Antimicrobial activity of the Flo peptide produced in Scenedesmus acutus and Nannochloropsis oculata.

The continuous increase of bacterial pathogen resistance to conventional antibiotics has challenged the research community to develop new antimicrobial strategies. Antimicrobial peptides (AMP) are a promising alternative to combat multidrug-resistant strains compared to conventional antibiotics because of their biocompatibility. In the present study, the Flo peptide, an AMP from the Moringa oleifera tree, was expressed in the chloroplast of the microalgae Nannochloropsis oculata and Scenedesmus acutus. The transgene insertion was verified by PCR amplification, and the homoplasmy was corroborated in spectinomycin-resistant lines. The identification and quantification of the peptide were performed using ELISA. The antimicrobial activity was studied against the Gram-negative Escherichia coli (ATCC 25,922) and Klebsiella pneumoniae (ATCC 700,603). The inflammatory response of the total soluble proteins of transplastomic N. oculata was assessed by measuring secretion of the cytokines IL-6, IL-10, and alpha-tumor necrosis (TNF-α), and cytotoxicity was assessed. These results provide a potential strategy to produce the Flo peptide in microalgae with antibacterial activities.

Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants.

Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens in developing countries. Some vaccine formulations containing the heat labile toxin B subunit (LTB) have been used in clinical trials; however, the induction of neutralizing antibodies against the heat-stable toxin (ST), a poor immunogenic peptide, is necessary, as most ETEC strains can produce both toxins. In this study, a plant optimized synthetic gene encoding for the LTB-ST fusion protein has been introduced into plastids of tobacco leaf tissues, using biolistic microprojectile bombardment, in an effort to develop a single plant-based candidate vaccine against both toxins. Transplastomic tobacco plants carrying the LTB-ST transgene have been recovered. Transgene insertion into the plastid was confirmed by both PCR and Southern blot analysis. GM1-ELISA revealed that the LTB-ST fusion protein retained its oligomeric structure, and displayed antigenic determinants for both LTB and ST. Western blot analysis, using LTB antisera, confirmed the presence of a 17-KDa protein in transplastomic lines, with the correct antigenicity of the fusion protein. Expression levels of this fusion protein in different lines reached up to 2.3% total soluble protein. Oral immunization of mice with freeze-dried transplastomic tobacco leaves led to the induction of both serum and mucosal LTB-ST specific antibodies. Following cholera toxin challenge, a decrease of intestinal fluid accumulation was observed in mice immunized with LTB-ST-containing tobacco. These findings suggest that tobacco plants expressing LTB-ST could serve as a plant-based candidate vaccine model providing broad-spectrum protection against ETEC-induced diarrhoeal disease.

Expression of an immunogenic F1-V fusion protein in lettuce as a plant-based vaccine against plague.

Yersinia pestis is a pathogenic agent that causes the bubonic and pneumonic plague. The development of an efficient and low-cost oral vaccine against these diseases is highly desirable. In this study, the immunogenic fusion protein F1-V from Y. pestis was introduced into lettuce via Agrobacterium-mediated transformation, and putative transgenic lines were developed. The presence of the transgene in these putative transgenic lines was determined using polymerase chain reaction (PCR), and transgene integration and transgene copy number were confirmed following Southern blot analysis. The presence of specific F1-V transcripts was confirmed by reverse-transcriptase (RT)-PCR. Using monoclonal antibodies, ELISA and western blot analysis revealed that the expected antigenic F1-V protein was successfully expressed in transgenic lines. Mice immunized subcutaneously with lettuce expressing the F1-V antigen developed systemic humoral responses as 'proof of concept' of using lettuce as a production platform for the F1-V immunogen that could be used as a candidate plant-based vaccine against plague.

Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer.

Expression of genes in plant chloroplasts provides an opportunity for enhanced production of target proteins. We report the introduction and expression of a fusion DPT protein containing immunoprotective exotoxin epitopes of Corynebacterium diphtheriae, Bordetella pertussis, and Clostridium tetani in tobacco chloroplasts. Using biolistic-mediated transformation, a plant-optimized synthetic DPT gene was successfully transferred to tobacco plastomes. Putative transplastomic T0 plants were identified by PCR, and Southern blot analysis confirmed homoplasmy in T1 progeny. ELISA assays demonstrated that the DPT protein retained antigenicity of the three components of the fusion protein. The highest level of expression in these transplastomic plants reached 0.8% of total soluble protein. To assess whether the functional recombinant protein expressed in tobacco plants would induce specific antibodies in test animals, a mice feeding experiment was conducted. For mice orally immunized with freeze-dried transplastomic leaves, production of IgG and IgA antibodies specific to each toxin were detected in serum and mucosal tissues.