efficient vaccine delivery system for the development of nasal vaccines against pneumococcal infection

Induction of PspA-specific systemic and respiratory antibody responses by intranasal immunization with PspA-C-CPE.

<p>Mice were nasally immunized with vehicle, PspA alone, or PspA-C-CPE (PspA; 5 μg) once weekly for 3 weeks. One week after the last immunization, PspA-specific serum IgG (A), nasal IgA (B), BALF IgG (C), and IgA (D) were measured by ELISA. Data are shown as mean ± SEM and are representative of two independent experiments. Vehicle, n = 4; PspA, n = 5; PspA-C-CPE, n = 5. Values were compared by using the non-parametric Mann–Whitney <i>U</i> test. *<i>P</i> < 0.01.</p

Construction and preparation of PspA-C-CPE.

<p>(A) Schematic illustration of PspA-C-CPE. PspA was fused with C-CPE at its N-terminus. A G4S linker was inserted between PspA and C-CPE. (B) PspA and PspA-C-CPE were expressed in <i>Escherichia coli</i> as His-tagged proteins and purified by Ni-affinity chromatography. The PspA and PspA-C-CPE recombinant protein were applied to SDS-PAGE followed by staining with Coomassie brilliant blue. Lane 1, size ladder; lane 2, PspA; lane 3, PspA-C-CPE.</p

PspA-C-CPE-mediated induction of protective immunity against pneumococcal infection.

<p>Mice were nasally immunized with vehicle, PspA alone, or PspA-C-CPE (PspA; 5μg) once weekly for 3 weeks. One week after the last immunization, mice were intrarespiratory challenged with <i>S</i>. <i>pneumoniae</i> (5.0 × 10⁶ CFU/mouse), and their survival was monitored for 14 days. Survival was compared between groups by using the non-parametric Mann–Whitney <i>U</i> test. **<i>P</i> < 0.05, *<i>P</i> < 0.01. Data were collected four experiments. Vehicle, n = 35; PspA, n = 45; PspA-C-CPE, n = 59.</p