Amebiasis, also defined as invasive intestinal and extra intestinal amebiasis, is caused by Entameoba histolytica, an invasive protozoan parasite. World Health Organization (WHO) has reported that approximately 50 million people are infected each year causing an estimated 40 to 100 thousand deaths annually. Entameoba histolytica ranks only second to malaria as a protozoan cause of death. Amebiasis occurs world wide but people living in Central and South America, Africa and Asia are the majority to suffer from morbidity and mortality. The enteric parasite has no zoonotic reservoirs and insect vectors for its transmission and infects humans and non-human primates. Therefore, anti-amebic vaccine could completely eradicate the disease. Entamoeba histolytica invades tissue and causes the disease in series of events. The disease is caused when the cyst form of the parasite is ingested with contaminated food or water. After excysting in the small intestine to form the trophozoite, the parasite adheres to the colonic mucus and epithelial cells through interaction of Gal/GalNAc lectin, an amebic surface adhesin with the host glycoconjugates. The parasite then secrets the proteolytic enzymes that disrupt the intestinal mucus and epithelial barrier facilitating tissue penetration. The trophozoite then kills the host epithelial and immune cells. Also, it resists the host's immune response causing the prolonged infection called the invasive amebiasis and causes colon or liver abscess. The symptoms include gradual onset of abdominal pain, diarrhea and bloody stools. Also, it can form cysts that are excreted with stools to start new cycle. The parasite recognition of the host glycoconjugates plays an important role in the pathogenesis. Therefore, the Gal/GalNAc lectin could be a possible vaccine candidate. The Gal/GalNAc lectin is composed of a 260-kDa heterodimer of disulfide-linked heavy (170 kDa) and light (35 kDa) subunits, which is non-covalently associated with an intermediate sub-unit of 150 kDa. The only recognized Carbohydrate recognition domain (CRD) was found in the heavy sub-unit. The CRD of the lectin is the potential target for colonization blocking vaccines and drugs. Preliminary studies have shown that the recombinant fragments of cysteine-rich region of LecA (lectin) containing the CRD (carbohydrate recognition domain) of the GalNAc lectin conferred protection against amebiasis. Therefore, production of LecA in plants using chloroplast genetic engineering would result in low cost vaccine because of high expression levels of vaccine antigens, and elimination of the cold-chain (low temperature, storage & transportation), hospitals and health professionals for their delivery. The LecA protein was expressed in transgenic chloroplasts of Nicotiana tabacum var. Petit havana by transforming the chloroplast genome using the LecA gene (1755 bp) by homologous recombination. The pLD-CtV has trnI and trnA genes that are used as flanking sequences for homologous recombination and the constitutive 16s rRNA promoter to regulate transcription. The aadA gene conferring spectinomycin resistance has been used for selection and gene10 regulatory sequence from T7 bacteriophage to enhance translation. The chloroplast integration of LecA was confirmed by PCR and Southern blot analysis. The expression of LecA protein in transgenic chloroplasts was analyzed by immunoblot analysis using anti-LecA antibodies. Maximum expression levels of LecA up to 6.3 % of the total soluble protein were observed in the old leaves. The evaluation of the immune response in animal model is underway. This is the first report of expression of LecA in a plant system.

The vast majority of valuable agronomic traits are encoded polygenetically. Chloroplast genetic engineering offers an alternate approach to multigene engineering by allowing the insertion of entire pathways in a single transformation event, while being an environmentally friendly approach. Stable integration into the chloroplast genome and transcription of the phaA gene coding for β-ketothiolase was confirmed by Southern and northern blots. Coomassie-stained gel and western blots confirmed hyperexpression of β-ketothiolase in leaves and anthers, with high enzyme activity. The transgenic lines were normal except for the male sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Transgenic lines followed an accelerated anther developmental pattern, affecting their development and maturation, resulting in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium and vacuolation, decreased the inner space of the locules, affecting pollen grain and resulted in the irregular shape and collapsed phenotype. Reversibility of the male sterility phenotype was achieved by exposing the plants to continuous illumination, producing viable pollen and copious amounts of seeds. This is the first report of engineered cytoplasmic male sterility and offers a new tool for transgene containment for both nuclear and organelle genomes. Detailed characterization of transcriptional, posttranscriptional and translational processes of heterologous operons expressed via the chloroplast genome is reported here. Northern blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons, revealed that in most cases, only polycistronic mRNA was produced or polycistrons were the most abundant form and that they were not processed into monocistrons. Despite such lack of processing, abundant foreign protein accumulation was detected in these transgenic lines. Interestingly, a stable secondary structure formed from a heterologous bacterial intergenic sequence was recognized and efficiently processed, indicating that the chloroplast posttranscriptional machinery can indeed recognize sequences that are not of chloroplast origin, retaining its prokaryotic ancestral features. Processed and unprocessed heterologous polycistrons were quite stable even in the absence of 3'UTRs and were efficiently translated. Unlike native 5'UTRs, heterologous secondary structures or 5'UTRs showed efficient translational enhancement independent of any cellular control. Finally, we observed abundant read-through transcription in the presence of chloroplast 3'UTRs. Such read-through transcripts were efficiently processed at introns present within native operons. Addressing questions about polycistrons, as well as the sequences required for their processing and transcript stability are essential for future approaches in metabolic engineering. Finally, we have shown phytoremediation of mercury by engineering the mer operon via the chloroplast genome under the regulation of chloroplast native and heterologous 5'UTRs. These transgenenic plants hyperexpress were able to translate MerA and MerB enzymes to levels detectable by coomassie stained gel. The knowledge acquired from these studies offer guidelines for engineering multigene pathways via the chloroplast genome.