The immunotoxin is one of our tools to block transmission of malaria parasite in mosquitos. It is composed of two main parts : The first one is a single-chain antibody fragment (scFv) directed to Pbs2l, which is a surface membrane protein of plasmodium. The second part is a lytic peptide, Shiva-1, which is thought to act by forming “pores” on the parasite’s membrane. The immunotoxin is supposed to specifically target and lyse the parasite.

We tested expression of the immunotoxin in E.Coli. In a western blot analysis of whole cell lysates we could see bands corresponding to full length immunotoxin and possibly degraded fragments of the protein (see figure). The immunotoxin contains a PelB sequence that targets it for secretion into the periplasm. We concentrated the supernatant of both the immunotoxin and a control culture by running it through a filtering device with a 5 kDa cut-off. Running a western blot with these samples (see figure) we concluded that the immunotoxin was secreted as expected.

Figures: The same experiments were conducted for the proteins p25 and p28. No bands were detected on the western blots. This leads us to the conclusion that these proteins were only very weakly expressed or not at all. This might be explained by the fact that we took the native sequence from plasmodium falciparum. The genome of plasmodium is very A-T-rich (put reference). We think that expression of p25 and p28 may be improved by codon optimizing it for expression in bacteria like E.Coli and Asaia like we did for the immunotoxin. Additional to the Western Blots we tried purifying the proteins using Ni-NTA columns. This still needs futher improvement.

As mentionned before, we decided on two different ways to target the parasite and prevent the malaria transmission in the mosquito. Our engineered bacteria could express either an immunotoxin, or two p-proteins, or even both for more efficiency.

Immunotoxin

The immunotoxin is composed of two main parts. The first is a single-chain antibody fragment (scFv) directed to Pbs2l, a surface membrane protein of P. berghei ookinetes. This fragment is linked to the second part, a lytic peptide, Shiva-1. Shiva-1 is a synthetic peptide which amino acids sequence is similar in terms of length (38 amino acids) and amino acid properties to Cecropin B, an antibacterial peptide. The purpose of the immunotoxin is to specifically target and lyse the parasite.

Previous experiments showed that feeding mosquitoes with recombinant E.Coli expressing this immunotoxin induced a significantly lower oocyst density. [1]

However E. Coli is not a natural symbiote of the mosquito. We would like to improve this experiment by making Asaia express the immunotoxin in the mosquitos intestinal tract, and run experiments to see if it significantly reduces the number of oocysts.

The sequence of the immunotoxin was taken from our reference paper [1], and the codons were optimized for E.Coli before ordering the sequence.

P-proteins

The ookinete has been intensively studied by scientists, looking for an ideal transmission-blocking vaccine target. IGEM EPFL team's interest goes toward the ookinete surface membrane proteins Pfs25 and Pfs28. In fact, it has been shown that the binding of specific antibodies to these surface proteins was fatal to more than 99% of the parasites[2]. Indeed, P25 and P28 seem to be essential to the ookinete survival, the crossing of the midgut epithelium and its transformation into an oocyst.

The team decided to design Asaia to express a soluble form of these 2 P-proteins. It could seems contra-intuitive to provide more of these proteins (by expression in Asaia) that are very important for the parasite survival, migration, and development. But our hypothesis is that adding a lot of these proteins in a soluble form will compete with the parasite surface protein and prevent the interaction necessary to its transmission. Because the functions of P25 and P28 are redundant, both proteins need to be inhibited or outcompeted to efficiently block the malaria transmission.

• results*

The p-proteins were not codon optimized for E.Coli, they have the native P.falciparum sequence. P. falciparum has an atipycal genome, very A-T rich. This could explain why there was some difficulties for E.Coli to express our P-proteins. A way to improve the expression could be to optimize the P-proteins codons for E.Coli.

References:

• [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T29-42JHDJD-9&_user=10&_coverDate=03%2F31%2F2001&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=5f4b78b08a1846e241faaed33bc76cb3&searchtype=a 1. Shigeto Yoshida, Bacteria expressing single-chain immunotoxin inhibit malaria parasite development in mosquitoes, Molecular and Biochemical Parasitology (2001)]
• [http://www.nature.com/emboj/journal/v20/n15/full/7593895a.html 2. Ana M. Tomas, Gabriele Margos, Robert E. Sinden, P25 and P28 proteins of the malaria ookinete surface have multiple and partially redundant functions, The EMBO Journal (2001)]
• [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1951121/?tool=pubmed 3. Ajay K. Saxena, Yimin Wu, and David N. Garboczi, Plasmodium P25 and P28 Surface Proteins: Potential Transmission-Blocking Vaccines, Eukaryot Cell (2007)]