Structure guided mimicry of an essential P. falciparum receptor-ligand complex enhances cross neutralizing antibodies.

Invasion of human erythrocytes by Plasmodium falciparum (Pf) merozoites relies on the interaction between two parasite proteins: apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2). While antibodies to AMA1 provide limited protection against Pf in non-human primate malaria models, clinical trials using recombinant AMA1 alone (apoAMA1) yielded no protection due to insufficient functional antibodies. Immunization with AMA1 bound to RON2L, a 49-amino acid peptide from its ligand RON2, has shown superior protection by increasing the proportion of neutralizing antibodies. However, this approach relies on the formation of a complex in solution between the two vaccine components. To advance vaccine development, here we engineered chimeric antigens by replacing the AMA1 DII loop, displaced upon ligand binding, with RON2L. Structural analysis confirmed that the fusion chimera (Fusion-FD12) closely mimics the binary AMA1-RON2L complex. Immunization studies in female rats demonstrated that Fusion-FD12 immune sera, but not purified IgG, neutralized vaccine-type parasites more efficiently compared to apoAMA1, despite lower overall anti-AMA1 titers. Interestingly, Fusion-FD12 immunization enhanced antibodies targeting conserved epitopes on AMA1, leading to increased neutralization of non-vaccine type parasites. Identifying these cross-neutralizing antibody epitopes holds promise for developing an effective, strain-transcending malaria vaccine.

Structure guided mimicry of an essential P. falciparum receptor-ligand complex enhances cross neutralizing antibodies.

Invasion of human red blood cells (RBCs) by Plasmodium falciparum (Pf) merozoites relies on the interaction between two parasite proteins, apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) 1,2 . Antibodies to AMA1 confer limited protection against P. falciparum in non-human primate malaria models 3,4 . However, clinical trials with recombinant AMA1 alone (apoAMA1) saw no protection, likely due to inadequate levels of functional antibodies 5-8 . Notably, immunization with AMA1 in its ligand bound conformation using RON2L, a 49 amino acid peptide from RON2, confers superior protection against P. falciparum malaria by enhancing the proportion of neutralizing antibodies 9,10 . A limitation of this approach, however, is that it requires the two vaccine components to form a complex in solution. To facilitate vaccine development, we engineered chimeric antigens by strategically replacing the AMA1 DII loop that is displaced upon ligand binding with RON2L. Structural characterization of the fusion chimera, Fusion-F D12 to 1.55 Å resolution showed that it closely mimics the binary receptor-ligand complex. Immunization studies showed that Fusion-F D12 immune sera neutralized parasites more efficiently than apoAMA1 immune sera despite having an overall lower anti-AMA1 titer, suggesting improvement in antibody quality. Furthermore, immunization with Fusion-F D12 enhanced antibodies targeting conserved epitopes on AMA1 resulting in greater neutralization of non-vaccine type parasites. Identifying epitopes of such cross-neutralizing antibodies will help in the development of an effective, strain-transcending malaria vaccine. Our fusion protein design is a robust vaccine platform that can be enhanced by incorporating polymorphisms in AMA1 to effectively neutralize all P. falciparum parasites.

Distinct transcriptional and processing regulations control miR167a level in tomato during stress.

Besides their definite role in plant developmental processes miR167 also serve as mediator of stress response. Although differential expression of miR167 occurs during stresses, the regulatory-mechanism of biogenesis remained elusive. Therefore, using tomato as the model plant we have explored the mechanism of regulation of miR167a expression during stresses. Fungus or virus infections and exposure to cold stress raised the level of miR167a expression. Whereas, salt, drought and heat treatments resulted in the downregulation, indicating different stresses activated alternative mechanisms for miR167a regulation. Interestingly, the relative expression level of precursors in control versus temperature stressed plants differed from the pattern observed in the mature miR167a expression, suggesting that both transcriptional and processing regulation were important for biogenesis. The promoter-regulatory sequence of the major isoform MIR167a harbours several development and stress-related regulatory sites. Accordingly, promoter assays using transient transformation and transgenic tobacco plants proved stress-dependent regulation of the promoter. Further analyses corroborated the role of tomato DREB2A protein in the transcriptional regulation during temperature stress. Finally, in vitro assays established the importance of processing factors in cold-stress dependent efficient processing of MIR167a precursors. These data confirm distinct role of transcriptional and processing machinery in stress-influenced regulation of tomato miR167a biogenesis.

Integrated miRNA and mRNA expression profiling reveals the response regulators of a susceptible tomato cultivar to early blight disease.

Early blight, caused by the fungus Alternaria solani, is a devastating foliar disease of tomatoes, causes massive yield loss each year worldwide. Molecular basis of the compatible host-pathogen interaction was elusive. We adopted next generation sequencing approach to decipher miRNAs and mRNAs that are differentially expressed during Alternaria-stress in tomato. Some of the interesting findings were also validated by alternative techniques. Our analysis revealed 181 known-miRNAs, belonging to 121 miRNA families, of which 67 miRNAs showed at least 2-fold change in expression level with the majority being downregulated. Concomitantly, 5,450 mRNAs were significantly regulated in the same diseased tissues. Differentially expressed genes were most significantly associated with response to stimulus process, photosynthesis, biosynthesis of secondary metabolites, plant-pathogen interaction and plant hormone signal transduction pathways. GO term enrichment-based categorization of gene-functions further supported this observation, as terms related to pathogen perception, disease signal transduction, cellular metabolic processes including oxidoreductase and kinase activity were over represented. In addition, we have discovered 102 miRNA-mRNA pairs which were regulated antagonistically, and careful study of the targeted mRNAs depicted that multiple transcription factors, nucleotide-binding site leucine-rich repeats, receptor-like proteins and enzymes related to cellular ROS management were profoundly affected. These studies have identified key regulators of Alternaria-stress response in tomato and the subset of genes that are likely to be post-transcriptionally silenced during the infection.

Mechanism of regulation of tomato TRN1 gene expression in late infection with tomato leaf curl New Delhi virus (ToLCNDV).

Tomato leaf curl disease caused by geminiviruses is manifested by curling and puckering of leaves and thickening of veins, resembling developmental defects. This is probably due to the long-term altered regulation of expression of development related gene(s). Our results show that in the infected leaves the transcript level of TORNADO1 (SlTRN1), a gene important for cell expansion and vein formation, increased significantly. SlTRN1 is transcribed from two start sites. The preferential usage of one start site governs its expression in viral-stressed plants. To investigate the role of specific promoter elements in mediating differential expression of SlTRN1, we performed SlTRN1 promoter analysis. The promoter-regulatory sequences harbor multiple W-boxes. The SlWRKY16 transcription factor actively interacts with one of the W-boxes. WRKY proteins are commonly induced by salicylic acid (SA), and consequently SA treatment increased transcript level of SlWRKY16 and SlTRN1. Further mutational analyses confirmed the role of W-boxes in mediating SlTRN1 induction during ToLCNDV infection or SA treatment. We postulate that the activation of SA pathway during stress-response in tomato induces WRKY16, which in turn modulates transcription of SlTRN1 gene. This study unravels the mechanism of regulation of a developmental gene during stress-response, which may affect the severity of symptoms.