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.

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)]

Effector molecules

Gwen: je vais supprimer ca demain, personne n'y voit d'inconvénient?

Candidate effectors molecules to block malaria propagation

• A lytic peptide called SB-37 similar (in term of length and amino acids property) to Shiva-1 (the lytic peptide of our ordered immunotoxin) was used to kill P. falciparum in vitro [1]. SB-37 is a slightly modified version of a Cecropin, the Cecropin B [1], the sequence is in the paper [1].

• Cecropins are peptides derived from the insect immune defence (a giant moth). Cecropin A was expressed in a simbiont of the bug Rhodnius prolixus in order to kill T. cruzi, a parasite that cause the Chagas disease [2]. More recently, It was also expressed in the mosquito’s gut (transgenic mosquitoes) to prevent the propagation of the malaria [3] (See [http://www.anaspec.com/products/product.asp?id=30699 Sequence]). Because of its hight homology the cecropins A and B, cecropin D could also be used (sequence: [4]). There exists 6 cecropins : cecropin A, … , F.

• The Glossina attacin is an antimicrobial peptide effective on both gram-negative bacteria and protozoa (Plasmodium is a protozoa!). It was expressed in a symbiont of the tsetse fly in order to kill the pasasite T.brucei; We should determine if it acts on Asaia, attacins kill E.Coli but [http://www.copewithcytokines.org/cope.cgi?key=Attacins do not act on many other gram-positive and gram-negative bacteria]. Sequence: [5].

Other effectors molecules against Plasmodium are cited by Jacobs-Lorena and al. [6]:

• The salivary gland/midgut peptide (SM1), it is a dodecapeptide (sequence: [7]), It was found by testing a number of random peptides, and it binds to both the gut and salivary gland bocking the Plasmodium at these two stages.

• An other attractive approach is to block chitinase present in the mousquito’s gut. This enzyme plays an important role in modelling of the peritrofic matrix (PM, that surround the bood meal) which is an important barrier for the Plasmodium. It has been shown that inhibiting chitinase make the PM thicker and efficiently blocks the plasmodium development. Chitinase is activated by cleaving an N-terminal propeptide, and it has been demonstrated, by feeding the mosquito with the peptide, that this same propeptide, called prochitinase peptide (13 amino-acids) blocks efficiently the chinase activity and the plasmodium developement in the mosquito’s gut (sequence: [8]).

• The Phospholypase A2 (PLA2) inhibits oocyst formation in the mousquito’s gut by feeding or expressing it in transgenic mousquitos (See [9] and [10]). Interestingly this enzyme comes from the snake or bee venom and its anti-malaria activity does not depend on its hydrolytic activity.

In summary we have those potential effector molecules:

• SB-37
• Ceropins (mainly A, B and D)
• Glossina attacin
• SM-1
• Prochitinase peptide
• PLA2

References

• [http://www.ncbi.nlm.nih.gov/pubmed/3049204 1. Jaynes et al. 1988]
• [http://www.ncbi.nlm.nih.gov/pubmed/9096383 2. Durvasula et al. 1997]
• [http://www.ncbi.nlm.nih.gov/pubmed 3. Kim et al. 2004]
• [http://www.ncbi.nlm.nih.gov/pubmed/7140755 4. Hultmark et al. 1982]
• [http://www.ncbi.nlm.nih.gov/pubmed/18477243 5. Wang et al., 2008]
• [http://www.ncbi.nlm.nih.gov/pubmed/15894187 6. Jacobs-Lorena et al., 2005]
• [http://www.ncbi.nlm.nih.gov/pubmed/11687659 7. Ghosh et al., 2001]
• [http://www.ncbi.nlm.nih.gov/pubmed 8. Bhatnagar et al., 2003]
• [http://www.ncbi.nlm.nih.gov/pubmed/11809789 9. Zieler et al., 2001]
• [http://www.ncbi.nlm.nih.gov/pubmed/12167627 10. Moreira et al., 2002]